molecules

Review
Mass-Spectrometry-Based Research of Cosmetic Ingredients
Alina Florina Serb 1 , Marius Georgescu 2,3, * , Robert Onulov 4 , Cristina Ramona Novaconi 4 , Eugen Sisu 1 ,
Alexandru Bolocan 2 and Raluca Elena Sandu 5,6

1 Biochemistry Discipline, Biochemistry and Pharmacology Department, Victor Babes University of Medicine
and Pharmacy Timisoara, Eftimie Murgu Sq. No.2, 300041 Timisoara, Romania; aserb@umft.ro (A.F.S.);
sisueugen@umft.ro (E.S.)
2 Physiology Discipline, Functional Sciences Department, Victor Babes University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Sq. No.2, 300041 Timisoara, Romania; bolocan.alexandru@umft.ro
3 Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babes, ” University of Medicine and
Pharmacy Timis, oara, Eftimie Murgu Sq. No. 2, 300041 Timisoara, Romania
4 Faculty of Medicine, Victor Babes University of Medicine and Pharmacy Timisoara, Eftimie Murgu Sq. No.2,
300041 Timisoara, Romania; robert.onulov@yahoo.com (R.O.); novaconi.ramona@gmail.com (C.R.N.)
5 Department of Neurology, University of Medicine and Pharmacy of Craiova, St. Petru Rares, No. 2-4,
200433 Craiova, Romania; raluca.sandu@umfcv.ro
6 Department of Biochemistry, University of Medicine and Pharmacy of Craiova, St. Petru Rares, No. 2-4,
200433 Craiova, Romania
* Correspondence: georgescu.marius@umft.ro

Abstract: Cosmetic products are chemical substances or mixtures used on the skin, hair, nails, teeth,
and the mucous membranes of the oral cavity, whose use is intended to clean, protect, correct body
odor, perfume, keep in good condition, or change appearance. The analysis of cosmetic ingredients is
often challenging because of their huge complexity and their adulteration. Among various analytical
tools, mass spectrometry (MS) has been largely used for compound detection, ingredient screening,
quality control, detection of product authenticity, and health risk evaluation. This work is focused
on the MS applications in detecting and quantification of some common cosmetic ingredients, i.e.,
preservatives, dyes, heavy metals, allergens, and bioconjugates in various matrices (leave-on or
rinse-off cosmetic products). As a global view, MS-based analysis of bioconjugates is a narrow
Citation: Serb, A.F.; Georgescu, M.; field, and LC- and GC/GC×GC-MS are widely used for the investigation of preservatives, dyes,
Onulov, R.; Novaconi, C.R.; Sisu, E.; and fragrances, while inductively coupled plasma (ICP)-MS is ideal for comprehensive analysis of
Bolocan, A.; Sandu, R.E. Mass- heavy metals. Ambient ionization approaches and advanced separation methods (i.e., convergence
Spectrometry-Based Research of chromatography (UPC2 )) coupled to MS have been proven to be an excellent choice for the analysis of
Cosmetic Ingredients. Molecules 2024, scented allergens. At the same time, the current paper explores the challenges of MS-based analysis
29, 1336. https://doi.org/10.3390/
for cosmetic safety studies.
molecules29061336

Academic Editors: Rosa Iacovino and Keywords: mass spectrometry (MS); cosmetic ingredients; LC-MS; GC-MS; fragrances; preservatives;
Gianluca D’Abrosca dyes; allergens; metals; cosmetics regulation

Received: 19 February 2024

Revised: 11 March 2024
Accepted: 15 March 2024
Published: 17 March 2024
1. Introduction
A cosmetic product is defined by European legislation as a substance or mixture
designed to be used in contact with the external parts of the human body. Cosmetic
products can be divided into two main categories: leave-on products, which remain in
Copyright: © 2024 by the authors.
contact with the skin surface after their application, and rinse-off products, which are
Licensee MDPI, Basel, Switzerland.
removed from the skin after a short time period [1,2].
This article is an open access article
Unlike pharmaceuticals, cosmetics do not play a role in curing diseases. However,
distributed under the terms and
modern cosmetics are often “functional” products for wrinkle care, whitening, hydration,
conditions of the Creative Commons
and treatment of pores, stains, etc., that are produced to meet consumers’ present needs.
Attribution (CC BY) license (https://
Thus, some cosmetics contain quasi-drugs; however, their effects on the body are mild.
creativecommons.org/licenses/by/
4.0/).
Some cosmetics manufacturers have begun to use the term “cosmeceutical” to refer to

Molecules 2024, 29, 1336. https://doi.org/10.3390/molecules29061336 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 1336 2 of 48

products that have similar benefits to drug use [3]. Still, the new term is not recognized
by the FDA (Food and Drug Administration). Instead, the FDA classifies a product as
either a drug, a cosmetic, or a combination of the two. Because cosmetics are used freely
by consumers who do not have daily exposure limits, the absorption of quasi-drugs (and
other ingredients) through the skin must be carefully controlled, making monitoring the
transdermal absorption of drugs one of the most essential topics in the field of cosmetic
sciences. A wide variety of raw materials that can come from synthetic, biosynthetic, or
natural extracts are used in the formulation of personal care products. Suppliers of natural
extracts often claim that their products are “organic”, “pesticide-free”, or “natural”, but
it is necessary to check whether these statements are, in fact, true. Moreover, these raw
materials can be associated with a wide range of contaminants, byproducts, and degradants
of the raw materials used in their forms.
Cosmetic formulations include an enormous variety of different types of ingredi-
ents, such as active principles, excipients, and additives. Constituents of cosmetics can
be generally grouped as ingredients for giving the product form (water, oils, silicones,
surfactants, polymers, polyhydric alcohols, inorganic and organic powders), ingredients
for stabilizing the products (antimicrobial agents, pH control chemicals, antioxidants, and
chelating agents), ingredients for giving efficacies, effects, and concepts (plant extracts and
herbal medicine components, microbial-derived ingredients, proteins and amino acids, ce-
ramides, and vitamins) and ingredients acting on the senses of users (coloring and scenting
agents) [4]. Ingredients of cosmetics are combined so as to achieve the targeted efficacies
and effects and be suitable for the body part on which the product is to be applied, the
purpose, and the method of use. For each ingredient, there is a limitation on the quantity
that can be included, depending on the body part the product is to be used on and the
method of use (to be left on or rinsed off). In addition, there are ingredients whose inclusion
is restricted or prohibited due to safety issues [4].
The analysis of cosmetics represents a challenge mainly because of the great variety
of ingredients and formulations leading to immense matrix complexity and variability. In
this field, many analytical techniques play a crucial role, each designed to meet specific
demands to understand the molecular nature of cosmetic products and the complexity of
their formulations: chromatography (liquid chromatography (LC) [5,6], gas chromatog-
raphy (GC) [7,8], supercritical fluid chromatography (SFC) [9], capillary electrophoresis
(CE) [10,11], spectroscopy [12–17], electrochemistry [18], colorimetry [19,20], mass spec-
trometry (MS) [21–31], interfacial methods [32,33], rheological assessment [34–36], olfac-
tometry, and electronic nose technology [37–39]. Recent advances in MS and ionization
techniques provide access to richer and deeper information on monitoring the molecu-
lar compositions of endogenous or exogenous compounds in or on the skin as well as
those used in cosmetic formulations, with less time and effort. In this context, in order
to provide safe and efficient products to customers in the cosmetics industry, MS is an
indispensable analytical tool. Chromatographic methods hyphenated to MS, such as liquid
chromatography–mass spectrometry (LC-MS) and gas chromatography–mass spectrometry
(GC-MS), offer the remarkable capability to separate and identify complex mixtures within
cosmetic formulations, allowing it to be used for qualitative and quantitative analysis with
good sensitivity. However, they often necessitate many instrumental methods for broad
coverage of analyte classes and various matrices.
Comprehensive preparation and chromatographic separation can considerably de-
crease sample throughput, making direct analysis techniques such as ambient MS exciting
alternatives to traditional methods [40–46], mainly when direct assessment of cosmetic
efficacy on living organisms (e.g., skin) is required.
In addition to the sensitivity and specificity of the MS method, it is applied to a wide
range of compounds, which makes it irreplaceable for the investigation of complex systems
required for the development of cosmetics. Analytical measurement for quality control is re-
quired to warrant that formulations of cosmetics are in accordance with legislation, with the
efficacy and safety of cosmetics being of vital importance and serious concern worldwide.
Molecules 2024, 29, 1336 3 of 48

In the present review, the publications related to the analysis of cosmetic ingredients
by MS are presented and examined. Most of the published research articles are based on the
hyphenation of chromatography and MS or tandem MS and focus on the assessment of cos-
metic ingredients in different formulations, including those restricted or banned. Moreover,
the effects of the exposure of human skin to different ingredients are also discussed.

2. Literature Research Methodology

The literature search incorporated peer-reviewed journal articles written in English
and published between 1992 and 2024, found in the PubMed and Web of Science databases.
The main keywords of the search were “mass spectrometry” and “preservatives” and
“cosmetic”, “mass spectrometry” and “colorants” and “cosmetic”, “mass spectrometry”
and “allergens” and “cosmetic”, “mass spectrometry” and “metals” and “cosmetic”, as
well as “mass spectrometry” and “cosmetic ingredients”.
In both databases, the term “All fields” was used, which searches titles, abstracts,
author keywords, and keywords plus. Only articles written in English, available full texts,
and articles containing publications dedicated to the analysis of cosmetic ingredients by
MS are included in this review. Publications containing the analysis of other ingredients
than those found in the investigated topic and other analytical methods except MS or MS
in conjunction to different separation methods were excluded. Associated data, preprints,
meeting abstracts, opinion letters, data papers, notes, editorial materials, and papers not
written in English were excluded as well.
Duplicates were removed, and, further, the selection of the publications included in
the manuscript was based on the qualitative and quantitative evaluation of articles from
the PubMed and Web of Science database, particularly the title of paper, the keywords, the
abstract, the year of publication, and then the main text.

3. MS Analysis of Cosmetic Ingredients

3.1. Analysis of Bioconjugates in Cosmetic Products
The literature data is scarce regarding the investigation by MS of the various bioconju-
gates existing in cosmetic formulations. A study presented the analysis by matrix-assisted
laser desorption/ionization (MALDI) MS of the lipid profile in different cosmetic products
(lipsticks and dermatograph pencils), which made it possible to differentiate new products
from those in use and those that have expired [47]. The analysis was performed without an
elaborate preparation of the samples. For lipstick application, a soft polymer-coated bar
was coated with lipstick samples and then “stamped” onto specific stainless steel plates
(targets) of the MALDI mass spectrometer. The samples corresponding to the dermato-
graph pencils were applied directly onto the surface of the plate. The matrix used was
α-cyano-4-hydroxycinnamic acid (CHCA) in a solution of concentration 10 mg/mL (50%
acetonitrile: methanol) and the samples were covered using a commercial brush [47]. All
samples were analyzed in the positive-ion mode. The assignment of chemical structures
was performed following the analysis of collision-induced dissociation (CID) MS/MS frag-
mentation spectra and calculations with Mass Frontier software (v. 6.0, Thermo Scientific,
Carlsbad, CA, USA). Analysis of the data obtained showed that lipsticks often have a
formula rich in a very complex lipid matrix (up to 90%), such as Ricinus communis oil
(castor oil), beeswax, Candelilla wax, Carnauba wax, lanolin, etc.
These lipid matrices are composed of a wide range of lipid classes, generally triacyl-
glycerols (TAG), sphingolipids (SP), free fatty acids, and fatty esters, among many others.
This great variability in composition is what gives these products particular characteristics
such as fineness, thickness, creamy texture, and even the thixotropic effect. When ana-
lyzing and comparing different types of lipsticks (new, in use, and expired), a different
composition of lipids was observed, especially in terms of oxidation of TAGs and the
presence or absence of lipids with higher complexity, such as ceramides: the presence of
cleaved species in expired lipsticks, such as diacylglycerols (DAG) which are usually in the
range m/z 500–600, for “in use” lipsticks showed several types of TAGs, while “new” ones
Molecules 2024, 29, 1336 4 of 48

contained sphingolipids (ceramides) as the main difference from the others [47]. Similar
observations were noted for dermatograph pencils: the only notable difference observed
was the presence of oxidized TAGs in expired products and, due to the introduction of
oxygen species into molecules (epoxy, keto, and hydroxy acids), the m/z range increases
(~900 and higher). The ions from m/z 700–800 demonstrate the presence of TAGs in both
new and “in use” samples. The difference between “in use” and expired samples of der-
matograph pencils and lipsticks in terms of their compositions may be due to the fact
that lipsticks are usually in contact with saliva and other compounds around the mouth
area; therefore, other chemical transformations are more likely to occur than oxidation,
compared to dermatograph pencils [47].
In order to determine the commercial ceramides (from natural extracts, Doosan Cor-
poration Bio, Seoul, Republic of South Korea) in cosmetics for the quality control of the
product formulation, a fast, sensitive and selective method was used that involves the
coupling of reverse-phase liquid chromatography (LC) with electrospray ionization (ESI)-
MS [48]. Using LC/ESI-MS with fragmentation at source by CID in both positive- and
negative-ionization modes, it was possible to separate and identify the structures of sph-
ingoid bases (phytosphingosine at m/z 267, 255 and 225 and sphingosine at m/z 263
and 237) and the N-acyl chains of ceramides, as well as an impurity. The VG MassL-
ynx MS software (https://www.waters.com/nextgen/us/en/products/informatics-and-
software/mass-spectrometry-software/masslynx-mass-spectrometry-software.html) used
could switch between positive- and negative-ion modes within the same HPLC run, and at
higher cone voltages, in both modes, they yielded fingerprint spectra providing comple-
mentary structural information about both the fatty acid and the sphingoid base moiety.
It was observed that ceramides were separated and detected with a higher degree of
sensitivity in the positive-ion mode than in the negative-ion mode, while the product ion
spectra in negative-ion mode of ceramide species provided more structural information
than those obtained in positive-ion mode [48]. This study is one of the few that investigates
ceramides present in cosmetics.
Ceramides are involved in the skin’s barrier function, with their low levels in the
intercellular lipid lamellae of the stratum corneum being associated with dry skin [49]. In
practice, certain ceramides applied individually or as an emulsion mixture synergistically
improve the skin barrier function in humans [50]. Moreover, some dominant emulsions
with ceramide or pseudoceramide can decrease the severity of pruritus and trans-epidermal
water loss in various subjects [51]. Recently, ceramides have also been reported as one of
the main constituents of topical formulations for rosacea [52] and atopic facial eczema [53].
Although the use of ceramides in cosmetics is widespread, more extensive studies on the
toxicity and effects of topical administration of cosmetics containing these types of lipids
are needed. MS-based methods are indispensable tools for cosmetic science, since the
molecular composition on the surface and in most of the skin is extremely complex and
difficult to elucidate.
In recent years, research in the field of biosurfactants has begun to intensify due
to the great potential for their use in various branches of the economy, industry, and
medicine. Biosurfactants can be used as emulsifiers, de-emulsifiers, softeners, dispersants,
foaming agents, active food ingredients, and detergents in various industrial sectors such
as oil, organic chemistry, food, cosmetics and pharmaceuticals, mining and metallurgy,
fertilizers, environmental protection, etc. In this sense, some glycolipids have particular
properties such as surfactant, gelling, and antimicrobial, and as a result, these glycolipids
are increasingly used not only in pharmaceutical applications but also in the cosmetics
industry [54,55]. Due to all the properties listed above, as well as biodegradability and
biocompatibility, sucrose ester-type glycolipids are used in many cosmetic applications.
These glycolipids include the following:
- Lipids with manosyl erythritol (MEL)—these are glycolipids composed of a fatty acid
ester, either 4-OD-manopyranosyl-erythritol or 1-OD-manopyranosyl-erythritol [56],
produced by yeasts of the genus Pseudozyma, which have been shown to have a
Molecules 2024, 29, 1336 5 of 48

