Methods for the Analysis of Aspirin and Caffeine Tablets

To jump-start the standard development process and have earlier stakeholder engagement, USP is piloting a new approach for developing and sharing information with our stakeholders. Through a collaboration between USP’s Small Molecules Department and Global Analytical Development Laboratory, methods are being developed and validated for drug substances and drug products for which no monographs are currently available. These Emerging Standards are intended to improve USP’s official standards elaboration process by increasing transparency and allowing for broader stakeholder participation by publishing on the USP website prior to formal notice and comment through publication in the Pharmacopeial Forum.

Aspirin and Caffeine Tablets has been evaluated and shown to be a suitable candidate for development under this new program. Additional method development and validation information is provided to justify the use of method parameters.

Certain commercial software, instruments, or materials may be identified in this document to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the software, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate.

This document is not a USP compendial standard and is intended to serve as a resource for informational purposes only. It does not reflect USP or USP’s Expert Body opinions of future revisions to the official text of the USP-NF. Parties relying on the information in this document bear independent responsibility for awareness of and compliance with any applicable federal, state, or local laws and requirements.

Aspirin is in a group of medications called salicylates. It works by stopping the production of certain natural substances that cause fever, pain, swelling, and blood clots.¹

Caffeine is a naturally occurring central nervous system stimulant of the methylxanthine class. The primary goal of caffeine consumption is to combat fatigue and drowsiness, but there are many additional uses. The FDA has approved caffeine for use in the treatment of apnea of prematurity and the prevention and treatment of bronchopulmonary dysplasia of premature infants. Non-FDA-approved uses of caffeine include treating migraine headaches and post-dural puncture headaches, enhancing athletic performance, especially in endurance sports, and treating depression and neurocognitive declines, such as those seen in Alzheimer and Parkinson disease.² It is mainly used as a eugeroic or as a mild cognitive enhancer to increase alertness and attentional performance.³

The USP-NF contains monographs for Aspirin⁴ and Caffeine drug substances⁵, respectively, and several monographs for drug products including Aspirin Tablets⁶, Butalbital, Aspirin, and Caffeine Tablets⁷, Acetaminophen, Aspirin, and Caffeine Tablets⁸, and Orphenadrine Citrate, Aspirin, and Caffeine Tablets⁹. Aspirin and Caffeine are the active ingredients in Aspirin and Caffeine Tablets. However, there is no monograph for Aspirin and Caffeine Tablets. As part of the Emerging Standards initiative, it was decided to develop methods for Aspirin and Caffeine Tablets.

The recently published USP General Chapter 〈1220〉¹⁰ provides a framework for the implementation of the analytical procedure life cycle approach that is consistent with the quality by design (QbD) concepts described in the ICH guidelines. Stage 1 of the life cycle includes systematic procedure development experiments that result in an understanding of the effect of procedure parameters on the procedure performance. Design of Experiment (DOE), which includes statistical multi-variate analysis and modeling, is an important tool in Stage 1. The knowledge acquired through DOE studies also enables the determination of robust operation regions for procedure parameters and, if desired, a method operable design region (MODR).

This document summarizes method development efforts assisted by the Fusion QbD® software, as well as robustness and forced degradation study results. It also describes final procedures that may be suitable for identification, strength, and purity determination of aspirin and caffeine in the presence of various impurities and excipients in Aspirin and Caffeine Tablets. A summary of validation data and representative chromatographic results are included.

3.1. Aspirin, Caffeine, and Impurities Standards

USP Reference Standards for aspirin, caffeine, and salicylic acid (IMP C) were used. Acetylsalicylsalicylic acid (IMP D) and salsalate (IMP E) were procured from another supplier. The chemical structures of aspirin, caffeine, and related impurities are shown in Figures 1 and 2.

Figure 1a. Aspirin

Figure 1b. Caffeine

Figure 1. Aspirin and Caffeine

Figure 2a. Salicylic acid

Figure 2b. Acetylsalicylsalicylic acid

Figure 2c. Salsalate

Figure 2. Impurities

3.2. Samples

Two lots of Aspirin and Caffeine Tablet samples from the same manufacturer were used to evaluate methods described in this document.

