Simultaneous determination of four active pharmaceuticals in tablet dosage form by reversed-phase high performance liquid chromatography

Purpose: To develop a single, low-cost and rapid analytical method for the simultaneous determination of four active components - chlorpheniramine maleate, paracetamol, phenylephrine hydrochloride and caffeine – in a tablet dosage form. Method: This method was based on reverse-phase high performance liquid chromatography (RP-HPLC) and involved the use of a C-18 column (250 × 4.6 mm, 5.0 μm), a mobile phase consisting of buffer solution and methanol at a flow rate of 1.00 mL/min, and gradient determination with UV detection at 220 nm. Results: Retention time was 4.33, 10.36, 13.85, and 17.35 min for phenylephrine hydrochloride, paracetamol, caffeine, and chlorpheniramine maleate, respectively. Specificity data showed no interference from the excipients, and accuracy of the method was close to 100 %. The method was validated as per the guidelines of International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and all the results met the acceptance criteria for accuracy, precision, linearity, specificity, limit of quantitation, limit of detection, and robustness. Conclusion: This method can successfully perform quantitative assessment of phenylephrine HCl, chlorpheniramine maleate, paracetamol, and caffeine in tablet combination dosage forms faster and more cost-effectively than conventional methods.


INTRODUCTION
Pharmaceutical companies are increasingly pursuing combination dosage forms that can treat multiple symptoms simultaneously. A widely-used combination dosage form for the treatment of upper respiratory infections is the combination of phenylephrine hydrochloride, chlorpheniramine maleate, paracetamol, and caffeine, which is utilized for its analgesic, antipyretic, antihistamine, and antitussive activity.

EXPERIMENTAL Apparatus
Prior to this study, we optimized our protocol using different ratios of methanol, water, and ammonium buffer solution. Our apparatus consisted of Dionex Gradient System with UV Detector 730D and pump SP930 D. (ThermoFischerScientific, USA). Chromatographic separation was carried out at room temperature with a Cosmosil C-18 column (250 × 4.6 mm, 5.0 μm). (Nacalai Tesque ,Japan). Instrumental settings included a flow rate of 1.00 mL/min, column temperature of 30°C, and detector wavelength of 220 nm.

Chemicals and reagents
PHE, CPM, PARA, and CAF were supplied from Wallace Pharmaceuticals, Goa, India; HPLCgrade methanol, sodium dihydrogen phosphate dihydrate, and analytical research-grade tetra butyl ammonium hydrogen sulfate were from Merck, Mumbai, India. The water used was deionized and double-distilled. Tablets containing PHE, CPM, PARA, and CAF were obtained commercially (Helpex Anticold tablets). ( Sava Healthcare ,India ). Each tablet contained 10 mg PHE, 2 mg CPM, 500 mg PARA, and 30 mg CAF.

Mobile phase A:
For mobile phase A, 1.56 g sodium dihydrogen phosphate dihydrate and 1.70 g tetra butyl ammonium hydrogen sulfate were transferred to a 1,000-mL volumetric flask and dissolved in water to reach a volume of 1,000 mL, then mixed. The solution was then filtered through a 0.45-µm filter and degassed.

Preparation of solutions
A working standard solution containing 10 μg/mL PHE, 2 μg/mL CPM, 500 μg/mL PARA, and 30 μg/mL CAF was prepared by dissolving PHE, CPM, PARA, and CAF standards in 1 L 50% methanol. The mixture was sonicated for 30 min or until the standard dissolved completely. Sample solutions were prepared by finely powdering and weighing 20 tablets of the retail combination dosage. An amount of powder containing 10 mg PHE, 2 mg CPM, 500 mg PARA, and 30 mg CAF was added to a 100-mL volumetric flask and diluted with 70 mL 50% methanol diluent, mixed well and sonicated for 30 min, cooled to room temperature, and made up to the mark with additional diluent. A 2.5-mL aliquot of this solution was added to a 25-mL volumetric flask and made up to the mark with additional diluent.

RESULTS
The HPLC procedure was optimized by developing a simultaneous assay method for PHE, CPM, PARA, and CAF. At a flow rate 1.00 mL/min, the gradient method of sodium dihydrogen phosphate dihydrate and tetra butyl ammonium hydrogen sulfate buffer solution with methanol gave acceptable retention time, number of theoretical plates, and good resolution for PHE, CPM, PARA, and CAF standards ( Figure 2). The system suitability tests revealed that numbers of theoretical plates were above 2,000 and the tailing factor was less than 2.

Method development and validation parameters
The reverse-phase HPLC (RP-HPLC) method was developed for simultaneous estimation of PHE, PARA, CAF and CPM in tablet dosage forms, with RTs of 4.33, 10.36, 13.85, and 17.35 min, respectively. All validation parameters were quantified as detailed below.

Linearity
Linearity was assessed by injecting five different concentrations of each standard in the mobile phase in triplicate into the chromatographic system, keeping the injection volume constant. The peak values of area were plotted against the corresponding concentrations to obtain the calibration graphs.

Precision
Precision parameters assessed the repeatability of measurement and were calculated by injecting each standard solution six times, then measuring the peak areas. The RSD were 0.0861, 0.1093, 0.0330 and 0.2621 for PHE, CPM, PARA, and CAF respectively (Table 3).

Limit of detection and limit of quantitation
The limit of quantification (LOQ) and limit of detection (LOD) were evaluated based on signalto-noise ratios by serial dilution of PHE, CPM, PARA, and CAF solutions. The LOD and LOQ values are tabulated in Table 4.

Robustness
The robustness of the developed RP-HPLC method was assessed by making small, deliberate variations in the optimized method parameters and monitoring flow rate. The method was found to be unaffected when flow rate was changed ±0.1 mL/min (Table 5).

Specificity
The specificity of this method was assessed by examining the chromatogram and identifying where complete separation of PARA, PHE, CAF and CPM occurred. We observed no potential interference from the presence of excipients. The chromatographic peaks obtained were wellseparated at the baseline and were sharp in nature.

Application of developed method to a commercial tablet formulation
The developed RP-HPLC method identified similar quantities of PHE, CPM, PARA, and CAF in tablets as that listed on the product's label, ranging from 98.84-100.90% of the label amounts (Table 7). These findings confirmed our specificity results, indicating that excipients did not result in interference. This method produced well-differentiated determinations, with resolutions consistently greater than 2. The mean peak area of the chromatograms was plotted against the concentration of PHE, CPM, PARA, and CAF to obtain the calibration curve. Repeatability and intermediate precision values were within the acceptable limits set by ICH, indicating that the method is precise. Specificity experiments indicated no interference; the peaks of excipients and diluents did not overlap with the main peaks of PHE, CPM, PARA, and CAF. The LOD and LOQ values obtained by the proposed method were low, which indicated the sensitivity of the method. The stability studies indicate that both standard and sample drugs were stable up to 24 h, and change in flow rate, temperature and mobile phase composition did not cause any significant changes in the results. RSD for precision was < 2 %, confirming that this method is sufficiently precise.

CONCLUSION
The developed method has a significant advantage over other methods of analysis because it is capable of simultaneous determination of multiple active components present in combination dosage form, resulting in a faster, more cost-effective analysis. The time required for sample analysis is a few hours. The method has been validated extensively as per ICH guidelines, and it is suitable for use in pharmaceutical firms and laboratories.