DOI 10.

1007/s11094-023-02844-x
Pharmaceutical Chemistry Journal, Vol. 56, No. 12, March, 2023 (Russian Original Vol. 56, No. 12, December, 2022)

OPTIMIZATION OF ANALYTICAL METHOD

FOR SIMULTANEOUS DETERMINATION OF ACETAMINOPHEN,
CAFFEINE, AND ASPIRIN IN TABLET DOSAGE FORM

Ayman Ismail,1 Moataz Gamal,2 and Maha Nasr3,*

Original article submitted August 25, 2021.

A combination of acetaminophen, caffeine, and aspirin in tablets is present in the United States Pharmaco-
poeia (USP42). A method of analysis for the simultaneous assay of the three analytes by high-performance
liquid chromatography (HPLC) recommended in the monograph by using benzoic acid as an internal standard,
which is chromatographically separated last in the chromatogram. In the current work, an optimization experi-
ment was conducted to validate the analytical method without the internal standard, in order to shorten the
chromatogram runtime. The mobile phase represented filtered degassed mixture of methanol, glacial acetic
acid, and water (28:3:69 v/v). The HPLC device was operated at a flow rate of 2.0 mL/min and a column tem-
perature of 45°C. The injection volume was 10 mL. Acetaminophen, aspirin, and caffeine were detected at
275 nm UV wavelength. A Symmetry C18 HPLC column with length 150 mm, internal diameter of 4.6 mm,
and particle size of 5 mm was used. The method is specific, with no interference between the analyte peaks,
and it is linear in the range of 0.625 to1.870 mg/mL for acetaminophen, 0.625 to 1.870 mg/mL for aspirin, and
0.1625 to 0.4875 mg/mL for caffeine. The overall accuracy of the method ranged within 99 – 101% for the
three drugs, and the instrumental and intermediate precision were demonstrated to be satisfactory. It can be
suggested that the pharmacopoeial method of analysis can be used without the aid of internal standard, and the
sample run time can be reduced, which will in turn save time of analysis and resources.
Keywords: caffeine; acetaminophen; aspirin; HPLC; validation.

1. INTRODUCTION neous assay of acetaminophen, caffeine, and aspirin by

high-performance liquid chromatography (HPLC) is enlisted
Acetaminophen is a well-known antipyretic and analge- in the monograph with the aid of benzoic acid as an internal
sic that is present in the market in different dosage forms standard. Chromatographic separation of the three analytes
such as tablets, suppositories, and drops. Acetaminophen- and benzoic acid showed that benzoic acid was eluted as the
containing dosage forms are enlisted in various pharmaco- last compound. Therefore, the current work was aimed to op-
poeial monographs [1].Caffeine is a xanthine derivative timize the pharmacopoeial method of analysis to eliminate
which acts as a metabolic and central nervous system stimu- the need for internal standard, in order to shorten the injec-
lant that is beneficial in fatigue reduction and maintaining tion runtime. The modified method was then subjected to
validation studies to ensure the fitness for purpose.
alertness [2]. Caffeine is also present in various dosage forms
in different combinations. Aspirin is a nonsteroidal anti-in-
flammatory and analgesic agent, which is widely used for the 2. MATERIALS AND METHODS
treatment of fever and pain [3].
A combination of acetaminophen, caffeine, and aspirin in 2.1. Chemicals
tablets is present in USP42 monograph [4], as well as in sev- Acetaminophen was purchased from Farmason com-
eral marketed products. A method of analysis for the simulta- pany, India. Caffeine was purchased from Siegfreid Evion-
naz company, Switzerland. Aspirin was purchased from
1
Technical Services Section, GlaxoSmithKline, Giza, Egypt. Shandong company, China. Methanol (HPLC grade) and gla-
2
Quality Control Directorate, GlaxoSmithKline, Giza, Egypt.
3
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Phar-
cial acetic acid (HPLC grade) were purchased from Merck,
macy, Ain Shams University, Cairo, Egypt. Germany. Symmetry C18 HPLC column with 150 mm
*
e-mail: drmahanasr@pharma.asu.edu.eg length, internal diameter of 4.6 mm, and particle size of

1682
0091-150X/23/5612-1682 © 2023 Springer Science+Business Media, LLC
Optimization of Analytical Method for Simultaneous Determination 1683

Fig. 1. Chromatogram of acetaminophen, aspirin, and caffeine mixture (a) without internal standard and (b) with internal standard.