3.3. Reagents

Trifluoroacetic acid (TFA) (ACS grade), acetonitrile (LC/MS grade), and methanol (LC/MS grade) were obtained from Fisher Chemical. Ultrapure water was obtained from a Milli-Q water purification system.

The goal of this work was to develop stability-indicating Assay and Organic Impurities (OI) methods for known degradation products and other potential degradants for Aspirin and Caffeine Tablets. The high-performance liquid chromatography (HPLC) conditions from a procedure¹¹ developed for the separation of more than 10 active pharmaceutical ingredients (APIs) found in over the counter (OTC) nighttime sleep aids and cough and cold products, including aspirin and caffeine, were used as the starting point for method development. The method utilized 2.1-mm × 15-cm; 1.8-µm C18 (L1) column maintained at 40° and a mobile phase consisting of 0.15% TFA in water (Solution A) and a mixture of acetonitrile and methanol (75:25) (Solution B) delivered in a gradient mode. First, the method conditions were transferred to an equivalent HPLC C18 column, 4.6-mm x 15.0-cm, 3.5-µm. Then, Fusion QbD® software was utilized for the DOE in an optimization study to explore the MODR and to select chromatographic conditions.

4.1. LC Conditions Optimization

The following HPLC conditions were maintained constant in the optimization study: aqueous moiety of the mobile phase (Solution A: 0.15% TFA in water), flow rate (1.0 mL/min), column temperature (40°), detection wavelength (220 nm), injection volume (4 µL), and column brand/type (Waters XSelect HSS T3). The initial ratio of Solution A and Solution B in the mobile phase was set at 90:10 as in the starting point method.

The effects of four method variables, initial hold time, gradient time, final hold time, and acetonitrile: methanol ratio in Solution B, on resolution between all peaks of interest were studied (Table 1). The software generated 30 runs for the design, with randomization and replicates considered. The injected solution contained about 0.02 mg/mL each of aspirin, caffeine, IMP C, IMP D, and IMP E in methanol.

Table 1. LC Conditions Optimization Design

The chromatographic results were imported back into the software for data modeling, analysis, and visualization to guide the selection of method conditions that provide the best selectivity and robustness. An in-silico robustness study was performed by the software with theoretical Monte Carlo simulations using the DOE-derived models. The areas in Figure 3 represent the MODR within which the method is predicted to be robust by meeting all response goals, including sufficient peak resolution of NLT 2.0 between aspirin, caffeine, and 3 specified impurities (IMP C, IMP D, and IMP E). The areas within the rectangles (initial hold time 1.0 – 3.0 min, gradient time 7 – 15 min, final hold time 3 – 10 min and organic solvent ratio, acetonitrile and methanol, (0:100 – 100:0)) include method conditions that can each be independently adjusted while still meeting response goals according to the model prediction. The proposed final method conditions are 2.0 min initial hold time, 11 min gradient time, 6.5 min final hold time, and Solution B consisting of acetonitrile and methanol at (50:50), which sits near the center of the MODR. Finally, the detection wavelength was changed to 270 nm and 0.05% of TFA was added to Solution B to improve the baseline slope.

Figure 3. Potential MODR represented by the unshaded areas

4.2. Robustness

The Robustness solution consisting of 2 mg/mL of aspirin, 0.16 mg/mL of caffeine, 19 µg/mL of IMP C, and 1.5 µg/mL each of IMP D and IMP E in methanol was analyzed under both the proposed LC conditions and deliberately changed LC conditions. The changes included flow rate ±10% (0.9 mL/min and 1.1 mL/min), column temperature ±3° (37° and 43°), initial isocratic hold time ±0.5 min (1.5 min and 2.5 min), percent of Solution B in the mobile phase ±2% absolute (68% and 72%), percent of TFA in Solution A (0.1%), and different column lot. Resolution criteria of NLT 2.0 and peak tailing of NMT 1.5 were met for all peaks in the Robustness solution across all evaluated conditions.

4.3. Forced Degradation

Forced degradation studies were performed by exposing the Sample solution (refer to section 7.2) prepared from Aspirin and Caffeine Tablets to acid, base, and oxidation; and by exposing the Tablets composite (NLT 20 tablets crushed into a fine powder) to heat, heat/humidity, and light (Table 2). The stressed samples were analyzed against freshly prepared Sample solution. The chromatograms were processed at 270 nm to detect the degradation products.