moisturizing action compared to natural ceramides on the skin. These glycolipids are
used in antiwrinkle and skin-smoothing cosmetics [57].
- Sophorolipids (SLP)—these are glycolipids composed of fatty acids of 16 or 18 carbon
atoms bound to a sophorose as a hydrophilic part, produced by several species of
Candida or other related yeast species. These glycolipids are used in detergents,
lipsticks, lip creams, and eyeshadow [58].
- Trehalose lipids—these are glycolipids composed of fatty acids linked to a disaccharide,
trehalose, which is a nonreducing disaccharide in which two glucose molecules are
linked in an α, α, 1,1-glycosidic bond.
With regard to polysaccharides, their use in cosmetics has been as pervasive as the
use of cosmetics. Historically, due to their rapid availability from common natural sources
and their varied and unique multifunctionality, polysaccharides have been included in
cosmetics for centuries; for example, the use of β-glucan derived from yeast extracts is used
as a natural healing agent. Today, polysaccharides play an even more significant role in
the technology of formulating cosmetics. The interaction of polysaccharides with other
ingredients in a formulation (e.g., actives, surfactants, salts, other polymers, etc.) and the
ease with which they can be chemically modified allowed their pre-eminent use in cosmetics.
In addition, polysaccharides, of natural origin and polymeric, are renewable and do not
have a safety profile, since that is not accorded to synthetic polymers. When considering
the many cosmetically acceptable polysaccharides, it is found that their morphology and
functionality cover the entire territory of polymer technology. Polysaccharides perform a
multitude of cosmetic functions. For example, they act as rheology modifiers, suspending
agents, hair conditioning agents, and wound healing agents. They moisturize, hydrate,
emulsify, and hemolyze. Trying to distinguish the influence of a single polysaccharide in
a formula is like trying to understand the action of a finger while ignoring the hand as
a whole.
Dextrins are a class of low-molecular-weight carbohydrates produced by the acidic
and/or enzymatic partial hydrolysis of starch or glycogen, with the structure α-(1 → 4)-
Glucose (Glc) of amylose and the branched structure α-(1 → 4)-Glc and α-(1 → 6)-Glc of
amylopectin, but with lower polymerization. Converting starch to dextrins is a simple and
inexpensive method of reducing thickening and accelerating its moisturizing properties.
Dextrin-containing solutions are clear but have a much lower viscosity than parental starch.
Dextrins have a variety of uses as absorbents, binders, fillers, adhesives, films, conditioning
agents, thickeners, or as a foundation for makeup and face powders. Dextrins are also used
as an aid for spray drying or encapsulation to provide new dosage forms, controlled-release
or tasteless drugs, and food flavorings. Dextrins are an accessible raw material, generally
considered safe [59]. Regarding dermal cosmetics and biomedical applications, the use
of dextrins is still relatively unexplored; they are used clinically as peritoneal dialysis
solutions that can also act as drug delivery solutions [60] and as a wound dressing agent.
Dextrins have a set of advantages that enhance their specific use in biomaterials: they are
biocompatible and nonimmunogenic materials, degradable in vivo by amylases, and their
molecular weight ensures renal elimination, thus avoiding their accumulation in tissues
due to repeated administration [61].
Regarding the analysis of carbohydrate polymers, MALDI-TOF MS has proven to be an
accurate technique for characterizing their molecular weight distribution [62,63]. Kazmaier
et al. [64], studied a set of maltodextrins with a degree of polymerization in the range of 2
to 13 and concluded that MALDI-TOF MS is the most suitable technique for detecting high-
molecular-weight species such as oligosaccharides. Thus, it was observed that the degree
of polymerization increases linearly with the molecular weight [64,65]. Silva et al. [66]
reported comprehensive structural characterization of several commercial dextrins, which
were used to produce adipose dihydrazide crosslinked oxidized dextrin hydrogels by
MALDI-TOF in the positive-ion reflector mode using delayed extraction in the mass range
between 600 and 4500 Da, and size exclusion chromatography (SEC). MS characterization
provided important data on the chemical structure of different maltodextrins, determining
 hydrogels by MALDI-TOF in the positive-ion reflector mode using delayed extracti
the mass range between 600 and 4500 Da, and size exclusion chromatography (SEC
characterization provided important data on the chemical structure of different m
Molecules 2024, 29, 1336 dextrins, determining the number of glucose oligomers (6–17) contained 6inof 48
carbohy
polymer chains (degree of polymerization, DP), which is essential to establish pote
applications for commercial maltodextrins [65].
the number of glucose oligomers (6–17) contained in carbohydrate polymer chains (degree
of polymerization, DP), which is essential to establish potential applications for commercial
3.2. Analysis of Preservatives
maltodextrins [65]. in Cosmetic Products
Parabens
3.2. Analysis are esters in
of Preservatives ofCosmetic
the Products
parahydroxybenzoic acid (methylparaben (
ethylparaben (EP),arepropylparaben
Parabens (PP), butylparaben
esters of the parahydroxybenzoic (BP), isobutylparaben
acid (methylparaben (MP), ethyl- (IBP)
propylparaben
paraben (EP), (IPP), benzylparaben
propylparaben (BeP), and
(PP), butylparaben (BP),heptylparaben (HP)isopropyl-
isobutylparaben (IBP), (Figure 1)), w
paraben (IPP), benzylparaben (BeP), and heptylparaben (HP) (Figure
due to their low volatility, high stability, and their antibacterial and antifungal1)), which, due to pr
their low volatility, high stability, and their antibacterial and antifungal properties, have
ties, have been the most used as preservatives in cosmetics, personal care, pharmac
been the most used as preservatives in cosmetics, personal care, pharmaceuticals, food, and
cals, food, andproducts.
industrial industrial products.

FigureFigure
1. Chemical
1. Chemical structures ofparabens
structures of parabens found
found in cosmetic
in cosmetic products.
products. (a) (a)(c)MP;
MP; (b) EP; (b)
PP; (d) EP; (c) P
IPP;
(e) BP; (f) IBP; (g) BeP; (h) HP.
IPP; (e) BP; (f) IBP; (g) BeP; (h) HP.
Parabens have traditionally been considered low-toxicity compounds. However, it
Parabens have traditionally
has been discovered been considered
that some parabens can function as low-toxicity compounds.
endocrine disruptors, leadingHowev
to a potential increase in the incidence of breast cancer in
has been discovered that some parabens can function as endocrine disruptors,women or the onset of ma- leadi
lignant melanoma. They have also been associated with contact dermatitis and rosacea.
a potential increase in the incidence of breast cancer in women or the onset of malig
These risks are further exacerbated by the ability of parabens to be absorbed by human
melanoma. Theybeing
skin without havedegraded
also been associated
by esterases withthey
[67]. Since contact dermatitis
are unregulated, theyand rosacea. T
can con-
risks are
tain further exacerbatedthat
levels of preservatives bypose
thea ability of parabens
health risk, which is doublyto betrueabsorbed by human
for counterfeit
cosmetics. Therefore, accurate methods must be used to determine
without being degraded by esterases [67]. Since they are unregulated, they can co the levels of these
compounds in cosmetics and personal care products. Current analytical methods based on
levels MS
of for
preservatives that pose a health risk, which is doubly true for counterfeit
the determination of preservatives in cosmetics and personal care products include
metics. Therefore, accurate
high-performance methods must
liquid chromatography be used
(HPLC) to determine the levels
and ultra-high-performance (UHPLC) of these
pounds in cosmetics and personal care products. Current analytical methods base
coupled with mass spectrometry (HPLC-MS and UHPLC-MS), as well as gas chromatogra-
phy coupled with mass spectrometry (GC-MS). For analyte quantitation, internal standards
MS for the determination of preservatives in cosmetics and personal care produc
(ISs) can be used. These are chemical substances which are added at the same concentration
clude tohigh-performance
all samples throughout liquid chromatography
a quantitative analysis. The main(HPLC)
criteria forand ultra-high-perform
choosing an internal
(UHPLC) coupled
standard is basedwith mass spectrometry
on resolution—the IS should not(HPLC-MS and the
be present within UHPLC-MS),
sample matrixas or well a
interfere with any other compounds present within the sample.
chromatography coupled with mass spectrometry (GC-MS). For analyte quantita Ideally, a compound which
is similar in nature to the target analyte(s) would be chosen, as this is likely to behave in
internal standards (ISs) can be used. These are chemical substances which are add
Molecules 2024, 29, 1336 7 of 48

a very similar way, giving a similar retention time, peak shape, and response. It is very
common in the GC-MS method for the deuterated form of the target analyte to be used.
For complex analysis with a large number of components, multiple ISs can be used to
calculate analyte concentrations throughout the method. Using an IS is a powerful tool
for minimizing the effects of random and systematic errors during analysis, helping to
improve the precision of results and reduce the need for repeat measurements.

3.2.1. HPLC-MS and UHPLC-MS

HPLC-ESI-MS using ionization in both positive- and negative-ion mode and scanning
in the mass range of m/z 100–1000 was applied to determine MP, EP, PP, and BP, butylated
hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), as well as α-tocopherol (α-t)
and α-tocopherol acetate (α-ta) in various cosmetics (a commercial lanoline cream, a com-
mercial skin milk, and a commercial cream) after supercritical fluid extraction procedure
(SFE) and separation of the analytes on a C18 reversed-phase column using methanol–water
as mobile phase [68]. Pseudomolecular ions [M − H]− or [M + H]+ were obtained as the
base ions. MP, EP, PP, BP, BHA, and BHT were easily deprotonated at the electrospray ion
source under the experimental conditions to form the negative molecular ions [M − H]− ,
and, therefore, ESI in the negative-ion mode was selected as the best ionization mode and
employed for four paraben preservatives, BHA and BHT. ESI in the positive-ion mode was
employed for α-tocopherol and α-tocopherol acetate. The base ions were used to quantify
each compound to increase sensitivity in the selected ion monitoring (SIM) mode. This
method is accurate and reliable for all analytes and made it possible to determine their
concentrations in real commercial cosmetics from not detected to 977 mg/kg.
Nevertheless, it is challenging to obtain data on the use of cosmetics and the biological
(e.g., human blood serum) concentrations of their active ingredients. Using LC-ESI-MS/MS,
it was possible to determine MP and PP concentrations in serum and correlate the results
with the routine application of oral cosmetics (lipstick) [69]. Tahan et al. [69] analyzed blood
samples (15 mL) of volunteer women for three phases: the women used paraben-containing
products according to their routine (phase 1), the women used lipsticks containing MP
and PP for five days in conjunction with the routine use of paraben-containing products
(phase 2), and in phase 3, the women routinely used paraben-containing products while
refraining from using lipstick for five days. A statistically significant difference between
serum parabens concentration in women who used lipstick containing these preservatives
compared to the absence of this cosmetic in their daily routine was demonstrated, and a
strong association between serum parabens and lipstick use was observed.
While the methods used for the preservative analysis of cosmetic products have
mainly focused on the determination of parabens, the analysis of more than one class of
preservatives is still a field under development. Using LC-ESI-MS in addition to MP, EP, PP,
and BP, four other preservatives, BeP, butylated hydroxyanisole (BHA), DL-α-tocopherol
acetate, and 2,6-di-tert-butyl-4-methylphenol (BHT), could be separated, identified, and
quantified in samples of 100 mg of cosmetic and personal care products (lipstick, foundation,
deodorant, hand lotion, soap, and toothpaste). BHT, MP, and EP were detected in most
samples, while BeP and BP were not detected in any of the tested samples. It is interesting
to note that the deodorant sample tested and one of the foundations had relatively higher
amounts of parabens (MP and EP) compared to the other products. PP was also detected in
the foundation sample. It was observed that some of the analyzed products also contained
peaks belonging to preservatives that were not listed on the list of ingredients on the product
label. In comparison with other literary references, this method combines a simplified
and inexpensive sample preparation procedure with a short analysis time (8 min) while
providing a similar, if not improved, degree of separation and sensitivity [70].
Methods based on UHPLC-ESI-MS have been used more to determine parabens (MP,
EP, PP, and BeP) as contaminants in ambient waters in only 9 min of chromatographic
separation [71].
 Molecules 2024, 29, 1336 8 of 48

Moreover, for monitoring long-term exposure to parabens, an improved analytical

method based on UHPLC-ESI-MS for rapid and direct determination of parabens in hair
samples was successfully developed and implemented using online extraction and neg-
ative ionization. Hair samples (n = 10) were cut into pieces of 1–2 mm and placed in
methanol/dichloromethane (1:1) solution, extracted by ultrasonication at room temper-
ature for 30 min twice, and subjected to centrifugation, after which the supernatant was
injected into the LC-MS/MS. Five parabens (MP, EP, PP, BP, and BeP) were, thus, de-
tected and quantified (Table 1) in hair samples by LC-MS/MS using online extraction
and were completely separated in MRM mode. All of the parabens were observed as the
deprotonated precursor ions of [M − H]− type, and for the generation of MS/MS spectra,
deprotonated precursor ions were fragmented at optimized parameters and the product
ions were generated from the loss of either the alkyl chain or benzyl groups from the ester
group (m/z 136 ion), followed by the loss of CO2 (m/z 92 ion)—the most abundant product
ion of MS/MS for all the parabens which was chosen for quantification, whereas m/z
136 ion (the second highest ion) was used as confirmation for the parabens [72].

Table 1. The parabens detected and quantified in hair samples by LC-MS/MS.

Monoisotopic Mass Concentration Range

Paraben
(Da) (ng/g, Mean ± SD) (ng/g, Mean ± SD)
MP 152.047348 123.6 ± 61.6 48.3–224.2
EP 166.062988 64.5 ± 43.5 11.5–158.3
PP 180.078644 136.9 ± 48.5 70.2–214.5
BP 194.094299 74.2 ± 27.5 25.4–111.1
BeP 228.078644 55.6 ± 24.3 15.3–100.2

All of the parabens were observed as deprotonated precursor ions [M − H]− , which
were subjected to fragmentation by MS/MS. The obtained product ions resulted from the
loss of either the alkyl chain or benzyl groups from the ester group (m/z 136 ion—the
second highest ion which was used as confirmation for the parabens), followed by the loss
of CO2 (m/z 92 ion—the most abundant product ion of fragmentation for all the parabens,
which was chosen for quantification).
The developed method has been shown to be sensitive, selective, and accurate and
was appropriate in hair sample analysis [72].
Other compounds used as common preservatives in many personal care products
Molecules 2024, 29, x FOR PEER REVIEW
(mascara, makeup remover, liquid soaps, body wash, hairspray, hair color, conditioner,
shampoo, lotion, baby shampoo, baby lotion, sunscreen, shaving cream, and detergents)
are methylisothiazolinone (MIT) and methylchloroisothiazolinone (MCI) (Figure 2).

Figure 2. Chemical structures of (a) MIT (C4 H5 NOS) and (b) MCI (C4 H4 ClNOS).
Figure 2. Chemical structures of (a) MIT (C4H5NOS) and (b) MCI (C4H4ClNOS).