5 mm was purchased from Waters company, United States. methanol, glacial acetic acid, and water (28:3:69 v/v). The
Polyvinylidene fluoride (PVDF) syringe filter was purchased HPLC device (Waters, Alliance) was operated at a flow rate
from Merck company, Germany. Manufacturer of the drug of 2.0 mL/min and column temperature of 45°C. The injec-
tablets is GlaxosmithKline Egypt, and in addition to the three tion volume used was 10 µl. Acetaminophen, aspirin, and
active substances (acetaminophen, caffeine and aspirin), they caffeine were detected at the UV wavelength of 275 nm [4]
contain stearic acid, avicel PH 101, hydroxypropyl cellulose, using Symmetry C18 HPLC column with length 150 mm, in-
Dri-klear FC system, white color concentrate, carnuba wax, ternal diameter 4.6 mm, and particle size 5 mm.
and FD&C blue color dye.
2.3. Preparation of Solutions
2.2. Chromatographic System and Conditions
Standard solution. Acetaminophen (125 mg), aspirin
Quantitative analysis of acetaminophen, aspirin, and caf- (125 mg), and caffeine (32.5 mg) were accurately weighed
feine tablets using HPLC method was originally developed into 100 mL volumetric flask and dissolved by sonication for
by USP42 [4]. In the current work, the method was modified 5 min in 50 mL of mobile phase solution. After sonication,
to eliminate the use of internal standard and reduce injection the flask was left to cool down to room temperature and
runtime. The modified method was validated to comply with completed to the final volume using mobile phase solution.
the validation parameters described in International Confer- Sample solution. Five tablets containing acetaminophen
ence on Harmonization (ICH) Quality Guidelines Q2 [5]. (250 mg), aspirin (250 mg), and caffeine (60 mg) were accu-
The mobile phase represented a filtered degassed mixture of rately weighed, put into 1000 mL volumetric flask, and dis-
1684 Ayman Ismail et al.

Fig. 2. Chromatograms of acetaminophen, aspirin, and caffeine mixture under acid, heat, alkali and peroxide stress conditions.
Optimization of Analytical Method for Simultaneous Determination 1685

TABLE 1. System Suitability Parameters for HPLC Analysis of sponse from five serial dilutions of acetaminophen, aspirin,
Acetaminophen, Aspirin, and Caffeine Mixture without Internal and caffeine covering the range from 50% to 150% of their
Standard nominal concentrations in the sample preparations. The
Parameter
equation of the best fitting straight line and the coefficient of
Acetaminophen Caffeine Aspirin determination were calculated [7].
(reference value)
The accuracy was assessed using nine determinations
Tailing factor (NMT 1.20) 1.08 1.16 1.02
over three different concentrations (50, 100, and 150% from
Resolution (NLT 1.5) NA 3.62 11.14
the nominal concentration). Samples were prepared by spik-
ing the placebo (excipients of the formulation) to the stan-
dard preparation and average recoveries from different con-
centrations were calculated [8, 9]. The instrumental precision
solved in 700 mL mobile phase solution by sonication for
was evaluated through calculating the % relative standard
15 min. The flask was then left to cool down to room temper-
deviation of acetaminophen, aspirin, and caffeine in five de-
ature and then completed to the mark volume using mobile
terminations of standard solution. System suitability of the
phase solution.
method of analysis was evaluated through calculating the
2.4. Validation Procedure tailing factor and the resolution of three analytes [8, 9]. The
intermediate precision was assessed by preparing twelve dif-
Validation of the previously mentioned analytical
ferent sample solutions and measuring them by two analysts.
method was conducted to comply with the validation param-
Percentage assays of three analytes were obtained and relative
eters described in International Conference on Harmoniza-
standard deviation (% RSD)% of assays was calculated [10].
tion (ICH) Quality Guidelines Q2 [5]. Validation parameters
including the specificity, linearity range, accuracy, instru-
mental precision, and intermediate precision were studied. 3. RESULTS AND DISCUSSION
The specificity was assessed by studying the behavior of
3.1. Optimization of Analytical Method
acetaminophen, aspirin, and caffeine solutions under stress
conditions, namely acidic, alkaline, oxidative hydrolysis and The instrumental and chromatographic conditions were
dry heat. The peaks were evaluated to ensure the lack of in- adopted from the USP42 monograph, with the difference that
terference between any peaks of interest [6]. The linearity the internal standard was not used for the determination of
range was evaluated by preparing and measuring the re- any of the analytes in the proposed method. The method of