*RRT is calculated with respect to aspirin peak retention time.

IMP C was observed in freshly prepared Sample solution (Control, unstressed) at about 0.1%; the amount grew to about 0.6% in the aged solution. IMP C was formed at all stress conditions except light. IMP D and IMP E were formed under Heat and heat/humidity. Formation of unknown degradants with more than 1.0% peak area observed under oxidative stress by AIBN and Heat. All unknown impurity peaks were separated from aspirin, caffeine, and specified impurities peaks. Photo Diode Array (PDA) purity analysis for the aspirin peak (235‒400 nm) and for the caffeine peak (210‒400 nm) showed homogeneity of the UV spectra across aspirin and caffeine peaks in control and all stress conditions, indicating a lack of coelution.

Identification (ID) of aspirin and caffeine in Aspirin and Caffeine Tablets was evaluated using the final HPLC conditions from the Method Development section, with PDA spectral match and retention time match. See the Assay section for method conditions and solution preparations.

5.1. PDA Spectral Match

The ID procedure, solutions, and results are summarized in Table 3, and representative UV spectra of aspirin and caffeine from the Standard solution (Refer to section 6.2) and Sample solution are shown in Figures 4 and 5, respectively.

Table 3. Summary of Procedure, Solutions, and Results for the Identification Test by PDA Spectral Match

Figure 4. UV spectra of caffeine and aspirin from the Standard solution

Figure 5. UV spectra of caffeine and aspirin from the Sample solution

5.2. Retention Time Match

The ID test, solutions, and results are summarized in Table 4, and representative chromatograms of the Standard solution and Sample solution are shown in Figures 6 and 7, respectively.

Table 4. Summary of Solutions and Results for the Identification Test by Retention Time Match

The final HPLC method from the Method Development section was validated for the assay of Aspirin and Caffeine Tablets following USP General Chapter 〈1225〉 Validation of Compendial Procedures¹². The method was found to be specific, linear, accurate, and precise for the samples evaluated.

6.1. Instruments and Method

The analysis of Aspirin and Caffeine Tablets was performed using Agilent 1260 and Waters Alliance instruments with PDA detectors, and the results were processed using Empower (Waters software). The Waters XSelect HSS T3, 4.6-mm x 15.0-cm, 3.5-µm column was used. The analysis was performed at a column temperature of 40°, with a flow rate of 1.0 mL/min and 4 µL as the injection volume. The autosampler was kept at 8°. The PDA detector was set at 190–400 nm wavelength and the detection for the chromatograms was at 270 nm. The separation was achieved by a mobile phase gradient program as listed in Table 5.

Table 5. Mobile Phase Gradient Program

6.2. Solutions

Solution A: Transfer 1.5 mL of trifluoroacetic acid and 1000 mL of water into a 1-L bottle and mix well.

Solution B: Transfer 0.5 mL of trifluoroacetic acid into a 1-L bottle containing 500 mL of acetonitrile and 500 mL of methanol and mix well.

Diluent: Methanol.

Preparation of Caffeine standard stock solution: A solution of 0.4 mg/mL of USP Caffeine RS was prepared in Diluent with an aid of sonication.

Preparation of Standard solution: A solution of 0.5 mg/mL of aspirin and 0.04 mg/mL of caffeine was prepared as follows. Weigh 25 mg of USP Aspirin RS and transfer to a 50-mL volumetric flask. Pipet 5 mL of Caffeine standard stock solution into the flask. Add about 35 mL of Diluent to dissolve the solids. Dilute to volume with Diluent and mix well.

Preparation of Sample stock solution: A solution with nominally 5.0 mg/mL of aspirin and 0.4 mg/mL of caffeine from Aspirin and Caffeine Tablets was prepared as follows. Grind NLT 20 Aspirin and Caffeine Tablets to a fine powder. Transfer an accurately weighed portion, equivalent to about 250 mg of aspirin and 20 mg of caffeine, to a 50-mL volumetric flask. Add about 40 mL of Diluent and sonicate for 10 min. Dilute to volume with Diluent and mix well.