MIT and MCI inhibit bacterial growth in cosmetic products on their

most commonly used as a mixture in products. Their presence in differ
products has been linked to allergic reactions, lung toxicity, and possible
Molecules 2024, 29, 1336 9 of 48

MIT and MCI inhibit bacterial growth in cosmetic products on their own, but are most
commonly used as a mixture in products. Their presence in different cosmetic products has
been linked to allergic reactions, lung toxicity, and possible neurotoxicity [73] and has been
investigated over time using GC-MS, LC-MS, and HPLC-UV. In recent years, an efficient
UHPLC-MS/MS method was developed and validated for the determination of MIT and
MCI in selected cosmetic products (shampoo/conditioners or skin care products, such as
body lotions, gels, moisturizers, and body cleansers) by Wittenberg et al. [74]. The method
used in this study optimizes the extraction and chromatography parameters of Lin et al. [75]
by using four columns (Waters Acquity BEH C18, Waters Acquity BEH Amide, Agilent
Poroshell 120 PFP, and Phenomenex Kinetex HILIC) in addition to the column used by Lin
et al. (Waters Acquity HSS T3) to increase the retention times of the analytes and achieve an
efficient determination of only MIT and MCI in cosmetic products. However, the Acquity
HSS T3 column in combination with H2 O w/0.1% formic acid (FA) and MeOH w/0.1% FA
as the mobile phases was selected for separation because it produced optimal peak shape,
sensitivity, and retention times. Thus, the mass spectrometer was operated in scheduled
multiple reaction monitoring (MRM) mode and used positive ESI as the ionization source. A
stock solution containing 5 g/mL of MIT and MCI in 50:50 H2 O/acetonitrile v/v and stock
IS solution containing 250 ng/mL of MCI-d3 in 50:50 H2 O/acetonitrile v/v were prepared
separately in different volumetric flasks. Ten calibration solutions were prepared using the
stock solution. The concentration ranges for the standard solutions were 0.1–500 ng/mL
for MIT and 0.1–1000 ng/mL for MCI. A constant concentration of 25 ng/mL of the IS was
added to each standard solution and was used for quantitation by plotting the ratio of
analyte signal to internal standard signal against the concentration of the analyte. Analyte
confirmation was determined by the primary transition to secondary transition ratio. Any
value within 15% of the theoretical ratio (0.734) was confirmed as a true-positive result.
The lower limit of quantitation was determined to be 0.1 g/g for both preservatives.
The concentrations of MIT and MCI ranged from not quantified, or below the lower limit
of quantitation, to 89.64 g/g and not quantified to 10.31 g/g, respectively.
The analytical method described by Wittenberg et al. [74] was proved to be the fastest
and most sensitive method for identifying and quantifying MIT and MCI in cosmetic
products and may be applied to a wide variety of cosmetic products, being suitable for
monitoring the frequency of incorrect labeling of MIT and MCI on cosmetic product
ingredient lists.

3.2.2. GC-MS
In recent years, GC-MS has been increasingly applied to paraben analysis, competing
with traditional HPLC-UV in terms of the number of publications and even surpassing
it in terms of application to environmental analysis. The proposed GC-MS methods for
the determination of parabens are based on a variety of mass analyzers: Q [76–80], triple
Q [81], IT [82], and TOF [83]. In general, GC-MS has the same advantages as HPLC-MS:
unambiguous identification of analytes and low detection limits that allow the determina-
tion of parabens present in low concentrations and their simultaneous determination with
other species of various natures. GC-MS also has some advantages over HPLC-MS, with
higher resolution, lower costs, and lower solvent waste production. On the other hand,
GC-MS usually requires derivatization of the analytes to obtain their corresponding volatile
derivatives. Thus, the GC-MS methods for the determination of parabens can be divided
into three groups: (a) based on derivatization by acetylation with acetanide—applied to
the determination of parabens and other preservatives in soaps, shampoos, makeup prod-
ucts, creams, body milk, etc. [82]; (b) based on derivatization by silylation with N, O-bis
(trimethylsilyl) acetamide—applied for the determination of parabens in water and cosmet-
ics [76]; (c) without derivatization, in the form of GC-MS [84] and GC-MS/MS [81]. In the
latter case, isotopically labeled versions of these parabens were used as IS for quantitation
to compensate for the fluctuation in instrument response and matrix effects in complex
matrices such as cosmetic products. A stock standard solution of parabens was prepared in
Molecules 2024, 29, 1336 10 of 48

MeOH to a concentration of 5000 g/mL. Working standard 1 was prepared by transferring

1 mL of the MP stock solution, 2 mL of the EP stock solution, 4 mL of the PP stock solution,
and 5 mL of the BP stock solution into a 20 mL scintillation vial. Working standards 2 and 3
were prepared by diluting working standard 1 8-fold and 50-fold with MeOH, respectively.
A stock IS solution was prepared by weighing each paraben-d4 standard into a volumetric
flask, to which MeOH was added up to a concentration of 4000 g/mL. A working IS was
prepared by transferring 1, 2, 4, and 5 mL of MP-, EP-, PP-, and BP-d4 stock standard
solution into a vial and mixing well. Both working standards 2 and 3 and the working IS
were used to prepare calibration standards in MeOH. Eight point calibration curves using
peak area ratios were created. The concentrations ranged from 50.0 to 10,000 ng/mL, 100.0
to 20,000 ng/mL, 200.0 to 40,000 ng/mL, and 250.0 to 50,000 ng/mL for MP, EP, PP, and
BP, respectively. IS concentrations in each calibration standard were 1000, 2000, 4000, and
5000 ng/mL for of MP-, EP-, PP-, and BP-d4, respectively.
The measurements were performed using dynamic monitoring of the selected reaction
(automatic selection of the optimal scanning parameters for each analyte during elution,
via software); this procedure led to an improvement in the peaks corresponding to the MS
analysis of the samples, further reducing the loss of resolution caused by the absence of a
derivatization reaction.

3.3. Analysis of Colorants in Cosmetic Products

Due to the hydrophilic nature of most dyes, LC is the usual choice in combination
with MS for their investigation. Chromatographic separation is often required to properly
identify and quantify dyes in cosmetic samples. Due to the ability of dyes to absorb in
the UV–Vis spectrum, diode-array (DAD) or UV–Vis detectors have traditionally been the
preferred detectors. In recent years, MS has become a valuable choice [23,85–88], given
the increased selectivity and sensitivity that are especially useful in the analysis of banned
compounds. MS exceeds DAD limitations, such as the overlap of UV–Vis spectra between
matrix ingredients.
Largely, acid (possessing acidic groups, such as SO3 H and COOH and of anthraquinone-,
azo-, and triarylmethane type), direct, and fiber-reactive dyes are manufactured as salt
compounds, are typically anionic, and ionize in negative-ion mode in MS, while basic dyes
(usually salts generated by aromatic bases reacting with acids) are cationically charged and
ionize in positive-ion mode in MS; disperse dyes (mostly polar molecules containing azo or
anthraquinone groups) generate positive ions during mass spectrometric analysis, but the
dye itself is nonionic [89].
In some studies, a comparison between LC-DAD and LC-MS/MS [85,88] was es-
tablished. In this way, Noguerol et al. [88] developed two LC methods: with UV–Vis
detection and MS in tandem with triple quadruple with ESI ionization performed in
positive-ion mode for routine control of 10 dyes (Fat Brown RR, Malaquite Green Carbinol
Base, Dimethyl Yellow, Sudan I, Sudan Orange G, Solvent Blue 35, Sudan II, Sudan Black,
Sudan III and Sudan IV), especially dyes banned in the cosmetics industry. LC-ESI-MS/MS
in full mass spectrum scanning mode allowed structural information to be obtained about
multiple peaks observed for some of the dyes studied by HPLC-UV/Vis analysis. The
presence of more than one peak for a compound was mainly due to possible isomerization
processes, impurities, or degradation products. Multiple reaction monitoring (MRM) mode
was used for quantification. In terms of sensitivity, there was a remarkable difference
between the two methods. The quantification limits were one or two orders of magnitude
smaller for the LC-MS/MS analysis, which is particularly appreciated given the degree
to which these dyes are restricted in commercial products. The combination of LC and
MS in the analysis of cosmetic dyes was first reported by Xian et al. [87] in 2013. Subse-
quently, Guerra et al. have developed several improved methods based on this analytical
technique [23,85,86].
Using sequential steps based on HPLC, LC-MS/MS, and LC-Q-TOF-MS, in Han
et al. [90], 13 banned synthetic colorants were detected and structurally characterized (Basic
Molecules 2024, 29, 1336 11 of 48

Blue 26, Basic Red 2, Disperse Brown 1, Disperse Orange 3, Disperse Yellow 3, HC Blue No.
2, HC Yellow No. 5, Solvent Orange 4, Solvent Yellow 1, Solvent Yellow 3, Solvent Orange
7, Solvent Red 24, and Basic Yellow 28) in 120 purchased long-lasting cosmetic samples
(classified as tattoo eyebrow, tattoo lipstick, and hair tint). First, sample solutions were
prepared with 100% methanol to a volume of 50 mL and subjected to ultrasonic extraction
at room temperature for 30 min, followed by centrifugation and filtration. As the next
step, the illegal colorants were detected by HPLC with diode-array detection (HPLC-DAD)
using an Agilent 1260 Infinity II LC system (Agilent, Santa Clara, CA, USA) equipped with
a DAD and separated on a Zorbax Eclipse XDB-C18 (4.6 mm × 150 mm, 5 µm; Agilent,
Santa Clara, CA, USA) column and by LC-MS/MS performed on a Waters ACQUITY
ultra-performance liquid chromatography coupled with a Xevo TQ-XS (Waters, Milford,
MA, USA) system using ESI as ionization in both positive- and negative-ion modes. The
mobile phase consisted of 5 mM/10 mM ammonium acetate in water containing 0.1%
formic acid and acetonitrile–MeOH (80:20, v/v). Disperse Yellow 3 and HC Yellow No. 5
were detected in the negative-ion mode, while the other 11 compounds were detected in
the positive-ion mode.
The MS fragmentation patterns, confirmed via LC-Q-TOF-MS (performed using an
Agilent 1290 Infinity II LC system (Agilent Technologies, Santa Clara, CA, USA) coupled
with an Agilent 6545XT Q-TOF-MS system (Agilent Technologies, Santa Clara, CA, USA)),
of 11 out of 13 prohibited dye species were reported by Han et al. [90] for the first time,
and among the 120 cosmetic samples, one was found to contain three illegal compounds:
Basic Blue 26, Basic Red 2, and Basic Yellow 28. Thus, the work of Han et al. [90] shows
future promise in view of more rigorous screening and control of the presence of illegal
ingredients in cosmetic products and to hinder/restrain their distribution.
Most dyes used in cosmetics are sodium or calcium salts which contain one or more
ionized groups in their structure, such as sulfonic groups. This implies the possible
formation of multicharged ions in the ionization source. In addition, the separation of ionic
compounds by reverse-phase LC is a difficult task that requires significant effort, especially
in the separation of neutral compounds. In this sense, the mobile phase (ionic strength,
pH, and composition) plays an important role. In some cases, a mobile phase without
additives, consisting of water and an organic modifier (acetonitrile or methanol), was used
with good results—good sensitivity and rapid analysis [86,87]. The use of UPLC-MS/MS
allowed the analysis of 11 dyes (including Acid Violet 49, Pigment Red 57, Pigment Red
53:1, Acid Yellow 36, Rhodamine B, Basic Violet 3, Disperse Yellow 3, Pigment Orange
5, Sudan I, Sudan II, Sudan IV, and Solvent Blue 35) in 4 min [18] and, respectively, of
12 dyes (including Tartrazine, Amaranth, Ponceau 4RC, Sunset Yellow, Allura Red AC,
Acid Red 2G, Ponceau SX, Brilliant Blue FCF, Orange I, Acid Black 1, and Acid Orange 7) in
6 min [91] in lip gloss, eyeshadow, lipstick, and other cosmetics. The results showed that
the UPLC-MS/MS method could be a fast, simple, sensitive, and quantitative technique
for the simultaneous determination and confirmation of dyes in oily cosmetics, cream
cosmetics, and powder cosmetics. Similar results were obtained for a mixture of nine dyes
with a conventional porous C18 column [86].
Although the use of MS allows selective identification of coeluted compounds, chro-
matographic separation is recommended. To this end, it is necessary to add volatile neutral
salts to the mobile phase to avoid interactions between negatively charged ionized com-
pounds and partially ionized residual silanols in the stationary phase. However, the
presence of salts in the ion source may cause a suppression of ionization. Thus, the compo-
sition of the mobile phase must be investigated to achieve a compromise between good
separation and performance. Therefore, the use of only 3 mM ammonium acetate in the
mobile aqueous phase is recommended [23,85]. This salt concentration was sufficient to
avoid “peak tailing”, while the improvement of chromatographic separation for a fairly
large number of analytes was within the satisfactory quantification limits. In another
study [85], other chromatographic parameters were optimized to separate dyes from preser-
vatives. The matrix effect is the suppression or amplification of the ionization of the target
Molecules 2024, 29, 1336 12 of 48

compound by others in the sample and is very common in LC-MS/MS analysis, especially
when ESI sources are used. In each method of dye analysis by MS/MS, a matrix effect study
was performed. The most comprehensive study was performed for 19 dyes in 7 cosmetic
matrices (lip balm, nail polish, hair spray, eyeshadow, toothpaste, blush, and gel) [23]. In
all cases, the optimized sample extraction procedure allowed a sufficiently clean extract to
perform the analysis with negligible matrix effects, except for certain compounds in several
matrices. Unlike conventional techniques of MS, Nizza et al. [92] investigated the use of
MS-coupled desorption electrospray ionization (DESI) for the analysis of semipermanent
hair dyes in two semisolid cosmetics: a blemish cream (BB cream) and a hair coloring
gel. As a novelty, the use of an environmental MS technique allowed a direct analysis
without prior sample preparation or chromatographic separation. A thin layer of sample is
deposited on the porous Teflon, and a pneumatically assisted ESI is used to release neutral
analytes present on this surface as secondary ions. To test the robustness of direct DESI-MS
analysis towards complex chemical matrices, a 10-component mixture was deposited onto a
surface and examined in both positive- and negative-ion modes. Thus, positive-mode DESI
mass spectrum resulting from this analysis yielded protonated molecules for o-toluidine
(m/z 108), p-phenylenediamine (m/z 109), resorcinol (m/z 111), 2,4-toluenediamine (m/z
123), 4-chloroaniline (m/z 128), benzidine (m/z 185), and benzyl salicylate (m/z 229), while
the azo dyes Ponceau SX (PSX), Sunset Yellow FCF (SY FCF), and Orange II (OII) were
readily detected in negative-ion mode, at m/z 217 [M − 2Na]2− , 435 [M + H − 2Na]− , and
457 [M − Na]− for PSX, m/z 203 [M − 2Na]2− , 407 [M + H − 2Na]− , and 429 [M − Na]−
for SY FCF, as these sulfonated azo dyes are disodium salts and only the nonsodiated anion
at m/z 327 for OII.
MALDI ionization coupled to MS has proven to be a fast and robust technique for the
evaluation and quantification of compounds of interest in various common cosmetic matri-
ces. In this sense, this has been taken as far as creating a new subdomain—“Cosmetomics”,
as a simple alternative, both for industrial and academic analysis, using the MALDI-MS
principles for the purpose of product analysis. By using MALDI-MS, in the work of Oliveira
et al. [47], the quantification in nail polishes of the dye Sudan III (with potential carcino-
genic risk), which is a common dye in cosmetics, was performed. It was declared as an
ingredient on all labels as CI 21600 (color index) and/or Solvent Red 23 (trade name). The
health risks associated with a possible carcinogen in a nail polish formulation are due to
accidental ingestion through nail biting or even during cooking or baking.
The analysis was performed without complex preparation of the samples, these being
applied directly on the surface of the MALDI board. The matrix used was α-cyano-
4-hydroxycinnamic acid (CHCA) (Sigma Aldrich, Allentown, PA, USA) in 10 mg/mL
solution (50% antonitrile: methanol) and the samples were coated using a commercial
brush [47]. MALDI-MS analysis was performed using an MALDI-LTQ-XL with imaging
feature (MSI) (Thermo Fisher, Pleasanton, CA, USA) and MS/MS experiments were per-
formed by collision-induced dissociation (CID) in negative-ion mode. The assignment of
chemical structures was conducted following the analysis of MS/MS spectra, as well as by
calculations with Mass Frontier software (see 6.0, Thermo Scientific, Carlsbad, CA, USA).
Image data were analyzed in triplicate using ImageQuest software (Thermo Scientific,
Carlsbad, CA, USA, https://www.thermofisher.com/order/catalog/product/10137985)
and quantification was performed using ImageJ (National Institutes of Health, USA-Open
Source) on grayscale images. The area was standardized in the number of pixels for all
reproductions, and ImageJ software (https://imagej.net/ij/) assigned a value for selection
based on the intensity of each pixel [47]. The results obtained proved that this approach
is a useful, fast, and easy tool for semiquantitative analysis. The results obtained were
promising, and consistent because the amount of Sudan III (m/z 351, [M − H]− ) on each
sample was directly influenced by the color of the nail polish (Table 2). The CCR sample,
which showed the highest relative concentration, was light blue. Sudan III is known to be
a red-to-brown dye under normal conditions, but when subjected to acidic conditions, it
turns blue. This special dye also helps give a thick and shiny appearance, which was to be
Molecules 2024, 29, 1336 13 of 48

expected from the CCR sample. The other two samples with the highest content (RCR and
ICR) are red to brown [47].