TABLE 2. Acetaminophen Determination Linearity TABLE 3. Aspirin Determination Linearity

Concentration, Acetaminophen Concentration, Aspirin

Run # % Mean Run # % Mean
mg/mL Area mg/mL Area
1 50 0.6250 3564782 3567582 1 50 0.6250 1508701 1509320
2 3568116 2 1509042
3 3570249 3 1510216
1 80 0.9979 5794754 5795734 1 80 0.9998 2467762 2467912
2 5794328 2 2467187
3 5798121 3 2468786
1 100 1.2474 7120828 7128014 1 100 1.2498 3033651 3036241
2 7137225 2 3049294
3 7125949 3 3034778
1 120 1.4969 8503060 8512020 1 120 1.4998 3654352 3656238
2 8508553 2 3656556
3 8524447 3 3657807
1 150 1.8700 10570771 10563568 1 150 1.8700 4549648 4546401
2 10528302 2 4530792
3 10591632 3 4558762
R2 0.9998 R2 0.9999
Y-intercept 1.7% Y-intercept 0.4%
1686 Ayman Ismail et al.

TABLE 4. Caffeine Determination Linearity TABLE 6. Caffeine Determination Accuracy

Concentration, Caffeine Actual

Run # % Mean Recovery Mean
mg/mL Sample # concentration
Area
mg/mL mg/mL % %
1 50 0.1625 2791865 2794097
1 (50 %) 0.1500 0.1471 98.3 99
2 2794006
2 (50 %) 0.1482 99.0
3 2796421
3 (50 %) 0.1479 98.8
1 80 0.2405 4549890 4550313
1 (100 %) 0.2990 0.3016 100.7 101
2 4548107
2 (100 %) 0.3024 101.0
3 4552942
3 (100 %) 0.3008 100.5
1 100 0.3006 5606002 5610588
1 (150 %) 0.4049 0.4486 99.9 100
2 5618506
2 (150 %) 0.4521 100.7
3 5607256
3 (150 %) 0.4519 100.6
1 120 0.3607 6718697 6724839
Mean (%) +100
2 6725650
3 6730171
1 150 0.4875 8391549 8383657
2 8353125 and caffeine, as illustrated by studying the behavior of the
3 8406297 sample solutions under stress conditions (Fig. 2). The sample
R2 0.9999 solutions were chromatographed and the peaks of the three
Y-intercept 0.7%
analytes were separated without any interference with any
peak. The resolution of each peak was determined to be more
than 1.5 [11]. The tailing factor for each peak was deter-
mined to be less than 1.2.
analysis yielded a chromatogram with shorter injection Linearity and range. The method was proven strongly
runtime (Fig. 1a ), as the internal standard was separated as linear in the concentration range from 0.625 to 1.870 mg/mL
the last peak in the chromatogram yielded by the original for acetaminophen, 0.625 to 1.870 mg/mL for aspirin, and
pharmacopoeial method (Fig. 1b ). Results of determining 0.1625 to 0.4875 mg/mL for caffeine (50 – 150% of their
the system suitability parameters are shown in Table 1. nominal concentrations in sample preparations). The coeffi-
cient of determination (r2) was calculated to be 0.9998 for
3.2. Method Validation acetaminophen, 0.9999 for aspirin, and 0.9999 for caffeine.
Specificity. The method was proven specific and stabil- The Y-intercept % was calculated to be 1.7% for acetamino-
ity indicating for determination of acetaminophen, aspirin, phen, 0.4% for aspirin, and 0.7% for caffeine. The coeffi-
cients of determinations and Y-intercepts shown in supple-

