Validated Stability-Indicating HPLC Method For Simultaneous Determination of Carisoprodol, Paracetamol, And Caffeine in A Combined Dosage Tablet

Carisoprodol (CAR) is a muscle relaxant that target the brain and spinal cord. It’s thought to reduce muscle spasms by inhibiting the multi-synaptic reflex, likely through its suppressive effect on inter-neuronal activity. This compound is officially listed in the IP, BP and USP. ^[1-3] (Figure 1[A]). Paracetamol is a widely used as analgesic and antipyretic agent, primarily through its inhibitory effect on prostaglandin synthesis within the brain. This action subsequently mitigates the release of chemical mediators that induce pain and elevate body temperature. This compound is officially listed in the IP, BP and USP. ^[4,5] (Figure 1[B]). Caffeine enhances the effectiveness of paracetamol. This attributed to caffeine’s ability to cross blood brain barrier and block adenosine receptor. Since adenosine play a crucial role in energy transfer and promoting sleep, its blockade by caffeine contributes to the potentiated analgesic effect. Caffeine is official in IP, BP and USP ^[6-8] (Figure 1[C]).

(A)

(B)

(C)

Figure 1: (A)Carisoprodol, (B)Paracetamol and (C)Caffeine

Carisoprodol, Caffeine and Paracetamol act synergistically to alleviate pain and muscle spasms. Consequently, the quantitative analysis of CAR, PCM and CAF in both isolated forms and combined with other therapeutic agents, has been a subject of numerous reported analytical method ^[9-25], despite extensive literature, a significant remains as no stability indicating HPLC method is currently available for the accurate quantification of carisoprodol, paracetamol and caffeine in their combined dosage form.

MATERIALS AND METHODS

Instrumentation and reagents

For the analytical procedure, a Shimadzu HPLC (LC 2010), featuring an Ultraviolet-visible detector and binary gradient system, was employed. LC Solution software facilitated data collection and processing. Separation occurred on a HIBAR ODS C18 column (250 mm × 4.6 mm, 5 µm). Yarrow chem Pvt. Ltd. and RMS scientific services provided API, chemical and reagents, all used in mobile phase preparation. A Mettler Toledo weighing scale (0.1 mg sensitivity) ensured precise material measurements, and a Labman LMPH-9 pH meter was used for pH adjustments.

Preparation of Standard Solution:

A precisely weighed mixture of CAR, PCM, and CAF (175 mg, 325 mg, and 32 mg respectively) was dissolved in methanol in a 100 mL volumetric flask, yielding a stock solution of 1750 µg/mL CAR, 3250 µg/mL PCM, and 320 µg/mL CAF. Subsequent dilutions were performed: 1 mL of the stock solution was diluted to 10 mL with methanol to achieve concentrations of 175 µg/mL CAR, 325 µg/mL PCM, and 32 µg/mL CAF. A further 1 mL from this intermediate solution was then diluted to 10 mL with methanol, resulting in final concentrations of 17.5 µg/mL CAR, 32.5 µg/mL PCM, and 3.2 µg/mL CAF.

Selection of Detection Wavelength:

The UV spectra of working standards for CAR (10 µg/mL), PCM (6 µg/mL), and CAF (6 µg/mL) were recorded across the 200-400 nm region. Upon overlapping these spectra, an iso-absorptive point was identified at 210 nm. This specific wavelength was then chosen as the analytical wavelength for the development of the RP-HPLC method for the estimation of CAR, PCM, and CAF.

Optimization of separation conditions:

Several elution conditions were tried in order to get optimum separation. Detection wavelength was kept constant at 210 nm, while flow rate, organic phase and buffer proportions were varied. The condition that gave perfect separation, along with system suitability parameters, is highlighted as table 1.

Table 1: Optimized chromatographic conditions

Figure 2: Optimized chromatogram of PCM+CAR+CAF (32.5+17.5+3.2 µg/ml)

RESULTS AND DISCUSSION

System Suitability:

All system suitability parameters, including retention time (Rt), tailing factor (T), resolution (Rs), and number of theoretical plates, were found to be within acceptable limits. This was confirmed after injecting a CAR+PCM+CAF (17.5+32.5+3.2 µg/mL) solution three times, and statistically evaluating the parameters presented in Table 2 against their prescribed ranges.