Table 2. Nail polish samples and the color for each. The third column refers to the aspect of
each product.

Sample Nail Polish Color Aspect

AHC Dark blue Creamy
CCR Light blue Creamy–shiny
HTT Brown Simple
ICM Red Metallic
ICR Orange-Golden Creamy
IPC Orange Simple
RCR Red Creamy
RMT Pink Metallic
SLB Golden Simple

A comprehensive MS-based analytical methodology for the simultaneous screening

of a large variety of coloring agents of great concern for regulatory control in cosmetics
was established by Chen et al. [93] using ultra-high-performance liquid chromatography
(UHPLC) coupled with quadrupole-orbitrap high-resolution mass spectrometry (Q-orbitrap
HRMS) and ESI in positive- and negative-ionization modes. The chromatographic elution
was performed with two binary mobile phase compositions for electrospray ionization
in positive (ESI+) and negative (ESI−) modes, respectively. A 5 mmol/L ammonium
formate solution at pH 5.5 and acetonitrile were paired for the ESI+ mode, and 5 mmol/L
ammonium bicarbonate solution at pH 9.0 and acetonitrile were paired for the ESI− mode.
The method was applied for the screening of the 63 coloring agents in 69 types of cosmetic
samples (16 of lipsticks, 13 of eyeliners, 14 of blushers, 16 of eye shadows, 4 of toothpastes,
and 6 of nail polishes), which were obtained from various sources.
In the initial stage, the cosmetic samples were subjected to the matrix solid-phase
dispersion (MSPD) sample preparation method using anhydrous sodium sulfate and sand,
then the MSPD column was further eluted with 2 mL of methanol and the extract was
further analyzed by UHPLC-Q-orbitrap HRMS, under synchronous full-scan MS and
data-dependent MS/MS (full-scan MS1 /dd-MS2 ) acquisition mode. The identification and
screening of target compounds were performed by using a self-built accurate-mass database
and a customized mass spectral library and based on accurate mass agreement, consistency
of retention time, characteristic ionic ratio, and isotopic distribution, the MS/MS spectra,
and comparisons between theoretical and measured isotopic patterns [93].
Eleven legally prohibited coloring agents (Basic Violet 1, Basic Violet 10, Basic Violet 3,
Pigment Red 3, Pigment Red 48:4, Solvent Blue 35, Solvent Red 23, Acid Orange 20, Pigment
Red 48:2, Pigment Red 49, and Pigment Red 53:1) (Table 3) were detected in 26 cosmetic
samples in total. In addition, some dye agents found in the studied samples were not
labeled on their cosmetic packing.
The UHPLC Q-orbitrap HRMS method exhibited great potential for routine high-
throughput, sensitive, and reliable screening of dye agents in cosmetic products for close
quality control and to protect consumer health.
 3,
3, Pigment
Pigment Red
Red 3,
3, Pigment
Pigment Red 48:4, Solvent Blue 35, Solvent Red 23, Acid Orange 20,
3, Pigment
3, Pigment Red
Red 3, Pigment Red
3, Pigment 48:4,
Red 48:4, Solvent
48:4, Solvent Blue
Solvent Blue 35,
Blue 35, Solvent
35, Solvent Red
Solvent Red
Red 23, 23, Acid
23, Acid Orange
Acid Orange
Orange 20,20,
20,
Pigment
Pigment Red
Red 48:2,
48:2, Pigment Red
Red 49,
49, and
and Pigment
Pigment Red
Red 53:1)
53:1) (Table
(Table 3)
3) were
were detected
detected in
in 26
26
Pigment
Pigment Red
Red 48:2,
48:2, Pigment
Pigment Red
Red 49,
49, and
and Pigment
Pigment Red
Red 53:1)
53:1) (Table
(Table 3)
3) were
were detected
detected in
in 26
26
cosmetic
cosmetic samples
samples in
in total.
total. In
In addition,
addition, some
some dye
dye agents
agents found
found in
in the
the studied
studied samples
samples
cosmetic
cosmetic samples
sampleson in total.
intheir In addition,
total.cosmetic
In addition, some dye agents found in the studied
some dye agents found in the studied samples samples
were
were not
not labeled
labeled on their cosmetic packing.
packing.
were not
were not labeled
labeled onon their
their cosmetic
cosmetic packing.
packing.
Molecules 2024, 29, 1336
The
The UHPLC
UHPLC Q-orbitrap
Q-orbitrap HRMS
HRMS method
method exhibited
exhibited great
great potential
potential for
for routine
routine
The UHPLC
The UHPLC Q-orbitrap
Q-orbitrap HRMS HRMS method
method exhibited
exhibited great
great potential
potential for
for routine
routine
14 of 48
high-throughput,
high-throughput, sensitive,
sensitive, and
and reliable
reliable screening
screening of
of dye
dye agents
agents in
in cosmetic
cosmetic products
products for
for
high-throughput, sensitive,
high-throughput, sensitive, andand reliable
reliable screening
screening ofof dye
dye agents
agents in
in cosmetic
cosmetic products
products forfor
close
close quality
quality control
control and
and to
to protect
protect consumer
consumer health.
health.
close quality control and to protect consumer
close quality control and to protect consumer health. health.
Table 3. Legally prohibited coloring agents detected in 26 cosmetic samples [93].
Table
Table 3.
3. Legally
Legally prohibited
prohibited coloring
coloring agents
agents detected
detected in
in 26
26 cosmetic
cosmetic samples
samples [93].
[93].
Table 3.
Table 3. Legally
Legally prohibited
prohibited coloring
coloring agents
agents detected
detected in
in 26
26 cosmetic
cosmetic samples
samples [93].
[93].
No.No. Dye
Dye Agent
Agent Molecular Ion (m/z) Molecular Formula Structural
Molecular Ion (m/z) Molecular Formula Structural Formula Formula
No.
No. Dye
Dye Agent
Agent Molecular Ion
Ion (m/z)
Molecular Ion (m/z) Molecular Formula
Molecular FormulaFormula Structural Formula
Structural Formula
Formula
No.
11 1 Dye
BasicAgent
Violet
Basic 11
Violet Molecular
358.22696 (m/z)
358.22696 Molecular
C CH HN Structural
Basic Violet 24
2428 2833N3
11 Violet 111
Basic Violet
Basic
358.22696
358.22696
358.22696
C
C 24 H 28
24H28N3
C24H28N3
N

22 2 Basic
Basic Violet
Violet
Basic 10
10
Violet 443.23227
443.23227 C
C CH
28
H HN
31
N3122O
O 3
22 Basic
Basic Violet
Violet 1010
10
443.23227
443.23227
443.23227
28
C28
C 28H2831
H3131N
N22O
N
O2333O3

33 Basic
Basic Violet
Violet 33 372.24277
372.24277 C
C 25H30N3
25H30N3
33 3 Basic
Basic Violet
Basic
Violet 33 3
Violet 372.24277
372.24277
372.24277 C25
C CH
25 H HN
30
2530 N
3033N3

44 Pigment
Pigment Red
Red 3 308.10236 C 17H13N3O3
44 4 Pigment Red 333
Pigment Red 308.10236
308.10236
308.10236
C 17H13N3O3
C17
C 17H13N3O3
Pigment Red 3 308.10236 CH N133O
1713H N33 O3

Molecules 2024, 29, x FOR PEER REVIEW 15 of 43

55 Pigment Red
Pigment Red48:4
48:4 421.02478
421.02478 C18CH HClN
1813 2O26O
13 ClN S 6S

6 Solvent Blue 35 351.20642 C22H26N2O2

 Molecules 2024, 29, 1336 15 of 48

Table 3. Cont.

No. Dye Agent Molecular Ion (m/z) Molecular Formula Structural Formula
66 Solvent
Solvent Blue3535 351.20642
Blue 351.20642 C22 CH
22 HN
2226 262N
O22O2
66 Solvent
Solvent Blue
Blue 3535 351.20642
351.20642 C
C22 HH 26N2O2
26N2O2

77 Solvent Red 2323 353.13892

353.13892 C2222H16N4O
77 Solvent Red
Red23 C HH
Solvent 353.13892 C2216HN O4 O
164N
Solvent Red 23 353.13892 C22 16N4O

8 Acid Orange
Orange 2020 327.0441 [M
[M −− Na
Na++]]−− C1616H11N2O4S
888 Acid
Acid Orange
Acid Orange
327.0441
20 20 327.0441 [M −[M
327.0441 Na− +]−Na+ ]− C
C16 HCH 11N2O4S
16N
11 ON
H211 4S2 O4 S

Molecules 2024, 29, x FOR PEER REVIEW 16 of 43

99 Pigment Red
Pigment 48:2
Red 48:2 419.01062
419.01062 C18CH1811HCClN 2O26O
11 CClN S 6S

10 Pigment Red 49 377.05936 C20H14N2O4S

11 Pigment Red 53:1 375.02057

Molecules 2024, 29, 1336 16 of 48

Table 3. Cont.

No. Dye Agent Molecular Ion (m/z) Molecular Formula Structural Formula
1010
10 Pigment
Pigment
Pigment Red
RedRed4949
49 377.05936
377.05936
377.05936 C20
C CH
20
H HN
2014
14 N SO4 S
O244S
N1422O

11
11 Pigment Red
Pigment Red 53:1 375.02057
375.02057
11 Pigment Red53:1
53:1 375.02057

3.4. Analysis
3.4. Analysis of of Allergens
Allergens in in Cosmetic
Cosmetic Products
Products
3.4. Analysis of Allergens in Cosmetic Products
Fragrances, next
Fragrances, next to to heavy
heavy metals
metals (nickel),
(nickel), preservatives,
preservatives, and and hair
hair dyes
dyes in in cosmetics,
cosmetics,
are Fragrances,
the most common next cause
to heavy of metals
skin (nickel), preservatives,
sensitization [94–99], which and hair adyes
implies in cosmetics,
life-long change
are
are the
themost
mostcommon
commoncause causeof ofskin
skinsensitization
sensitization[94–99],[94–99],whichwhichimplies
impliesaalife-long
life-longchange
change
in the
in the immune system system specificity. Skin Skin allergy is is clinically manifested
manifested as allergic allergic contact
in the immune
immune system specificity. specificity. Skin allergy allergy is clinically
clinically manifested as as allergic contact
contact
dermatitis. Once
dermatitis. Once sensitized,
sensitized, this this condition
condition rapidly
rapidly develops
develops upon upon re-exposure
re-exposure to to aa suffi-
suffi-
dermatitis. Once sensitized, this condition rapidly develops upon re-exposure to a sufficient
cient
cient amount
amount of the product containing the allergen. Based on several studies [99,100],
amount of theof the product
product containing containing the allergen.
the allergen. Based onBased several onstudies
several[99,100],
studiesperfumes,
[99,100],
perfumes, deodorants,
perfumes, deodorants, and and aftershaves
aftershaves were were the the riskiest
riskiest product
product categories
categories regarding
regarding
deodorants, and aftershaves were the riskiest product categories regarding sensitization
sensitization and
sensitization and contact
contact allergies.
allergies. Moreover, in in addition
addition to to contact
contact dermatitis,
dermatitis, other other
and contact allergies. Moreover, in Moreover, addition to contact dermatitis, other adverse effects
adverse effects
adverse effects like like asthma,
asthma, allergic
allergic rhinitis,
rhinitis, migraine
migraine and and photosensitivity,
photosensitivity, possible possible ac-
like asthma, allergic rhinitis, migraine and photosensitivity, possible accumulation in ac- the
cumulation
cumulation in the human body (associated with genotoxicity, which could lead to mu-
mu-
human bodyin(associated
the humanwith body (associatedwhich
genotoxicity, with genotoxicity, which could
could lead to mutagenic or lead to
carcinogenic
tagenic or
tagenic or carcinogenic
carcinogenic effects),effects), and and other
other side
side effects
effects can can also
also bebe developed.
developed.
effects), and other side effects can also be developed.
Fragrances,
Fragrances, such as perfumes
such asasperfumes
perfumes and
and deodorants,
deodorants, aftershave
aftershave products, shampoos,
products, shampoos,
Fragrances, such and deodorants, aftershave products, shampoos, condi-
conditioners,
conditioners, laundry
laundry products,
products, cleaning
cleaning products,
products, etc., are
etc.,utilizedutilized
are utilized in every
in every aspect
aspect of our
our
tioners, laundry products, cleaning products, etc., are in every aspect of ourofdaily
daily
daily lives.
lives. lives. Currently,
Currently,
Currently, more thanmore3000
more thanchemical
than 3000 chemical
3000 chemical
substances, substances,
substances, either natural
either
either natural natural
fragrance fragrance
fragrance
materials ma-
ma-or
terials
terials or synthetic
or synthetic
synthetic fragrance
fragrancefragrance
chemicals, chemicals,
chemicals,
are responsibleare responsible
are responsible
for odorous for odorous
for properties properties
odorous properties of scented
of scentedofproducts.
scented
products.
products.
At the same Attime,
At the same
the same
a mixture time,
time, ofaa20mixture
mixture
to over 200 of 20
of 20 to over
to
constructsover the200fragrance
200 constructs
constructs the fragrance
the
compounds fragrance com-
com-
(including
pounds
pounds (including fragrance/aroma
(includingcomponents,
fragrance/aroma fragrance/aroma solvents, components,
components, solvents,
colorants, solvents, colorants,
fixatives, colorants,
and UV filters) fixatives,
fixatives, and
and UV
[100,101]. UV
filters)
filters) [100,101].
[100,101].
Natural fragrances are divided into two major classes: aroma (obtained from plants/
Natural
Natural
essential fragrances
oils)fragrances
and musk are are divided (extracted
divided
compounds into two
into two from majoranimal
major classes:
classes: aroma[102]).
aroma
sources (obtained
(obtained Because from
fromof
plants/essential
plants/essential
the high prices of oils)
oils) and
and musk
essential musk compounds
compounds
oils, dealers (extracted
(extracted
are tempted from animal
from animal
to adulterate sources [102]).
sources [102]).
the products by addingBe-
Be-
cause of the
lower-cost
cause of the materials,
high prices
high pricesand,of essential
of essential oils, dealers
thus, synthetic
oils, dealers are tempted
aromatics
are tempted to adulterate
can reduce
to adulterate
perfume thecosts
the products
and are
products by
by
adding
often used
adding lower-cost
lower-cost materials,
as an alternative
materials,source and, thus,
and, thus, synthetic
of compounds aromatics
that are not
synthetic aromatics can
can reduce
easily
reduce perfume
obtained
perfume from costs and
natural
costs and
are oftenorused
sources
are often used as an
not found
as an alternative
alternative
in nature. source source of of compounds
compounds that that are
are notnot easily
easily obtained
obtained from from
natural
naturalDue sources
to theor
sources oradverse
not found
not found in nature.
effects
in nature.
of fragrances, they are considered an emerging health
Due
and Due to the
environmental adverse effects
concern of
[103].
to the adverse effects of fragrances, fragrances,
For thosethey they are considered
substances
are considered an emerging
responsible,
an emerging
or suspectedhealthto
health and
andbe
environmental
responsible, for concern
causing [103].
allergicFor those
reactions, substances
their use responsible,
has
environmental concern [103]. For those substances responsible, or suspected to be re- to be or
limited suspected
and/or to be
strictly re-
reg-
sponsible,
ulated. The
sponsible, forInternational
for causing allergic
causing allergic reactions,
Fragrance
reactions, their use
Association
their use (IFRA)
has to
has to be
be limited and/or
periodically
limited and/or strictly
publishes
strictly regu-
a regu-
list of
prohibited or restricted fragrance substances recognized by the IFRA expert panel [104].
In the EU, Regulation No. 1223/2009 [105] on cosmetics listed 26 fragrance ingredients,
including two natural extracts (oak moss and tree moss) and 24 volatile chemicals that are
considered to be more likely to cause allergic reactions in sensitive individuals (Table 4).
These 26 fragrance substances are subject to specific labeling requirements if any individual
concentration exceeds 10 µg/g for leave-on and 100 µg/g for rinse-off products. Such
regulatory requirements necessitate consistent reference analytical methods suitable for
routine quality control.
Molecules 2024, 29, 1336 17 of 48

Table 4. Chemical characterization of 24 individual fragrance ingredients associated with allergic

reactions (according to EU, Regulation No. 1223/2009 [105]).

