EVALUATION OF THE IMPACT OF PHYSICO-CHEMICAL
PROPERTIES ON BIOAVAILABILITY OF DRUG PRODUCT AND
ESTIMATION OF ANALYTE, DEGRADATION IMPURITIES BY LIQUID
CHROMATOGRAPHY IN AN ANTIMICROBIAL LEAD MOLECULES TO
ENSURE THE ROBUST PHARMACEUTICAL PRODUCT
DEVELOPMENT
Thesis submitted to
(GITAM DEEMED TO BE UNIVERSITY)
In partial fulfilment for the award of degree of
DOCTOR OF PHILOSOPHY
IN
CHEMISTRY
By
GIRISH KISHANRAO DESHPANDE
[Reg. No. 122062404017]
DEPARTMENT OF CHEMISTRY
SCHOOL OF SCIENCE
GITAM DEEMED TO BE UNIVERSITY
Visakhapatnam-530045
July 2024
i
Recommendations
Evaluation of Physico-chemical properties, quantification of analyte and degradation impurities
plays an important role in pharmaceutical product development. Thatincludesthe study on
biological performance of drug product in human bodyand ensure the quality of drug product
with respect to potency, purity and efficacy. A study of physico-chemical properties of drug
molecule is prerequisite for the formulation development and helpful to optimizestability profile,
biological performance, storage condition, etc.
Pre-formulation is the key process between application of lead chemical entity with inactive
ingredients and formulation development stage to ensure the physical and chemical
compatibility. Hence it is very essential to provide the complete pathway of drug formulation
development. That includes Solubility analysis, bulk characterization and stability studies are the
principal areas of formulation development.
In drug substance characteristic, crystallinity and polymorphism of drug molecule play crucial
role in in-vitro performance of drug development. Environmental factors encounteredduring the
manufacturing process or physical modifications in material may leads to different crystalline
hydration states for the active ingredient.
These hydrates exhibit diverse physicochemical properties, such as solubility differences,
chemical stability, and nature of particle. Water in active/inactive ingredient hydrates can be
categorized based on three different structural classes that include those residing in remote lattice
sites, lattice channel sites, or ion-coordinated sites.
In the case of isolated lattice sites, water molecules are secluded from each other due to contact
with drug molecules. Water molecules forming lattice channel sites are in contact with other
water molecules of adjoining unit cells along an axis of a unit cell called channel water. Ion-
coordinated water takes part in an ion water bond which is usually much stronger than the
hydrogen bonds present, called crystal-bound water. It has been shown that under low relative
humidity (RH), some channel water containing hydrates may undergo dehydration or absorb
water under high humidity conditions. In addition to the generation of different hydrates, a single
hydrate form of active or inactive pharmaceutical ingredients may contain more than one
structural type of water.
Moxifloxacin hydrochloride is available in three forms: monohydrate, anhydrous and
amorphous. Thermal and spectroscopic data indicates that the Moxifloxacin hydrochloride lattice
undergoes an adjustment upon removing channel water and is a little bit compromised in the
crystal integrity. This study affirms that removing one mole of channel water through heat results
in dehydrated Moxifloxacin hydrochloride with specific physical and chemical changes. These
changes are reversible as sample rehydrates and crystal lattice returns to its original state over the
period.
Understanding the physicochemical behavior of active ingredients in the manufacturing process
helps to optimize the finished product, enhancing the dissolution and clinical performance in
human plasma. Governing the unacceptable properties of active substances during the
manufacturing process will help to control the related substances, residual solvents and
genotoxic as well as elemental impurities in the final product. This study empathizes with the
rate of hydration /dehydration phenomena to control the quality of the artefact during the
blending and compression stages of the manufacture.
Analytical methods for the determination of lead molecule in drug product formulation is being
developed and validated using RP-HPLC. The newly developed method demonstrates the
characteristics of stability indicating method. In the pharmaceutical industry, analytical
techniques play a pivotal role in establishing the quality, safety, and efficacy attributes of drug
products. Furthermore, widespread use of AQbD has demonstrated its value in driving
innovation in the pharmaceutical sector. The ICH has proposed a new regulatory guideline, Q14
Analytical procedure development, which will be made public soon.
The newly innovated and synthesized lead molecule Methyl-Ester-Toluene-Sulfonamide is the
combined derivative of Sulphonamide-anthranilate. In this method estimation was achieved by
gradient elution pattern with a flow rate of 0.8 ml/minutes. 0.1% Triethylamine in water and with
pH 2.0 buffer as mobile Phase-A and mixture of acetonitrile and tetrahydrofuran in the ratio of
(975:25) v/v as mobile phase B. 210 nm wavelength is used for detection of compound on
Agilent 1260 infinity series HPLC system equipped with DAD detector. The stationary phase
(column) used was ACE 3 C18 PFP (250 mm × 4.6 mm) 3 μm at 40 °C temperature.
The gradient program is time (min)/% B: 0.0/50, 3.0/50, 15.0/70, 25.0/90, 30.0/90, 31/50, &
38/50.
The method is simple, specific, precise, linear, accurate, rugged, rapid, and selective. The
method validation data and robustness data through QbD based approach indicates that the
proposed method is enough robust and suitable for it’s intended purpose. Therefore, the newly
developed method is readily available to pharmaceutical industry for new drug development and
commercial use in quality control.
For the estimation of known and unknown degradation products in Baricitinib marketed product,
a simple, specific, optimum run time, sensitive method was developed with reversed-phase high-
performance liquid chromatography. When employing premixed water and methanol in the
proportion of 950: 50 v/v as mobile phase A and premixed acetonitrile, methanol and water in
the proportion of 750: 200: 50 v/v/v as mobile phase B, the suggested high-performance liquid
chromatographic method achieved optimal separation of 11 impurities in 42 minutes by using
gradient program at a flow rate of 1.0 ml/min. The Agilent Zorbax SB-C18 (150 cm × 4.6 mm)
3.5 µm was utilized. The detection was carried out at 225nm. The procedure's dependability was
established, and its experimental design was examined. The proposed method has proven useful
in the quality control laboratories.
To separate and quantify all the seven related compounds of Molnupiravir, a novel, simple,
specific, rapid, LC-MS compatible reverse phase high-performance liquid chromatographic
method was developed with stationary phase YMC-Pack ODS-AQ (250 mm × 4.6 mm) 5 µm
HPLC column. The method is duly validated as per ICH guideline. More than a 3.5 resolution
was established between the Molnupiravir peak and closely eluted Impurity F peak. All
impurities and Molnupiravir peak separation is achieved within 45min. The methodology was
challenged by deliberate changes in method parameters like column temperatures, varying flow
rate, change in pH of the buffer solution used for the preparation of mobile phase A, collecting
data at different wavelength, etc. In all these variations, method performance was as per
acceptable system suitability criteria. Moreover, proposed methodis LC-MS compatible and can
be applied for potential characterization work. As methos is suitable for quantification hence can
be employed effectively in the quality control of bulk drug production and pharmaceuticals.