Table 2: System suitability parameters for CAR, PCM and CAF

Forced Degradation studies:

A forced degradation study was performed to assess the impact of degradation products under various stress conditions, including acidic, basic, oxidative, and dry heat environments. For this purpose, an initial stock solution was prepared: an accurately weighed equivalent of 175 mg Carisoprodol, 325 mg Paracetamol, and 32 mg Caffeine was dissolved in methanol in a 100 mL volumetric flask, achieving concentrations of 1750 µg/mL CAR, 3250 µg/mL PCM, and 320 µg/mL CAF. Following 10 minutes of sonication and filtration through Whatman filter paper, this stock solution was utilized for the comprehensive degradation experiments.

Acid Degradation

To assess acid degradation, 0.1 mL of Solution A was pipetted into a 10 mL volumetric flask, to which 5 mL of 1 N HCl was added. The solution was refluxed at 50°C for 60 minutes, then cooled to ambient temperature, neutralized with 1 N NaOH, and diluted to 10 mL with methanol, resulting in CAR+PCM+CAF (17.5+32.5+3.2 µg/mL). A 20 µL injection was performed, and the extent of degradation was determined by comparing the treated sample to a 0-hour control. A reagent blank (without stock solution) was also processed.

Figure 3: Chromatogram of Acid Hydrolysis [Blank]

Figure 4: Chromatogram of Acid Hydrolysis [Treated] [1N HCl, 50°C, 60 min]

Base Degradation

To assess base-induced degradation, 0.1 mL of Solution A was added to a 10 mL volumetric flask, followed by 5 mL of 1 N NaOH. This solution was refluxed at 50°C for 60 minutes. After cooling to ambient temperature, it was neutralized with 1 N HCl and diluted to 10 mL with methanol, yielding CAR+PCM+CAF (17.5+32.5+3.2 µg/mL). A 20 µL sample was injected onto the column, and the extent of degradation was determined by comparing the treated sample against a 0-hour control. A reagent blank (without stock solution) was also processed.

Figure 5: Chromatogram of Base Hydrolysis [Blank]

Figure 6: Chromatogram of Base Hydrolysis [Treated] [1N NaOH, 50°C, 60 min]

Oxidative Degradation

To assess oxidation-induced degradation, 0.1 mL of Solution A was pipetted into a 10 mL volumetric flask, followed by the addition of 5 mL of 3% v/v H?O?. The solution was heated at 50°C for 40 minutes. After cooling to ambient temperature, it was diluted to 10 mL with methanol, yielding CAR+PCM+CAF (17.5+32.5+3.2 µg/mL). A 20 µL sample was injected onto the column, and the extent of degradation was determined by comparing the treated sample against a 0-hour control. A reagent blank (without stock solution) was also processed.

Figure 7: Chromatogram of Oxidative Stress [Blank]

Figure 8: Chromatogram of Oxidative Stress [Treated] [3 % V/V H2O2, 50°C, 40 min]

Thermal Degradation

To assess dry heat degradation, a precise mixture of 175 mg Carisoprodol, 325 mg Paracetamol, and 32 mg Caffeine was spread in a petri dish and subjected to 70°C in a hot air oven for 2 hours. After this thermal exposure, the contents were quantitatively transferred to a 100 mL volumetric flask and diluted to volume with methanol, forming a stock solution of CAR+PCM+CAF (1750+3250+320 µg/mL). A 0.1 mL aliquot of this stock was then diluted to 10 mL with methanol to prepare the working solution at CAR+PCM+CAF (17.5+32.5+3.2 µg/mL).