No. Fragrance Allergen Molecular Formula MW

1 Alpha isomethylionone C14 H22 O 206.32
Amyl cinnamal
2 C14 H18 O 202.29
(Jasmonal A)
3 Amyl cinnamyl alcohol C14 H20 O 204.31
4 Anisyl alcohol C8 H10 O2 138.16
5 Benzyl alcohol C7 H8 O 108.14
6 Benzyl benzoate C14 H12 O2 212.24
7 Benzyl cinnamate C16 H14 O2 238.28
8 Benzyl salicylate C14 H12 O3 228.24
9 Butylphenyl methylpropional (Lilial) C14 H20 O 204.31
10 Cinnamal C9 H8 O 132.16
11 Cinnamyl alcohol C9 H10 O 134.17
12 Citral C10 H16 O 152.24
13 Citronellol C10 H20 O 156.26
14 Coumarin C9 H6 O2 146.14
15 Eugenol C10 H12 O2 164.20
16 Farnesol C15 H26 O 222.37
17 Geraniol C10 H18 O 154.25
Hexyl cinnamal
18 C15 H20 O 216.32
(Jasmonal h)
19 Hydroxycitronellal C10 H20 O2 172.26
Hydroxyisohexyl 3-cyclohexene
20 C13 H22 O2 210.31
carboxaldehyde (Lyral)
21 Isoeugenol C10 H12 O2 164.20
22 Limonene C10 H16 136.23
23 Linalool C10 H18 O 154.25
24 Methyl 2-octynoate C9 H14 O2 154.21

The Scientific Committee for Consumer Safety expanded, as of July 2023, the existing
list of 26 regulated fragrance allergens with 56 new substances [106]. The contact allergens
are classified as established, likely, and possible contact fragrance allergens. Among the
82 established contact allergens for humans (including the previous 26 regulated allergens),
28 are natural extracts and 54 are single chemicals. In order for cosmetic products to comply
with the current legal requirements, the transition periods of three or five years must be
taken into account.
The determination of fragrance substances in cosmetic products is challenging pri-
marily because of the complexity of cosmetic formulations and the chemical similarity of
the fragrance substances with other ingredients. This complexity makes it challenging to
develop a universal method to cover all classes of cosmetic products [107].
Sample preparation is an essential step to remove interfering compounds and con-
centrate fragrance compounds in the sample before their analysis. Various preparation
methods have been introduced, from direct analysis to methods with multiple clean-up
steps. Liquid products (eau de toilettes and perfumes) are directly injected, without sample
preparation, except dilution using different solvents or filtration [107–109] (if the amount of
nonvolatile constituents is low when submitted to GC, as the performance of the GC system
Molecules 2024, 29, 1336 18 of 48

is rarely hampered by other constituents of the sample). If the analytes occur in more com-
plex media, such as creams, lotions, foundations, and lipsticks, they need to be extracted
from their matrix, prior to their analysis, via different established methods: by a fluid (in
one step extraction technique [110–113] and improved variants such as MSPD: matrix solid-
phase dispersion [23,93,114,115], QuEChERS (quick, easy, cheap, effective, rugged, and safe
extraction) [116–119], or energy-assisted extraction techniques: vortex-assisted extraction
(VAE) [120,121], ultrasound-assisted extraction (UAE) [121,122], and pressurized liquid
extraction (PLE) [121,123], by liquid–liquid extraction (LLE) techniques [120,124–128], by
solid-phase extraction techniques (µSPE: micro solid-phase extraction, dSPE: dispersive
solid-phase extraction; SBSE: stir bar sorptive extraction) [129–138], and gas-phase extrac-
tion (headspace solid-phase microextraction (HS-SPME) [139–143].
MS, as a highly sensitive and selective analytical technique, has been used in qualita-
tive and quantitative analysis of cosmetic products for ingredient screening and compound
identification. Commonly, GC, as well as LC, hyphenated to MS or MS/MS were employed
for routine evaluation of the analytes in different cosmetic matrices.
GC-MS is the most popular technique for fragrance analysis since they usually have
low boiling points. Currently, GC is extensively used for sample infusion in EI-MS and
has been greatly applied for the determination of allergens [28,31,98,107,144–166] and
other risky components in cosmetic analysis (Table A1). In addition to EI, other ioniza-
tion techniques like chemical ionization (CI) and photoionization (PI) are employed for
GC-MS investigations in this field. Shibuta et al. [148] used multiphoton ionization (MPI)
as the ion source by means of an fs laser, which emitted at 200 and 267 nm for the de-
termination of 26 allergenic compounds in perfumes. This ion source proved suitable
for the selective ionization of analytes by optimizing the wavelength of the light source,
and the obtained limit of detection (LOD) values were all below 100 pg/µL. In fragrance
analysis, GC was interfaced/coupled usually with quadrupoles (Q) [167–184], ion traps
(IT) [153,157,185–187], and a few cases with time-of-flight (TOF) [148,188] mass analyzers,
and in some situations, MS/MS analysis was also performed [107,189] for fragrance finger-
prints. MS data acquisition was achieved typically in scan mode (full scan mode in different
mass ranges) [190] selected ion monitoring (SIM)—in order to distinguish target fragrance,
with known MS features and maximum sensitivity, making it ideal for quantification
and validation [31,107,152,155,156,158–160,163,165,170,188], selected reaction monitoring
(SRM)/multiple reaction monitoring (MRM) (resulting in increased selectivity, sensitivity,
and signal/noise ratio [107,189], and selected ion storage (SIS), with a reduced LOD, due
to the removal of parasite ions and more unrestricted spaces to store more ions of interests
in the analyzer [162].
As the core problem for analyzing fragrance products is the large/abundant number
of ingredients, up to several hundred different ones, and a broad concentration range,
using a single column in the GC system could cause inadequate separation of analytes
of interest [28]. In this view, to improve selectivity in complex cosmetic matrices, sev-
eral methodologies based on multidimensional comprehensive approaches have been
developed by a few working groups [107,109,167,170,173,174,191], and the recent recom-
mendation [192] is to use 2D GC systems for fragrance investigation to avoid obtaining
negative false or positive results.
LC-MS is used in the analysis of fragrance components that are challenging to analyze
by GC, because of their decreased volatility and/or their thermostability (Table A1, [193,194]).
C18 columns with various characteristics were generally used [195–198], while in some
particular situations, C8 columns were preferred [199]. The use of a single mobile phase
hampered the separation of all components of the multicomplex formulation of cosmetics,
making it necessary to use chromatography with various gradient profiles [195,197].
ESI, atmospheric pressure chemical ionization (APCI), and atmospheric pressure
photoionization (APPI) are ionization procedures frequently coupled with LC-MS systems.
Mainly, ESI shows good versatility in the ionization of various substances, and LC ESI-MS
is quite ideal for the analysis of large, fragile molecules [200]. In contrast, APCI is usually
Molecules 2024, 29, 1336 19 of 48

used for weakly polar compounds with a mass below 1500 Da [201], and APPI is more
appropriate for nonpolar compounds that are difficult to charge [202]. Single (Q) [195]
or triple (3Q) quadrupoles [196,197] are primarily used in conjunction with LC-MS in
the determination of fragrance allergens. MS data were acquired in full scan and SIM
modes [195], as well as in SRM mode in other experiments [197].
Convergence chromatography (CC), a separation technique used to bridge LC and GC,
uses carbon dioxide as the primary mobile phase with the option (if necessary) of using an
additional cosolvent, such as acetonitrile or methanol, to obtain selectivity similar to that
of the normal phase LC. This method was developed as ultra-performance convergence
chromatography (UPC2 ) with MS detection for the analysis of scented allergens in perfumes,
cosmetics, and personal care products in a very short time of 7 min [203] (Table A1). It is
an ideal alternative to both HPLC and GC analysis, offering a few advantages such as the
following:
(a) The ability to investigate compounds suitable for LC and GC in a single analysis;
(b) A higher selectivity and specificity compared to HPLC or GC analysis only;
(c) An analysis time at least six times faster than for HPLC and GC;
(d) Solvent use is 95% lower than in existing HPLC methods.
Because of the challenging sample preparation and chromatographic separation, GC-
and LC-MS-based methods cannot provide rapid and high-throughput analysis. Thus, the
development of advanced ionization techniques like ambient ionization mass spectrometry
(AIMS) offers simplicity, reproducibility, and efficiency in cosmetic analysis, but its usage
in allergen identification is scarce to date. Liu et al. [204] used dielectric barrier discharge
(DBD)-MS to detect the presence of fragrance allergens in commercially available perfume
products, obtaining a reasonable linear range and low LOD, sometimes at ppt levels. DBD
ion sources contain very few components (two electrodes, one a stainless-steel needle or
wire and the other a copper strip separated by an insulating barrier such as glass, between
which, upon application of a high voltage, a low-temperature plasma form that can be
applied directly to the surface of liquid or solid samples, or into which gas state samples
can be introduced) and can be created very economically compared to commercialized
sources, and has been shown to be a potent instrument in detecting airborne allergens [205].
The MS technique can be used alone to explore fragrance fingerprints, essential for
counterfeiting discovery. Marques et al. [206] used direct infusion by ESI of perfume
samples (original, counterfeit, and “inspired”) diluted by methanol/water (1:1) in Q-TOF-
MS, and the data were acquired in positive-ion mode. The peak lists were analyzed further
by principal component analysis (PCA) so that ESI-MS fingerprinting allowed for the fast
and reliable detection of perfume falsification. Quite similar profiles in positive-ion mode
were obtained by Haddad et al. [207] by easy ambient sonic-spray ionization (EASI) Q-
Trap MS and PCA analysis for counterfeit recognition of perfume samples, while Chingin
et al. [208] used ESI-Q-TOF-MS in positive-ion mode without PCA analysis for rapid
fingerprinting of various perfumes and forgery identification. In the above-presented
reports, many m/z peaks detected with considerably higher signal levels in MS spectra
from the counterfeit perfume are attributable to the low-purity materials used for their
production, which can be responsible for some known side effects as allergies and toxicity.
Identification of fragrance compounds based on their relative retention times in GC,
as well as their mass spectra, depends directly on the quality and comprehensiveness
of the library used. Only libraries of EI mass spectra are efficient, and other ionization
techniques yield spectra that are much too dependent on the instruments and experimental
conditions [155]. The most common mass library is National Institute of Standards and
Technology (NIST), composed of three primary parts: the first part is “NIST/EPA/NIH
Mass Spectral Library (EI)” and contains “Main EI MS Library” and “Replicate EI MS
Library”; the second part is “Tandem (MS/MS) Library”, which comprises small molecules
and biologically-active peptides sections; and the last one is titled “GC Method/Retention
Index Library” [209]. This library is typically used in the fragrance industry, e.g., various
research groups [140,174,181,182,185] identified different suspected fragrance allergens
Molecules 2024, 29, 1336 20 of 48

by comparison of the experimental spectra with those of the NIST database. Also, Wiley,
National Bureau of Standards library, was prepared by McLafferty et al., in which the
registry spectra for 108,173 compounds are available [155]. This library was also used for
qualitative analysis of fragrances in cosmetics and perfumes [140,168,174,185]. The main
disadvantage of such libraries is the inability to distinguish between compounds with
identical mass spectra due to the lack of retention data. While the retention times vary
with the different chromatographic system, the retention index is a system-independent
constant calculated by a formula that varies with the polarity of a column and is used in
certain libraries, which makes them more reliable. For example, the “Flavour & Fragrance
Natural & Synthetic Compounds” (FFNSC) GC-MS library, prepared by the group of
Mondello et al. [28], contains retention indexes compatible with three types of columns,
including nonpolar column (EquityTM-1), micropolar column (SLBTM-5 ms), and highly
polar column (SUPELCOWAXTM 10). Mondello and coworkers identified the allergens
by using the FFNSC mass spectral library database [28]. The third edition of this library
(FFNSC 3) is registered with 3462 natural and synthetic chemical compounds relating
to flavors. Additionally, Tranchida et al. employed this library to detect the recently
highlighted fragrance allergens (54) in cosmetics (by the Scientific Committee on Consumer
Safety) [107].
Other libraries, like Adams and MassFinder, are dedicated to essential oils and com-
prise the retention index of each compound measured on a nonpolar column [155]. In some
situations, when analysis of fragrance is a major activity, the best method is to build a home
library, e.g., the in-house fragrance MS library, which was built on an HP Chemstation
platform by Liu et al. [187], and another example is the in-house “Baser Library of Essential
Oil Constituents”, which contains MS and retention data of over 3500 genuine compounds
found in essential oils [155].
Another custom-made accurate-mass database was constructed for the comprehensive
identification of 100 multiclass regulated ingredients in cosmetics [209]. The cosmetic
samples were analyzed by UHPLC Q-orbitrap HRMS, using individual standard solu-
tions of analytes at a concentration of 100 ng/mL, under synchronous full-scan MS and
data-dependent MS/MS (full-scan MS1/dd-MS2) acquisition mode. The custom-made
accurate-mass database was made by inputting the exact mass information of the 100 target
compounds and their characteristic fragments together with their respective chemical name,
molecular formula, and chromatographic retention time using a TraceFinder version 4.1
software supplied with the instrument (Thermo Fisher Scientific, Waltham, MA, USA). In
addition to the accurate-mass database, a mass spectral library was built by importing the
acquired mass spectra containing both precursor ions and all characteristic fragment ions
for the 100 target compounds using the TraceFinder software. The mass spectral library was
employed in combination with the accurate-mass database to search and identify the target
compounds of interest from the UHPLC-Q-orbitrap HRMS raw data based on retention
time, precursor and MS/MS fragment ions, ionic ratio, isotope pattern, and mass accuracy
tolerance [209].

3.5. Analysis of Heavy Metals in Cosmetic Products

Since cosmetic products are complex mixtures of texture and coloring agents, a variety
of metals can be present in them. Heavy metals can occasionally be found as contaminants
in cosmetic products (Table 5) due to impurities in raw materials or the manufacturing
procedure, processing aids, and contamination in the supply chain. Even if their presence as
impurities in cosmetic products is inevitable due to the ubiquitous nature of these elements,
they should be removed wherever technically possible. Thus, the regulatory agencies (Food
and Drug Administration (FDA), Federal Office of Consumer Protection and Food Safety
(BVL), etc.) make efforts to establish limits on the levels of heavy metals in cosmetics,
varying by product and country. Nevertheless, even the very perilous heavy metals are
often not explicitly regulated or entirely banned. Guidance on the safety assessment of
cosmetic ingredients has been published by the EU Scientific Committee on Consumers
Molecules 2024, 29, 1336 21 of 48

Products (SCCS, 2012). The Annex II (“List of substances which must not form part of the
composition of cosmetic products”—[210]) of the Directive lists more than 1000 chemical
substances that cannot be used in cosmetic products due to their toxicological properties.
According to this Annex, several metals, such as antimony (Sb), arsenic (As), cadmium
(Cd), chromium (Cr), cobalt (Co), mercury (Hg), nickel (Ni), and lead (Pb), are prohibited
ingredients in cosmetics because they are considered unsafe and are designated as having
toxic and/or allergological concern. However, there are currently no international standards
for heavy metal impurities in cosmetics. Limits for some dangerous heavy metals in
cosmetics have been established in Germany and USA (Table 6, [211,212]), but also in other
countries worldwide [213–217].

Table 5. Heavy metals contaminants in cosmetic products.