Preparation of Sample solution: A solution with nominally 0.5 mg/mL of aspirin and 0.04 mg/mL of caffeine was prepared from the dilution of the Sample stock solution in Diluent. Centrifuge a portion of the solution at NLT 3000 rpm for 10 min. Use the clear supernatant for analysis.

6.3. System Suitability Parameters, Validation Performance Characteristics, and Results

The system suitability parameters and results are summarized in Table 6. The validation performance characteristics and results are summarized in Table 7. Representative chromatograms of the Standard solution and Sample solution are shown in Figures 6 and 7, respectively.

Table 7. Summary of Validation Performance Characteristics, Solutions, and Results for the Assay Test

The final HPLC method from the Method Development section was validated as the OI procedure following 〈1225〉¹². The method was found to be specific, linear, accurate, precise, and free from interferences for the samples evaluated.

7.1. Instruments and Method

Same as in the Assay section, except that the detection of chromatogram was at UV 270 nm without a need for 3D PDA spectra.

7.2. Solutions

Prepare Solution A, Solution B, and Diluent as per the Assay procedure.

Preparation of individual Stock solutions: Five Stock solutions consisting of 0.2 mg/mL each of aspirin, IMP D or IMP E, 0.16 mg/mL of caffeine, or 0.24 mg/mL of IMP C, were individually prepared by dissolving appropriate amounts of each standard in Diluent.

Preparation of Standard solution: A solution consisting of 0.32 μg/mL (0.2%) of caffeine, 2.0 µg/mL (0.1%) of aspirin, 3.0 µg/mL (0.15%) each of IMP D and IMP E, and 60 μg/mL (3.0%) of IMP C, was prepared by combining appropriate volumes of individual Stock solutions in Diluent.

Preparation of Sensitivity solution: A solution consisting of 0.08 μg/mL (0.05%) of caffeine and 1.0 μg/mL (0.05%) each of aspirin, IMP C, IMP D, and IMP E was prepared by combining appropriate volumes of individual Stock solutions in Diluent.

Preparation of Sample solution:

A solution with nominally 2.0 mg/mL of aspirin and 0.16 mg/mL of caffeine from Aspirin and Caffeine Tablets was prepared as follows. Grind NLT 20 Aspirin and Caffeine Tablets to a fine powder. Transfer an accurately weighed portion, equivalent to about 200 mg of aspirin and 16 mg of caffeine, to a 100-mL volumetric flask. Add about 80 mL of Diluent and sonicate for 10 min. Dilute to volume with Diluent and mix well. Centrifuge a portion of the solution at NLT 3000 rpm for 10 min. Use the clear supernatant for analysis.

7.3. System Suitability Parameters, Validation Performance Characteristics, and Results

The system suitability parameters and results are summarized in Table 8. The validation performance characteristics and results are summarized in Table 9. Representative chromatograms of Diluent, Sensitivity solution, Standard solution, Sample solution, and Sample solution spiked with impurities at 100% level are presented in Figures 8–13, respectively. Linearity was established for caffeine, aspirin, and related impurities, whereas accuracy and repeatability were established for related impurities.

The limit of quantitation (LOQ) estimated from the USP Signal-to-Noise ratio was 0.05% for all three specified impurities, IMP C, IMP D, and IMP E, with respect to the nominal concentration of aspirin (2.0 mg/mL). The method was validated in the range of 1.5% ‒ 4.5% for IMP C and 0.075% ‒ 0.225% for IMP D and IMP E.

Table 8. Summary of System Suitability Parameters, Solutions, and Results for the Organic Impurities Test

Table 9. Summary of Validation Performance Characteristics, Solutions, and Results for the Organic Impurities Test

Table 10. Relative Response Factor

RRF values of the impurities were calculated by dividing the slope of the linearity curve for each impurity by the slope of the linearity curve for aspirin.

Figure 8. Chromatogram of Diluent (blank).

Figure 10. Chromatogram of Sensitivity solution.

Figure 11. Chromatogram of Standard solution.

Figure 12. Chromatogram (at expanded scale) of Sample solution.

Figure 13. Chromatogram (at expanded scale) of Sample solution spiked with specified impurities at the Standard solution level.