TABLE 5. Acetaminophen Determination Accuracy

TABLE 7. Aspirin Determination Accuracy
Actual
Recovery Mean
Sample # concentration Concentration Recovery Mean
Sample #
mg/mL mg/mL % % mg/mL mg/mL % %
1 (50 %) 0.6240 0.6169 98.9 99 1 (50 %) 0.6240 0.6120 98.1 98
2 (50 %) 0.6214 99.6 2 (50 %) 0.6165 98.8
3 (50 %) 0.6198 99.3 3 (50 %) 0.6150 98.5
1 (100 %) 1.2480 1.2560 100.6 101 1 (100 %) 1.2480 1.2549 100.5 101
2 (100 %) 1.2585 100.8 2 (100 %) 1.2591 100.9
3 (100 %) 1.2526 100.4 3 (100 %) 1.2513 100.2
1 (150 %) 1.8720 1.8509 98.9 99 1 (150 %) 1.8720 1.8779 100.3 101
2 (150 %) 1.8694 99.9 2 (150 %) 1.8882 100.8
3 (150 %) 1.8643 99.6 3 (150 %) 1.8879 100.8
Mean (%) 100 Mean (%) 100
Optimization of Analytical Method for Simultaneous Determination 1687

TABLE 8. Instrumental Precision TABLE 10. Caffeine Intermediate Precision

Injection Acetaminophen Caffeine Aspirin Results
1 7309343 6102895 3144853 Sample # % LC
2 7312792 6105637 3145566 Analyst A Analyst B
3 7315216 6108785 3146917 1 101 99.17
4 7312556 6104374 3145833 2 97.81 98.32
5 7316051 6118250 3147202 3 100.66 99.07
Average 7313192 6107988 3146074 4 100.25 98.90
RSD% 0.0 0.1 0.0 5 102.78 99.24
6 98.81 97.61
Mean Individual 100.05 99.72
mentary Tables 2 – 4 indicated strong linearity for the given SD 1.94 0.64
method of analysis [12]. RSD 1.9 0.6
Accuracy. The method was proven accurate in the con- Mean Total 99.38
centration range of 0.6240 to 1.8720 mg/mL for acetamino- SD 1.54
phen, 0.6240 to 1.8720 mg/mL for aspirin, and 0.1500 to
RSD 1.6
0.4049 mg/mL for caffeine. The mean recovery percentages
from nine standard preparations at three different concentra-
tions spiked with placebo of the tablet excipients were calcu-
lated to be 99%, 101%, and 99% for acetaminophen, 99%, Intermediate precision. The method was proven precise
101%, and 100% for caffeine, and 98%, 101%, and 101% for as tested by different analysts. The RSD% for acetamino-
aspirin (supplementary Tables 5 – 7). The average recoveries phen assay within six replicates of the first analyst and the
values demonstrated high accuracy of the method of analysis second analyst were calculated to be 1.1% and 0.9% respec-
[11, 12].
tively. The RSD% for aspirin assay within the six replicates
Instrumental precision. The method was proven precise
of the first analyst and the second analyst were calculated to
as demonstrated after calculating the RSD% of acetamino-
be 0.8% and 0.9% respectively. The RSD% for caffeine as-
phen, caffeine, and aspirin peak area responses from five
standard injections (Supplementary Table 8). The RSD% say within the six replicates of the first analyst and the sec-
was 0.0% for acetaminophen, 0.1% for caffeine, and 0.0% ond analyst were calculated to be 1.9 % and 0.6% respec-
for aspirin.

TABLE 11. Aspirin Intermediate Precision

TABLE 9. Acetaminophen Intermediate Precision Results
Results Aspirin
Sample #
Sample # % LC % LC
Analyst A Analyst B Analyst A Analyst B
1 99.35 99.95 1 100.19 99.67
2 100.11 98.35 2 99.65 101.10
3 98.35 100.19 3 100.77 98.98
4 98.85 100.52 4 100.23 98.96
5 100.53 98.91 5 98.65 98.84
6 101.21 100.16 6 98.89 98.74
Mean Individual 99.73 99.68 Mean Individual 99.73 99.38
SD 1.08 0.85 SD 0.83 0.90
RSD (%) 1.1 0.9 RSD 0.8 0.9
Mean Total 99.71 Mean Total 99.56
SD 0.93 SD 0.85
RSD (%) 0.9 RSD 0.8
1688 Ayman Ismail et al.

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guidelines of the International Conference on harmonization Proceedings of the International Conference on Harmonization,
(ICH) yielding satisfactory results. Therefore, it can be con-
Geneva (2005).
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Badawey, BMC Chem., 13, 1 – 15 (2019).
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