No. Heavy Metal Cosmetic Products

1 Mercury (Hg) Creams (antiseptic, skin-lightening) and some mascaras
2 Lead (Pb) Lipsticks, eyeliners, lip glosses, and hair dyes
3 Cadmium (Cd) Blush, eyeshadow, and face powders
4 Arsenic (As) Blush, eyeshadow, and face powders
5 Nickel (Ni) Foundations, eyeshadow, and mascaras
6 Chromium (Cr) Eyeshadow, lipsticks, and face powders
7 Aluminum (Al) Foundations, eyeshadow, and mascaras
8 Copper (Cu) Blush, eyeshadow, and some lipsticks
9 Antimony (Sb) Blush, eyeshadow, and mascara
10 Zinc (Zn) Foundations, sunscreens, and face powders
11 Manganese (Mn) Blush, eyeshadow, and some lipsticks
12 Cobalt (Co) Hair dyes and some eyeshadows
13 Selenium (Se) Hair dyes and some face powders
14 Barium (Ba) Blush, eyeshadow, and face powders
15 Beryllium (Be) Eyeshadow and face powders
16 Thallium (Tl) Hair dyes and some face powders

The meticulous analysis of heavy metal compounds found in cosmetics holds paramount
importance due to the potential health risks associated with exposure to these elements
(Table 6). Despite abundant research on metal detection in cosmetic products [218–228],
very few of these studies focused on estimating human exposures and health risks from
cosmetics, highlighting the need for comprehensive risk assessments.
Employing advanced techniques like MS for accurate determination allows for the
identification and quantification of trace amounts of heavy metals in cosmetics. This not
only ensures compliance with regulatory standards but also helps in establishing a robust
framework for consumer protection. The significance of such analyses becomes particularly
pronounced given the widespread use of cosmetics and the potential impact on diverse
consumer demographics, emphasizing the need for stringent quality control measures in
the cosmetic industry.
Molecules 2024, 29, 1336 22 of 48

Table 6. The permissible limits of heavy metals in cosmetic products and their adverse health effects.

No. Heavy Metal Limits for Limits for Cosmetics Effects of Exposure on the Human Body
Cosmetics (EU, (USA)
Germany)
1 Mercury (Hg) 0.1 ppm * 1 ppm (colorants) Renal, neurologic, and dermal toxicity [229],
cutaneous changes reported include burning of the
face, contact dermatitis, grey or blue–black facial
discoloration, flushing, erythroderma, purpura, and
gingivostomatitis.
2 Lead 2 ppm 20 ppm Affects the fetus and the central nervous system in
(Pb) (colorants) Children [230,231], probably carcinogenic to
10 ppm humans [232,233], neurotoxic, nephrotoxic, and
(lipsticks, lip glosses) hepatotoxic and can also produce effects on the
reproductive system, and can also affect fetal
development through its passage via the
placenta [234–239].
3 Cadmium (Cd) 0.1 ppm - Damage of the kidneys, fragility of the bones,
carcinogenic in humans [240–242].
4 Arsenic (As) 0.5 ppm 3 ppm Skin eruptions, alopecia, and striation of the nails,
(colorants) but also skin cancer [243], circulatory and peripheral
nervous disorders, an increased risk of lung cancer,
and a possible increase in the risk of gastrointestinal
tract and the urinary system cancers [244].
5 Nickel (Ni) 10 ppm - Contact allergy, eyelid dermatitis, as well as irritation,
eczema
6 Chromium (Cr) - - contact allergy [245], carcinogenic in humans
(Cr(VI)).
7 Antimony (Sb) 0.5 ppm - Pneumoconiosis, alterations in pulmonary function,
bronchitis, emphysema, gastrointestinal effects
(abdominal pain, vomiting, diarrhea, and ulcers),
dermatoses, and skin lesions [246–248], probably
carcinogenic to humans (Sb trioxide).
8 Cobalt (Co) - - Skin allergen causing allergic contact dermatitis
(ACD) and eczema, possibly carcinogenic to humans.
Note: * ppm—parts per million.

Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful analytical

technique widely used for determining the presence of heavy metals in various substances,
including cosmetics. In ICP-MS, a high-temperature plasma torch is used to ionize the sam-
ple, creating charged particles, which are further sorted and measured based on their m/z
ratio. The ionization process is considered a ”hard process”, unlike other MS techniques,
such as ESI-MS, which is considered a ”soft” ionization technique [249].
ICP-MS is incredibly useful in cosmetics testing because it provides highly sensitive
and precise results, even at trace levels of heavy metals. Its utility lies in its ability to
detect and quantify multiple elements simultaneously, offering a comprehensive analysis
of the sample composition. This makes ICP-MS an essential tool for ensuring the safety
and compliance of cosmetics, as it helps identify potential contaminants and ensures that
products meet regulatory standards, safeguarding consumer wellbeing [250]. ICP-MS was
used in many studies related to heavy metal analysis in cosmetics and proved to be a good
choice for determining low analyte concentrations, thus allowing the evaluation of toxic
and potentially toxic components in cosmetics [222–226,228,251–259].
Grosser et al. [228] showed that the levels of several heavy metals (Pb, As, Cd, Hg, Cr,
Se, Sb) in cosmetics such as lipsticks, nail polishes, and skin creams could be determined by
ICP-MS after preparation of the samples by microwave digestion in order to obtain clear
Molecules 2024, 29, 1336 23 of 48

solutions (except for the determination of Hg) and the obtained results indicated values
below the risk limits according to Canadian regulations.
Bobaker et al. [253] studied traditional plant-based beauty products marketed in Libya,
respectively, henna (derived from the Lawsonia inermis) and walnut tree bark (also known
as souak—used both as a dental care product and as a popular enhancer for henna colors)
and raised concerns about the potential presence of heavy metals such as Pb, Cd, and
As in these cosmetics, posing health risks (due to their narrow safety margins) through
skin absorption, and their accumulation in internal organs, which can result in toxicity.
Before heavy metal analysis by ICP-MS, the samples were processed (walnut tree bark
samples), oven-dried, and subjected to microwave-assisted digestion after the addition
of a nitric acid (HNO3 )/hydrogen peroxide (H2 O2 ) mixture [260–262]. ICP-MS analysis
(Perkin Elmer SCIEX Elan 9000 ICP-MS, Perkin-Elmer SCIEX, Norwalk, CT, USA) of the
samples revealed the presence of Pb, Cd, and As, with mean concentrations (Pb > Cd >
As) having abnormally high standard deviations, which can be attributed to differences
in product manufacturing, contamination levels, and the diverse sources of the products,
influenced by varying climatic conditions. Walnut tree bark samples exhibited higher heavy
metal levels compared to henna samples, and higher Pb concentrations were observed
in black henna compared to green henna samples. Additionally, most henna and walnut
tree bark products exceeded recommended limits for Pb and Cd, while As levels were
generally lower.
Rubio et al. [254] conducted similar research on henna and jagua traditional cos-
metic products, whose composition can be influenced by several factors, including post-
processing and contamination. The metal composition (including 11 elements, i.e., Al,
Cu, Zn, Ba, Mn, Co, Ni, Pb, Cr, Cd, and As) of henna and jagua commercial samples was
investigated using ICP-MS (Agilent 8800 Triple Quadrupole(QQQ) ICP-MS device, Agilent
Technologies, Hachioji, Tokyo, Japan), equipped with a MicroMist nebulizer and a quartz
spray chamber, after initial steps of predigestion and digestion, using HNO3 and H2 O2
solutions. Several henna samples showed high concentrations of Mn, Zn, and Pb, while Cu
and Cr levels were elevated in certain jagua samples. Banned elements, including Cr, Ni,
As, Cd, Co, and Pb, were found in trace amounts, raising concerns about unintentional con-
tamination. The method demonstrated strong linearity coefficients, accuracy, and precision
in determining heavy metal content in henna and jagua samples.
The impact of heavy metals present in homemade traditional cosmetic products
(lipstick, lip moisturizer, eye mascara, eyeliner, henna, tattoo, and spray hair dye) on human
and environmental health was investigated by Killic et al. [255]. The study used ICP-MS
on an Elan DRC-e (Perkin Elmer SCIEX, Norwalk, CT, USA) to assess the metal content in
homemade cosmetic products, comparing them to WHO’s (World Health Organization)
permissible limits. This research involved the collection of cosmetic products from the
market, with hair spray dye serving as a matrix for method validations. The samples
collected from the market were subjected to microwave digestion using HNO3 and H2 O2
digestion, followed by ICP-MS analysis of the diluted samples in order to determine As,
Cd, Co, Cr, Cu, Ni, and Pb levels. The optimized methods have proven high selectivity and
sensitivity, with detection limits LOD of 0.1–0.2 µg/L and LOQ of 0.2–0.8 µg/L for metal
assessment. Pb was found in all samples, surpassing the WHO-specified range, while As,
Co, Cd, Cu, Cr, and Ni were present in variable concentrations in certain products. The
average heavy metal levels differed across cosmetic categories, with Pb being constantly
identified. Pb and Ni concentrations in the smear exceeded the permissible WHO limits,
while Pb concentrations in other products were below the limit. Metal levels in spray hair
dye, smear, and tattoo usually remain at trace levels. The health risk was evaluated using
the target hazard quotient (THQ), which is the ratio of exposure to the toxic element to
the dose at which adverse health effects are expected to occur. The THQ index for lipstick
showed varying values, in descending order of Cr > Pb > Ni, while for other cosmetics, it
was Pb > Cr > Ni. The recommendation is that despite THQ values being lower than 1 for
Molecules 2024, 29, 1336 24 of 48

all tested samples, which will not result in immediate health risks, persistent and excessive
use of these products could possibly lead to long-term health problems for users.
Salama et al. [251] focused on evaluating the concentrations of 10 heavy metals (Pb, Al,
Cd, Co, Cr, Cu, Mn, Ni, Hg, and As) in various cosmetic products (beauty cream, hair cream,
skin cream, hair gel, hair food formula, etc.) available in the Saudi Arabian market using
ICP-MS (NexION 300 D, Perkin-Elmer, Inc., Shelton, CT, USA) method. Before MS analysis,
cosmetic samples were processed by dry-ashing method, but for particular samples like
creams and lotions, wet digestion using HNO3 and HClO4 was accomplished. The results
indicated that the levels of heavy metals varied considerably across different product types:
in shampoo products, the highest mean concentration was obtained for Al, followed by Pb,
Cu, Cr, Mn, Ni, Hg, As, Co, and Cd; in cream products, the highest average concentrations
were for Al, followed by Cu, Mn, Pb, Cr, Ni, Hg, Co, As, and Cd; while soaps exhibited
the highest mean levels for Al, followed by Cu, Pb, Cr, Mn, Ni, As, Co, Hg, and Cd; and
toothpaste products displayed the highest mean levels for Al, followed by Cu, Mn, Pb,
Cr, Co, Ni, Cd, As, and Hg. Overall, Al was shown to be the major heavy metal in most
samples, with exceptions in several skin, beauty, and shaving creams. Cu followed Al in
concentration, except in certain shampoo products, and Cd was identified as having the
lowest levels, except in toothpaste, where it was replaced by Hg for position in ranking.
Perez et al. [256] investigated the exposure (via incidental ingestion and via dermal
uptake) to metals in costume cosmetics for three subject categories: child (2–3 years), adult
(≥18 years) with infrequent use (12 times per year), and adult with daily occupational use.
Costume cosmetic products in different forms (lotion, spray, stick, eyeshadow), containing
both water-based and oil-based commercial costume makeup, were prescreened for metal
content using a handheld X-ray fluorescence (XRF) detector, and those with detectable
metals were subjected to a concentrated HF/HCl/HNO3 digestion and analyzed further
by ICP-MS (Agilent 8800) using a triple quadrupole analyzer (ICP-QQQ-MS). A SkinPerm
absorption model was employed to estimate dermal exposure concentrations and absorbed
doses, while to evaluate possible metal exposure via incidental ingestion, the measured
concentrations were used to calculate the potential exposure from direct ingestion (i.e.,
from lipstick) or from ingestion via hand-to-mouth contact, using exposure assumptions,
many of which are based on the statistical data [263]. The daily intake doses from oral
exposure were estimated using the method described in [264]. The results showed the
presence of Sb, Pb, Ni, Co, Cr, Hg, and Ag from below detection to 9.3 mg/kg wet weight in
various samples. This was found in one body paint sample, while Cd and methylmercury
(MeHg) were not detected. Oral ingestion accounted for over 99% of all metal intake. The
Pb dose from body paint was predicted to raise blood lead levels above baseline in all
users, with an increase of less than 1 µg/dL amongst the child and adult-intermittent
users, while occupational usage raised blood lead levels by 1.0 and 1.9 µg/dL for mean
and maximum Pb concentrations, respectively. Overall, the study revealed that costume
cosmetics with varying levels of As, Co, Ni, Pb, and Sb do not pose unnecessary health
risks to intermittent consumers; however, occupational exposures may exceed recognized
health-based control values.
Similarly, Salles et al. [257] focused on the presence of metallic-based pigments, in-
cluding heavy metals in liquid, cream, or pancake face paints, of various colors, commonly
applied to the head and trunk, with specific attention to occupational exposure (in adults)
and children’s vulnerability (related to costume cosmetics) in Brazil. A set of potentially
toxic elements (Al, As, Ba, Cd, Co, Cr, Cu, Ni, Pb, Sb, Sn, Sr) was determined through
HNO3 digestion, followed by ICP-MS analysis (Agilent 7900, Hachioji, Japan). The dermal
exposure evaluation involved calculating cancer risk and dermal hazard quotient based on
dermal absorption during product use. Incidental ingestion exposure assessment estimated
potential exposure via hand-to-mouth contact.
The study revealed statistically significant differences in sample concentrations be-
tween colors, especially for As, Ba, Cd, Co, Cu, and Pb, with variations in mean concentra-
tions, i.e., white, brown, and lilac paints showed elevated mean levels for As, red colors for
Molecules 2024, 29, 1336 25 of 48

Ba, and white, purple, and blue displayed the highest means for Pb and Cd. Yellow and
brown colors had the highest average concentrations for Co, while lilac, blue, and green
colors showed the highest means for Cu.
In general, for nearly all elements, pancakes and liquid samples had higher means.
Cream samples and professional pancakes had higher average concentrations for Cd, Cr,
and Pb, while those of Sr were higher in fluorescent and liquid paints. The levels of Cu
did not differ between types of costume cosmetics. High cancer risk was identified in both
children and occupational exposures to potentially toxic elements in costume cosmetics.
For children, the risks from accidental ingestion exceed those from dermal exposure due to
hand-to-mouth behavior. In contrast, for adults, the risk is higher through dermal exposure,
highlighting the need to monitor these elements in cosmetic products globally to safeguard
human health, particularly for continuous professional use and child consumption during
festive events.
Domeradzka-Gajda et al. [258] used ICP-MS to explore the impact of various cosmetic
ingredients, including parabens (methylparaben), phthalates (dibutyl phthalate), and (alu-
minum salts AlCl3 ) on the percutaneous absorption of silver nanoparticles (AgNP) using
an in vitro model based on isolated pig skin. AgNP, the most prominently advertised nano-
material, is present in numerous consumer products, including cosmetics, personal care,
health, and food items. The Nanodatabase and ANEC/BEUC inventory [265] list numerous
nanosilver-containing products related to skincare: crèmes, moisturizers, washing lotions,
cleansers, antiperspirants, soaps, shoe deodorants, or foot balms. The majority of AgNP
applications results from their strong antibacterial properties [266] primarily linked to the
release of Ag ions, but their percutaneous absorption is a subject of concern. Studies on
medical products containing AgNP indicate possible skin penetration, especially at the
level of injured skin [267–270].
Scarce research exists on the percutaneous absorption of AgNP through normal skin,
but evidence suggests detectable penetration [271–273]. The absorption may vary when
AgNP interacts with other cosmetic ingredients, especially on microabraded skin.
The authors used, for the purpose of the study, AgNP of different conventional sizes
(15 nm or 45 nm) and surface modification, and as citrate or PEG stabilized nanoparticles
and various matrices—pig skin sections and receptor fluid (Franz chambers). The deter-
mination of Ag content in receptor fluid was conducted by ICP-MS using a PerkinElmer
Elan1 DRC-e device, Perkin Elmer, SCIEX, Framingham, MA, USA. The sample intro-
duction system included a quartz cyclonic spray chamber, Mainhard nebulizer, and a
peristaltic four-channel pump. Triplicate measurements were performed for each sample,
with deionized water analyzed between replicates to check for memory effects. For skin
samples, laser ablation (LA)-ICP MS was applied by using skin disks, post 24 h exposure
in the Franz chambers, washed and fixed in 10% neutral buffered formalin, embedded
in paraffin, and sectioned at 20 mm. LA-ICP-MS data were processed into 2D elemental
images using Applied Spectra, Inc.’s ((West Sacramento, CA, USA) Data Analysis Software
(http://www.appliedspectra.com/downloads.html).
ICP-MS measurements after 24 h in receptor fluid indicated low but detectable sil-
ver absorption and no correlation with concentration, nanoparticle size, or the mode of
nanoparticle stabilization. Furthermore, the chosen cosmetic ingredients (methylparaben,
dibutyl phthalate, and AlCl3 ) did not exert a statistically significant influence on silver
absorption, with the highest amount of Ag that penetrated (0.45 ng/cm2 ) being measured,
such as for PEG stabilized Ag of 15 nm + methylparaben.
In addition to many studies that are focused on trace metal analyses in cosmetic
matrices like colored make-up products (as a lot of pigments contain metal compounds),
Rujido-Santos et al. [259] analyzed the metal content in the most commonly used moisturiz-
ing creams on the Spanish market, to verify their degree of compliance with [274] regarding
the presence of metals in cosmetics, as they have various functions in formulations of these
cosmetic products [275]. The study used a validated methodology based on microwave-
assisted acid digestion of the samples followed by ICP-MS and showed that most of the
Molecules 2024, 29, 1336 26 of 48

analyzed products did not comply with Regulation (EC) No. 1223/2009 [276] on cosmetic
products, while for several cases, speciation studies were necessary. Only one moisturizing
cream fulfilled the stated regulation.