Figure 9: Chromatogram of Thermal Stress [Treated] [70ºC for 2 hrs]

Table 3: Evaluation table of Forced Degradation Study

Validation of Optimized method:

Linearity

Compliance with ICH Q2(R2) guidelines for linearity was confirmed for the developed HPLC method. Mean representative calibration curves (n=5) for CAR, PCM, and CAF displayed strong linearity, with regression coefficients consistently above 0.995. The established linear ranges were 8.75-43.75 µg/mL for CAR, 16.25-81.25 µg/mL for PCM, and 1.6-8 µg/mL for CAF. For visual reference, calibration curves are presented in (Figure 10) CAR, (Figure 11) PCM, and (Figure 12) CAF, and an overlay chromatogram demonstrating separation is available in (Figure 13).

Table 4: Linearity data for CAR

Figure 10: Calibration curve for CAR (8.75-43.75 µg/mL)

Table 5: Linearity data for PCM

Figure 11: Calibration curve for PCM (16.25-81.25 µg/mL)

Table 6: Linearity data for CAF

Figure 12: Calibration curve for CAF (1.6-8 µg/mL)

Figure 13: Overlay chromatogram for Linearity of CAR (8.75-43.75 µg/mL), PCM (16.25-81.25 µg/mL) and CAF (1.6-8 µg/mL)

Repeatability

Repeatability was evaluated by performing five replicate injections for each concentration of CAR, PCM, and CAF across the entire calibration range, ensuring short time intervals between injections. The peak areas generated for CAR, PCM, and CAF were then used to calculate the Relative Standard Deviation (RSD), providing an indication of method precision.

Table 7: Repeatability data of CAR

n= 5 determinations

Table 8: Repeatability data of PCM

n= 5 determinations

Table 9: Repeatability data of CAF

n= 5 determinations

Limit of detection and quantitation

Determination of the Limit of Detection (LOD) and Limit of Quantitation (LOQ) was achieved primarily through statistical calculation and visual detection techniques. Leveraging the linearity data, LOD and LOQ were statistically computed using the mean of the slope (S) and the standard deviation of the standard error (σ).

Table 10:  LOD and LOQ data for CAR, PCM and CAF

Intraday and Inter-day Precision

To establish method precision, both intra-day and inter-day precision were investigated. Samples containing CAR, PCM, and CAF at concentrations spanning the calibration range (8.75+16.25+1.6, 26.25+48.75+4.8, and 43.75+81.25+8 µg/mL) were prepared. Intra-day precision involved analyzing these mixtures multiple times on the same day, at different intervals, while inter-day precision required analyses on separate days. For each level, three determinations (n=3) were performed in both studies.

Table 11: Intraday and inter-day precision data of CAR

Table 12: Intraday and inter-day precision data of PCM

Table 13: Intraday and inter-day precision data of CAF

Accuracy

The accuracy of the analytical method was assessed through a standard addition (spiking) technique. Samples were fortified with known amounts of standard analytes at 50%, 100%, and 150% of the target concentration. Three determinations were performed at each spiking level.

Table 14: Accuracy data of CAR

Table 15: Accuracy data of PCM

Table 16: Accuracy data of CAF

Robustness

Robustness was determined by incrementally changing key method parameters and comparing the results to a standard preparation to observe any significant effects. For this study, the mobile phase flow rate and mobile phase composition were specifically varied.

Table 17: Robustness data of CAR+PCM+CAF by RP-HPLC method

The assay sample was prepared by calculating the average weight of 10 tablets. A quantity of tablet powder equivalent to 655.45 mg (175 mg CAR + 325 mg PCM + 32 mg CAF) was transferred to a 100 mL volumetric flask, and the volume was made up with methanol, resulting in a 1750+3250+320 µg/mL stock solution. After 10 minutes of sonication and filtration through a 0.45 µm Whatman filter paper, 0.1 mL of this filtrate was diluted to 10 mL with methanol, preparing the working sample solution of 17.5+32.5+3.2 µg/mL CAR+PCM+CAF. Three 20 µL injections of this solution were then made onto the column under optimized chromatographic conditions.

Table 18: Determination of CAR.PCM and CAF from its marketed formulation

Table 19: Summary and Conclusion of RP-HLPC method

Table 20: Forced degradation study