4. Conclusions
Cosmetics and personal care products are complex formulations comprising a vast
number of ingredients with diverse physicochemical properties. As a primary concern,
cosmetic products that are commercially available must be safe for users. Providing
cosmetics safety and implementing compliance with standards, regulatory requirements,
and quality management systems requires efficient and robust analytical methods. Recent
advances in MS have contributed to many scientific findings in the cosmetic industry by
investigating very complex mixtures of chemical ingredients and their interactions with the
human body. Still, many directions have not yet been examined. Analysis of bioconjugates
in cosmetics by MS-based methods is a narrow field, mainly limited to the analysis of
different lipids, including ceramides, for the quality control of the product formulation
by MALDI-MS and ESI-MS, respectively, and CID used to carry out the fragmentation of
analytes. Investigation of dextrins in dermal cosmetics is relatively unexplored, and in this
area, MALDI-MS proved suitable for the characterization of their chemical structure in order
to determine their prospective applications. Both LC (HPLC, UHPLC)- and GC-MS (which
commonly requires derivatization of the analytes) are widely used for the quantification of
preservatives in cosmetics and personal care products, especially in the case of counterfeit
cosmetics, as they are unregulated and may contain levels of preservatives that can lead
to health risks associated with long-term exposure. Given the increased selectivity and
sensitivity, MS hyphenated to different LC variants (LC, HPLC, and UHPLC) is the usual
choice for accurately identifying and quantifying dyes in cosmetic samples due to their
hydrophilic nature, especially in the analysis of banned compounds. In addition, ambient
ionization techniques such as DESI interfaced to MS have been used for the direct analysis
of the sample without prior preparation or chromatographic separation, even for personal
care products directly on the cell surface, while MALDI-MSI usage in this direction was
limited to the analysis of dyes in nail polish samples.
Moreover, the input of HR detectors allowed comprehensive UHPLC-ESI-HRMS si-
multaneous screening of a great diversity of coloring agents of significant concern for
regulatory control in cosmetics. Usually, GC as well as LC coupled to MS or MS/MS and
using diverse ion sources were employed for routine evaluation of analytes having aller-
genic properties in different cosmetic matrices, mainly perfumes. Since fragrance products
consist of a large number of ingredients, methodologies based on multidimensional GC-MS
approaches are preferred by many researchers. With the exception of perfumes, the investi-
gations of most cosmetic matrices imply steps of sample preparation and chromatographic
separation in advance and, thus, cannot provide rapid and high-throughput analysis.
Ambient ionization approaches (AIMS) yield efficiency, reproducibility, and simplicity in
cosmetic analysis, but their utilization in allergen determination has been scarce up to
the present. The introduction of convergence chromatography (UPC2 ) with MS detection
has been proven to be an excellent choice in addition to HPLC and GC for the analysis of
scented allergens because of its ability to investigate compounds suitable for LC and GC in
a single analysis, with a reduced volume of the used solvent, and of good analysis time.
“Hard ionization” ICP-MS preceded by microwave-assisted digestion is ideal for detecting
and quantifying multiple elements simultaneously, offering a comprehensive analysis of
heavy metals present in different samples and being able to evaluate human exposure and
possible health impacts. In the future, there is no doubt that MS-based methodologies will
continue to be improved to meet the increasing complexity of regulatory constraints in the
cosmetic field, and expanding their use will undoubtedly increase the efficiency of cosmetic
quality and safety assessment to a significant level.
Molecules 2024, 29, 1336 27 of 48

Author Contributions: Conceptualization, A.F.S., M.G., E.S. and R.E.S.; methodology, A.F.S., C.R.N.,
R.O. and E.S.; software, R.O. and A.B.; validation, A.F.S., E.S. and M.G.; formal analysis, A.F.S., R.O.,
C.R.N. and E.S.; investigation, A.F.S., M.G., E.S., A.B., R.O., C.R.N. and R.E.S.; resources, C.R.N., R.O.,
A.B. and R.E.S.; data curation, A.F.S., M.G., E.S. and A.B.; writing—original draft preparation, A.F.S.,
M.G., R.O., C.R.N. and R.E.S.; writing—review and editing, A.F.S., M.G., E.S. and R.E.S.; visualization,
A.F.S., E.S., M.G. and R.E.S. supervision, A.F.S., M.G. and R.E.S.; project administration, A.F.S., M.G.
and E.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors declare no conflicts of interest.

Appendix A

Table A1. MS-based analysis of allergens in cosmetic products.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
Simultaneous determination of 30 fragrances
A total of 42 cosmetic products (including 24 listed allergens)—18 leave-on products
(12 rinse-off and 30 leave-on) contained at least one fragrance substance > 10 µg/g
1 GC-MS [166]
purchased from nine different and 5 of the 12 rinse-off
countries (USA and EU). products contained at least one fragrance
substance > 100 µg/g.
The method was tested in order to identify the
A total of 166 leave-on presence of 24 regulated allergens and 21 prohibited
cosmetic products purchased GC-MS substances in the cosmetic products: 2–17 allergens
2 [144]
within three years and stored and GC-MS/MS were identified per sample, and only safrole (of the
at room temperature. prohibited substances) was present in a concentration
> LOQ in 12 out of 166 tested samples.
GC HR-MS A total of 35 “difficult fragrance allergens” were
3 Six fragrance compositions. [145]
and GC-LR-MS quantified best by GC-orbitrap HR-MS.
A “Lily” matrix—a
combination of about 50 raw The validation of the GC-MS method
materials, mainly constituted for the quantification of the extended list of
4 GC-qMS [146]
of essential oils, aromatic plant 57 fragrance allergens (which led to the updated
extracts, synthetic ingredients, NF EN 16274 standard in 2021 [276]).
water and ethanol.
Validating a method to identify and quantify the
GC×GC qMS
5 One tested fragrance (CC02). suspected allergens (24) limited by EU regulations in [109]
(SIM mode)
fragrances by GC×GC-qMS.
Initial screening of 5 cosmetics
(3 creams and 2 lotions) and Simultaneous screening of 100 restricted ingredients
UHPLC-q-orbitrap
6 subsequent screening of in cosmetics (39 antibodies, 40 glucocorticoids, [209]
HR-MS
123 cosmetics (71 creams and 9 androgens, 8 progestogens, 4 antifungal agents.
52 lotions).
Molecules 2024, 29, 1336 28 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
The 1460 and 1910 WatercolTM columns can reliably
be used for the GC-MS analysis of EU-regulated
Two commercial perfumes
7 GC–EI-MS volatile allergens in commercial perfumes [150]
(“eau de toilette”).
(14 allergens identified in perfume 1 and 4 allergens
in perfume 2) and showed complementary selectivity.
A total of 26 allergens were identified (superior
performance of MPI/MS over EI/MS for more
Three commercially reliable determination of the allergy compounds); the
available perfumes (Sakura concentrations of methyl-2-octynoate (not written in
8 Eau de Toilette, Moroccan GC-MPI-TOF-MS the label of the bottle), citronellol, [148]
Rose, White hexylcinnamaldehyde, and linalool in Sakura Eau de
Musk). Toilette, and methyl-2-octynoate in White Musk (no
label) is larger than the concentration specified by the
Cosmetics Directive (0.001% for a leave-on sample).
A total of 20
A total of 58 allergens were identified (natural
commercial-scented plush toys Headspace
9 extracts, which were unsuitable for a [149]
(preserved in sealing packages (HS)-GC-MS
chromatography-based method, were not detected).
before analysis).
Detection of hydroxiperoxides of limone and linalool
(limonene-2-hydroperoxide
(Lim-2-OOH), linalool-6-hydroperoxide (Lin-6-OOH),
A total of 10 perfume products and linalool7-hydroperoxide (Lin-7-OOH, with
(7 eau de toilette, 2 aftershaves, 2D strong sensitization
10 [208]
and 1 eau de cologne) from the HPLC-ESI-MS/MS potency), the highest concentrations of the measured
Swedish market. hydroperoxides (445 ± 23 ppm of total linalool
hydroperoxides) being observed in one after-shave
product, which is likely able to elicit skin reactions in
already sensitized individuals.
A total of 7 different matrices:
5 were homemade (DWL,
fabric softener, liquid laundry
The standard addition protocol allowed the analysis
detergent, milky hair shampoo,
of suspected allergens in the investigated matrices
day cream), 1 (powder
11 GC-MS and allowed the quantification of all compounds [147]
detergent) was provided by a
(15 allergens) except farnesol and Lyral, within a
detergent manufacturer, and 1
concentration range of 50–100 mg/L.
was a natural raw material
(Peru balsam, Nelixia, Antigua,
Guatemala).
A total of 56 (69 analytes including isomers)
suspected chemically defined fragrance allergens in
A total of 62 commercialized
12 GC×GC-qMS perfumes were investigated (the majority of the [170]
perfumes.
analytes could be determined under or above the
10 mg/kg regulated limit (88–100%).
13 Citrus oils. LC-MS Quantification of 15 furocoumarins. [209]
The analysis of the 24 regulated and 6 additional
compounds (4 nonregulated cosmetic allergens and
2 potential carcinogenic compounds, methyl eugenol
and 4-allyl anisole) was achieved using the Xevo
Various cosmetic and personal
14 UPC2-MS/MS TQD in MRM mode with APCI ionization (+/−), [201]
care products.
coupled to an ACQUITY UPC2 System, in a 7 min
run. The method is more than six times faster than
existing HPLC and GC methods, with 95% less
solvent usage than existing HPLC methods.
Molecules 2024, 29, 1336 29 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
The chromatographic procedure seemed to be
slightly longer; however, the conditions showed
good resolution for about 200 terpenoid compounds
determined in general essential oil studies.
From the 25 standard allergens studied, 19 showed a
A total of 7 oils issued from
15 GC-MS retained DL (detection limit) < 13 mg/L, and [151]
plants.
5 were = 30–50 mg/L. These variations are well
explained by the form of the peaks.
GC-MS is considered a good technique for the
determination of volatile substances. Results were
obtained with good repeatability.
The GC-MS method described is a rapid (<5 min)
and effective screening tool in the determination of
26 allergens contained in mediumly complex
perfumes. The twin-filtered MS library search
procedure was shown to be a powerful tool for
reliable compound identification. As for all
monodimensional methods, it may fail when the
A total of 4 commercial number of sample volatiles greatly exceeds the peak
16 perfumes purchased in a local GC-MS capacity of a single column: the reliable [28]
store (Messina, Italy). qualitative/quantitative determination of 10–20 skin
sensitizers amongst 500–1000 other volatiles would
be an arduous task. A good result may be attained
for a 200-component fragrance and a bad one for a
150-compound perfume. The most appropriate
approach to be used, if a multiple-choice exists,
strictly depends on the analyst’s experience and
judgment.
The GC-MS method has been developed for the
Randomly chosen 18 cosmetic
routine analysis of 11 fragrance substances in
products—5 shampoos,
cosmetics: cinnamic alcohol, cinnamic aldehyde,
7 creams and lotions, 2 eau de
17 GC-MS eugenol, hydroxy citronellal, a-amyl cinnamic [168]
toilette, 1 deodorant spray,
aldehyde, geraniol, isoeugenol, coumarin,
1 lipstick, 1 face powder and
dihydrocoumarin, citronellal, and citral. DL of all of
1 soap bar.
the target fragrance substances were ~1 ppm.
The FM GC×GC–qMS method is sufficiently
sensitive for all the 54 allergens considered.
Moreover, and if required, the HR untargeted
analysis of perfume constituents can be performed.
A total of 5 commercial All FM
18 The FM model proposed is a low-cost and effective [107]
perfumes (P1–P5). GC×GC–qMS
alternative to cryogenic modulation; both the
hardware and operational costs are somewhat
limited, with many of the well-known benefits of
GC×GC maintained.
Molecules 2024, 29, 1336 30 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
To determine a more realistic LOQ (limit of
quantitation) in the context of a fragrance
concentrate, a fragrance concentrate (FT) was spiked
with all allergens at various levels between 10 and
500 mg/L and analyzed by a single laboratory. The
accuracy profile shows that the mean bias remains
less than 20% at all spiking levels down to 10 mg/L.
For 90% of determinations, the expected bias should
Fragrance concentrates
GC-MS be less than 35% down to a level of 20 mg/L and
19 provided in blind by [188]
GC×GC-MS between −49 and 77% at 10 mg/L (i.e., between 5
IFRA-member companies.
and 18 mg/L). This range remains acceptable to set
the LOQ at 10 mg/L, in view of the suspected
allergens analysis complexity.
Fragrance concentrates are very complex mixtures;
the occurrence of coelutions is frequent—the
two-columns × three-ion option best minimizes the
consequences of coelution on the determination of
suspected allergens.
Contents of 52 cosmetic ingredients belonging to
4 different types of ingredients: 6 preservatives,
12 synthetic musks, 26 fragrance allergens, and
8 phthalates can be determined in a single run. All
samples contained some of the target ingredients.
Several samples do not comply with the regulations
concerning the presence of phthalates. Musk’s data
confirmed the trend of the replacement of nitromusks
by polycyclic musks, as well as the noticeable
A total of 70 commercial introduction of macrocyclic musks in the perfume’s
20 GC-MS [153]
perfumes and colognes. composition. The prohibited musk moskene has been
detected in one sample in an appreciable
concentration. The average number of fragrance
allergens is 12 per sample; values > 1% have been
found in some samples. Preservatives data show that
parabens, although ubiquitous in other cosmetic
products, are not widely used in perfumery. In
contrast, the presence of BHT is indeed widespread.
Only about 38% of the perfumes were adequately
labeled for the allergens tested.
Molecules 2024, 29, 1336 31 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
Profiling and fingerprinting of medium- to highly
complex samples of interest in the flavor and
fragrance field was investigated. Capillary flow
technology reverse-inject differential flow modulator
was implemented with different column
configurations (lengths, diameters, and stationary
phase coupling) and detector combinations (MS and
FID) to evaluate its potential in the quantitative
profiling and fingerprinting of medium- to highly
A model mixture of volatiles complex essential oils, and a parallel dual-secondary
and essential oils of different GC×GC MS column dual-detection configuration that has shown
21 [173]
complexity (mint, lavender, GC×GC FID to improve the information potential also with
and vetiver essential oils). thermally modulated GC×GC platforms (MS for
identification FID for quantitation) was tested.
Experimental results demonstrate that careful tuning
of column dimensions and system configurations
yields improved (a) selectivity, (b) operable carrier
gas linear velocities at close-to-optimal values, (c) 2D
separation power by extending the modulation
period, and (d) handling of overloaded peaks
without dramatic losses in resolution and
quantitative accuracy.
Accuracy, precision, linearity, and LODs were
evaluated to assess the performance of the proposed
method. Quantitative recoveries (>75%) were
obtained, and RSD values were <10% in all cases.
The quantification limits were well below those set
Different cosmetics (leave-on by the international cosmetic regulations, making
and rinse-off products) from this multicomponent analytical method suitable for
22 national and international GC-MS routine control. A total of 25 fragrance allergens were [176]
brands were purchased from identified All the samples contained several of the
local sources. target cosmetic ingredients, with an average number
of seven. The total fragrance allergen content was, in
general, relatively high, even in baby care products,
with values close to or up to 1%, for several samples,
although the actual European Cosmetic Regulation
was fulfilled.
Molecules 2024, 29, 1336 32 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
Concentrations of HHCB, AHTN, and HHCB-lactone
A total of 60 household in consumer products ranged from <5 ng/g to over
commodities, including 4000 µg/g, <5 ng/g to 451 µg/g, and <5 ng/g to
perfumes, lotions, hair care 217 µg/g, respectively. The
23 products, and household GC-MS highest concentrations were found in perfumes, body [177]
cleaners, were purchased from creams, lotions, and deodorants. The results suggest
retail stores in Albany, New that a wide variety of source materials exist for
York. HHCB and AHTN and that these materials are used
on a daily basis.
The analysis of suspected volatile allergens in
products containing high-molecular-weight or
nonvolatile compounds such as plant extracts, solid
and liquid detergents, and shampoos was performed.
Based on PTV injection with ALEX and GC-MS,
nonvolatile matrices are retained in the liners filled
with PDMS foam, while good analytical performance
for the target solutes is preserved. This approach
drastically shortens and simplifies the sample
preparation step. The method also gives
quasi-quantitative analyte recoveries for all solutes
with the exception of methyl-2-octynoate and
methyl-2-nonynoate. For various nonvolatile
matrices, a single external calibration can be used,
while for the two mentioned esters, internal
standardization is presently carried out.
Analyzing all target compounds in the different
matrices with one single method is impossible;
therefore, we proposed at various meetings to
24 Different cosmetic products. GC-MS [156]
classify the different matrices into four classes.
Class I consists of samples that contain volatile or
semivolatile solutes, typically eluting on an apolar
column between n-decane (retention index 1000) and
n-docosane (retention index 2200).
Class II also consists of samples containing only
volatile and semivolatile solutes, but their complexity
is very high (A100 solutes) and/or the concentration
range is very broad (e.g., very low concentration of a
target compound next to a very high concentration of
matrix compound).
Class III comprises nonvolatile samples (solutes
eluting after n-hexacosane).
Class IV matrices are finished products like soaps,
liquid and solid detergents, etc. In these samples, the
solutes are typically present at relatively low
concentrations, while the matrix can be quite
complex due to the presence of glycols and
surfactants.
Molecules 2024, 29, 1336 33 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
A new method based on solid-phase
dispersion-pressurized liquid extraction (PLE)
followed by GC-MS has been developed for the
A total of 10 samples (several
determination of 26 suspected fragrance allergens (all
moisturizing and antiwrinkle
the regulated in the EU Cosmetics Directive
25 creams and lotions, hand GC-MS [157]
amenable by GC, as well as pinene and
creams, and sunscreen and
methyl-eugenol) in cosmetic samples.
after-sun creams).
The study revealed the presence of suspected
allergens in all the analyzed samples, and half of the
samples contained an elevated number of them.
MSPD and GC-MS were used for the rapid
determination of 18 plasticizers (phthalates and
adipates), 7 polycyclic musks, and 5 nitromusks,
which makes a total of 30 targets in both rinse-off and
A total of 26 cosmetic products
leave-on cosmetic formulations.
(creams, emulsions, lotions,
A total of 25 out of 30 targets were detected in the
gels for the skin, bath and
samples. The most frequently found compounds
shower preparations,
were galaxolide and tonalide, reaching
26 deodorants, hair-setting, GC-MS [154]
concentrations above 0.1% (1000 g·g−1 ) and diethyl
hair-cleansing, and
phthalate (between 0.7 and 357 g·g−1 ). The presence
hair-conditioning products,
of banned substances such as dibutyl phthalate,
shaving products, and
diisobutyl phthalate, dimethoxyethyl phthalate,
sunbathing products).
benzylbutyl phthalate, diethylhexyl phthalate,
diisopentyl phthalate and dipentyl phthalate, musk
ambrette, and musk tibetene was confirmed in 16 of
the 26 personal care products (62%).
A practical, simple, and low-cost sample GC-MS and
GC-MS/MS method has been developed for the
rapid simultaneous determination of 38 cosmetic
ingredients, 25 fragrance allergens, and
13 preservatives.
The final miniaturized process required the use of
only 0.1 g of sample and 1 mL of organic solvent for
the final extract ready for analysis.
The concentration levels ranged from the sub-parts
per million to the parts per million. Several
A broad range of cosmetics
fragrances (linalool, farnesol, hexylcinnamal, and
and personal care products
benzyl benzoate) have been detected at levels >0.1%
(shampoos, body milk,
(1000 g·g−1 ). With regard to preservatives,
27 moisturizing milk, toothpaste, GC-MS [98]
phenoxyethanol was the most frequently found
hand creams, gloss lipstick,
additive, reaching a relatively high concentration
sunblock, deodorants, and
(>1500 g·g−1 ) in 5 cosmetic products. BHT was
liquid soaps, among others).
detected in 8 samples, in 2 of them (a baby care
product and a lipstick) at high concentrations
(>1000 g·g−1 ). In 3 leave-on samples, methyl
paraben was also found at high levels (>1700 g·g−1 ).
Finally, triclosan was found at the maximum
concentration limit (0.3%) laid down by the European
regulation in 2 deodorant samples, and the total
paraben concentration was close to the maximum
concentration permitted (0.8%) in one leave-on
sample (body milk).
Molecules 2024, 29, 1336 34 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
An overview of the synthetic musk levels in
6 different personal care product categories was
performed. Especially body lotions, perfumes, and
Personal care products and deodorants contain high levels of synthetic musks.
sanitation products (n = 82) Maximum concentrations of HHCB, AHTN, MX, and
were obtained through the MK were 22 mg·g−1 , 8 mg·g−1 , 26 µg·g−1 , and
cooperation of several 0.5 µg·g−1 , respectively. By combining these results
volunteers. The samples were with the average usage of consumer products, low-,
divided into six categories: medium-, and high-exposure profiles through
28 GC-MS [159]
sanitation products (n = 14), dermal application could be estimated. HHCB was
perfumes (n = 19), deodorants the highest contributor to the total amount of
(n = 4), hair care products synthetic musks in every exposure profile
(n = 12), shower and bath (18–23,700 lg·d1). Exposure to MK and MX did not
products (n = 18), and body increase substantially (10–20-fold) between low- and
lotions (n = 15). high-exposure profiles, indicating that these
compounds cover a less broad range. In comparison,
exposure to HHCB and AHTN increased up to
10,000 fold between low and high exposure.
Occurrence and concentrations of macrocyclic-,
polycyclic-, and nitro musks in cosmetics and
A total of 73 household household commodities collected from Japan. The
commodities were purchased high concentrations and detection frequencies of
in Kumamoto, Japan: Musk T, habanolide, and exaltolides were found in
perfumes (n¼13), fabric commercial products, suggesting their large
softeners (n¼11), shampoos production and usage in Japan. Polycyclic musks,
(n¼11), body lotions (n¼9), HHCB and OTNE, also showed high concentrations
body soap (n¼5), in cosmetics and products. The estimated dairy
29 GC-MS [160]
antiperspirants (n¼5), laundry intakes of Musk T and HHCB by the dermal
detergents (n¼4) toilet exposure to commercial products were 7.8 and
deodorants (n¼4), body 7.9 µg/kg/day in humans, respectively, and perfume
fragrances (n¼2), hair liquid and body lotion are dominant exposure sources.
(n¼2), sunscreen (n¼2), dish The dairy intakes of HHCB by dust ingestions were
cleaner (n¼2), tooth powder 0.22 ng/kg/day in humans, which were
(n¼2), and bath cleaner (n¼1). approximately 5 orders of magnitude lower than
those of dermal absorption from commercial
household commodities.
SEC combined with GC-MS was developed for the
Fragrance-free cosmetic
quantitation of 24 restricted allergenic fragrance
samples (creams, body lotions,
compounds in cosmetic samples.
oils) were bought from
Fragrance calibration has to be performed with
commercial shops in Basel and
propyl acetate as a solvent containing a constant
stored at room temperature
proportion of matrix components. With the exception
until preparation for recovery
of hydroxycitronellal (66 ± 5%), all compounds
experiments (adding allergens
30 GC-MS showed good recovery rates in the range of 90–120%. [180]
in the range of 10 mg/kg).
The mean accuracy (relative error) was 1 ± 10% for
Additionally, for quality
all 24 compounds in five spiked creams (10 mg/kg
control, a hand cream (oil in
per allergen) and 8 ± 34% in a reference sample
water emulsion) of known
(4–15 mg/kg). The most significant benefit compared
fragrance content in the range
to other methods is the flexible clean-up with SEC,
of 4–15 mg/kg was used as a
which allows the determination of an extensive range
reference sample.
of compounds in difficult matrices with GC-MS.
Molecules 2024, 29, 1336 35 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
Personal care products were
purchased from retail stores in The developed and validated method using
Porto, Portugal: body and hair QuEChERS extraction followed by GC-MS was
31 washes (n = 5), toilet soaps GC-MS applied to the analysis of 12 samples, which revealed [186]
(n = 1), skin moisturizers musk concentrations ranging from 2 ng/g
(n = 4), roll-on deodorants (toothpaste) to 882,340 ng/g (perfumed body lotion).
(n = 1), and toothpaste (n = 1).
A total of 7 synthetic musks (musk ambrette, musk
tibetene, musk moskene, musk ketone, musk xylene,
phantolide, and tonalide) are extracted and
prepurified by a mixture solution of water and
Cosmetic samples from isopropanol from cream and separated and purified
national and international by tandem columns containing SLE column and
32 GC-MS [189]
brands were purchased from LC-Alumina-N SPE column.
local markets in Beijing. This pretreatment method combined with
GC-MS/MS technology has been proved to be
precise, accurate, and applicable to the routine
analysis of 7 synthetic musks residues in cream
samples.
A simple and quick sample preparation method was
A total of 12 commercially developed and used for preconcentration and
available essential oils were extraction of six phenylpropenes, including anethole,
purchased from local chemical estragole, eugenol, methyl eugenol, safrole, and
material stores in Taiwan. myristicin, from oil samples by dual dispersive
A total of 5 culinary herbs liquid–liquid microextraction. GC-MS was used for
(holy basil, sweet basil, thyme, the determination and separation of compounds.
laurel, and rosemary) and Several experimental parameters affecting extraction
5 spices (cumin, cinnamon, efficiency were evaluated and optimized. For all
nutmeg, cardamon, and clove) analytes (10–1000 ng/mL), the limits of detection
33 GC-MS [163]
were purchased from local (S/N 3) ranged from 1.0 to 3.0 ng/mL; the limits of
food retail stores in Kaohsiung, quantification (S/N 10) ranged from 2.5 to 10.0
Taiwan. ng/mL; and enrichment factors ranged from 3.2 to
Samples of 4 commercially 37.1 times. Within-run and between-run relative
available aromatherapy standard deviations (n = 6) were less than 2.61% and
massage oil products were 4.33%, respectively. Linearity was excellent, with
obtained from randomly determination coefficients (r2) above 0.9977. The
selected cosmeceutical stores experiments showed that the proposed method is
in Taiwan. simple, effective, and environmentally friendly for
analyzing phenylpropenes in oil samples.
Qualitative and quantitative analysis of 24 volatile
compounds listed as suspected allergens in cosmetics
by the European Union was performed. The
A total of 3 perfumes,
applicability of a headspace (HS) autosampler in
2 anti-hair loss products,
combination with GC equipped with a
1 post-depilation mousse,
34 GC-qMS programmable temperature vaporizer (PTV) and a [181]
1 cream deodorant, and
qMS detector is explored.
3 different cream samples
The method showed good precision and accuracy,
(body, sun, and hand creams).
and it is rapid, simple, and highly suitable for the
determination of suspected allergens in different
cosmetic products.
Molecules 2024, 29, 1336 36 of 48

Table A1. Cont.

Observations/
No. Samples MS-Based Analysis Ref.
Comments
Suspected fragrance allergens were determined in
cosmetic products using a combination of whole
evaporation-dynamic headspace (FEDHS) with
selectable GC/GC×GC-MS using capillary flow
technology (CFT) and low thermal mass GC
Commercial perfume samples (LTM-GC). The FEDHS approach allows the
and a cosmetic product (body nondiscriminating extraction and injection of both
35 GC/GC×GC-MS [182]
cream) were obtained from a apolar and polar fragrance compounds without
supermarket. contamination of the analytical system by
high-molecular-weight nonvolatile matrix compounds.
The system is highly flexible and easy to use, and
was applied to all classes of cosmetic samples,
including water-containing matrices such as shower
gels or body creams.
A new headspace GC-MS method was capable of
The samples, analyzed for
analyzing 24 volatile allergenic fragrances in complex
their content on illegal skin
cosmetic formulations, such as hydrophilic and
bleaching agents, were taken
lipophilic creams, lotions, and gels. This method was
by inspectors affiliated with
successfully validated using the total error approach.
the Belgian federal public
The trueness and precision deviations for all
36 service “Animal, Plant and GC-MS [165]
components were smaller than 8%, and the
Food Directorate-General”
expectation tolerance limits did not exceed the
(DG4) and the Belgium Federal
acceptance limits of ±20% at the labeling limit—used
Agency for Medicinal and
to analyze 18 cosmetic samples that were already
Health Products (FAMHP) and
identified as being illegal on the EU market for
were also used for this study.
containing forbidden skin-whitening substances.
There is an advantage of the Direct EI LC-MS
interface for the quantitation of principal
components, as well as for the identification of
unknown/undeclared ingredients Commercially
available products were diluted with methanol and
Different personal care
injected directly into a nano-LC column. Limonene,
products: a hand cream
linalool, and citral were selected as target
water-in-oil (w/o), an eau de
compounds because of their use as fragrances in
37 perfume, a shower gel, and an LC-MS [195]
toiletry and detergent products.
orange oil, and they were
Selected compounds are not detected with ESI
purchased from a local
because of their poor or very low response. No
drugstore.
matrix effects were observed, and the repeatability
was excellent even after several weeks of operation.
The product’s composition was investigated in full
scan mode to determine the presence of unknown or
nonlisted ingredients.
An LC-MS method for quantitative analysis of the
potential oak moss allergens atranol and
chloroatranol in perfumes and similar products
was developed and validated.
LOD for atranol and chloroatranol were 5.0 ng/mL
and 2.4 ng/mL, respectively; the method based on
A total of 10 randomly selected LC-MS and LC-MS/MS with ESI in negative mode
38 LC-MS [197]
perfumes and similar products. and SRM allowed the identification of these
compounds at concentrations below those causing
allergic skin reactions in oak-moss-sensitive patients.
The recovery of chloratranol from spiked perfumes
was 96 ± 4%. Low recoveries (49 ± 5%) were
observed for atranol in spiked perfumes, indicating
ion suppression caused by matrix components.
Molecules 2024, 29, 1336 37 of 48

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