Optimization of HPLC methods for the development of quality control methods of combined powder formulations with paracetamol, phenylephrine hydrochloride, pheniramine maleate

Abstract

Our main goal was to optimize HPLC methods for the determination of a combined powder containing paracetamol, phenylephrine hydrochloride, and pheniramine maleate based on the indicators «Quantitative determination», «Uniformity of dosage units», and «4-aminophenol». Precise HPLC methods were developed for determining paracetamol, phenylephrine hydrochloride, pheniramine maleate, and the impurity of paracetamol (4-aminophenol). The method was optimized by selecting the chromatographic column and gradient elution to balance component separation and chromatographic time. Separation was achieved using a diode array detector at 273 nm («Quantitative determination», «Uniformity of dosage units») and 225 nm (4-aminophenol) with a gradient mobile phase consisting of a 1.1 g/L sodium octanesulfonate solution (pH 3.2), methanol, and a chromatographic column Zorbax SB-Aq. The method showed a linear response in the range of 160–360 µg/mL for paracetamol, 5–11 µg/mL for phenylephrine hydrochloride, and 10–22 µg/mL for pheniramine maleate. The optimized method, validated according to ICH guidelines, reduced run times to 20 min for impurity analysis and 10 min for active ingredients, which is twice as fast as the official pharmacopeial method. The developed methods were successfully applied for the quality control of this powder.

Introduction

The role of combined medicinal products in treating various diseases is well-recognized in modern pharmaceutical practice [1]. These products are widely used to manage symptoms of colds and flu, such as headaches, nasal congestion, sore throat, fever, and general weakness. Given their broad application, ensuring their safety, efficacy, and quality through reliable analytical methods is of paramount importance.

Paracetamol, phenylephrine hydrochloride, and pheniramine maleate are commonly used in combined pharmaceutical formulations for the treatment of symptoms associated with colds and flu. Paracetamol (acetaminophen) is an analgesic and antipyretic drug with a well-established safety profile and is widely used to relieve pain and reduce fever. Its chemical name is N-(4-hydroxyphenyl)acetamide. Paracetamol is readily absorbed from the gastrointestinal tract and metabolized in the liver. It has a low solubility in water, with a pKa of 9.38 and Log P of 0.46 [2, 3], indicating its hydrophilic nature.

Phenylephrine hydrochloride is a sympathomimetic drug used as a decongestant to relieve nasal congestion. Its chemical name is (1R)-1-(3-Hydroxyphenyl)-2-(methylamino)ethanol hydrochloride. Phenylephrine hydrochloride is freely soluble in water and in ethanol (96 per cent) [3]. It works by stimulating alpha-adrenergic receptors, leading to vasoconstriction and reduced swelling in nasal passages.

Pheniramine maleate is an antihistamine used to relieve symptoms of allergy, such as runny nose and sneezing. Its chemical name is (3RS)-N,N-Dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1-amine (Z)-butenedioate. Pheniramine maleate is very soluble in water, freely soluble in ethanol (96 per cent), in methanol and in methylene chloride [3].

Analytical methods for the simultaneous determination of these compounds in pharmaceutical formulations must meet high standards of sensitivity, specificity, and efficiency. Existing methods, however, often suffer from significant limitations, including long analysis times, high solvent consumption, and difficulties in detecting impurities. The monographs of paracetamol, phenylephrine hydrochloride, and pheniramine maleate from the European Pharmacopoeia (Ph. Eur.) [3] prescribe titrimetric methods for the assay of these active substances: cerimetry for paracetamol, alkalimetry for phenylephrine hydrochloride, and acidimetry for pheniramine maleate. However, a significant limitation of these methods is their inapplicability to dosage forms containing these active pharmaceutical ingredients.

The US Pharmacopoeia (USP) [4] monograph for phenylephrine hydrochloride includes an HPLC method for its assay. While this method is more versatile, it is not designed to control other components in combination dosage forms, and it has a relatively long runtime (about 16 min) for a single component, making it inefficient for comprehensive analysis. Similarly, the monographs of paracetamol (acetaminophen) from both the Ph. Eur. [3] and USP [4] prescribe HPLC methods for impurity testing. These methods are applicable to various dosage forms but suffer from significant drawbacks, including extended runtimes (up to 70 min) and high consumption of mobile phase, reducing their suitability for routine quality control.

Earlier studies have focused on the development and validation of various analytical methodologies for the determination of active pharmaceutical ingredients and their impurities in complex pharmaceutical formulations. In particular, HPLC methods have been developed and applied for the analysis of phenylephrine hydrochloride, pheniramine maleate, paracetamol, dextromethorphan, chlorpheniramine, and other components in single-component and multi-component drug products [7–23]. These studies include stability-indicating methods [7], methodologies for liquid formulations [9, 13, 20], tablet dosage forms [12, 14, 15], as well as impurity and degradation product analysis, such as 4-aminophenol [16–18]. Furthermore, modern approaches, including chemometric analysis and the combination of spectroscopic and chromatographic methods, have been employed [10, 18]. These works have provided a solid foundation for further improvements in the analysis of complex pharmaceutical matrices.

Previous research, as detailed in the qualification scientific work “The Theoretical and Experimental Substantiation of Creation and Research of Combined Solid Dosage Forms” [5], proposed HPLC methods for the assay of paracetamol, phenylephrine hydrochloride, and pheniramine maleate, as well as for the impurity 4-aminophenol in combined powder formulations. However, these methods also involve long runtimes (22 and 38 min). Specifically, a runtime of 22 min for quantitative determination is too lengthy for in-process control in industrial production, where rapid results are crucial to ensure efficient workflow and timely product release.

The aim of this study was to optimize HPLC methods for the determination of combined powder containing paracetamol, phenylephrine hydrochloride, and pheniramine maleate based on the indicators «Quantitative determination», «Uniformity of dosage units», and «4-aminophenol» for routine use in quality control laboratories, providing faster and more efficient analysis suitable for industrial production needs. The optimization of the HPLC method was based on several key criteria, including reducing analysis time, improving method sensitivity, specificity, and reproducibility. In addition to time efficiency, we focused on minimizing reagent consumption and solvent usage, ensuring a more cost-effective and environmentally friendly approach. The optimization process also aimed to enhance the overall reliability of the method for routine quality control in pharmaceutical applications, providing a balance between speed and accuracy. These considerations make the method suitable for industrial use while adhering to environmental and economic standards.

Experimental

Instrumentation

The following HPLC columns were used: Zorbax SB-Aq 50 mm × 4.6 mm, 5 µm. The used columns were purchased from Agilent Technologies, Inc. Santa Clara, CA, United States. In this research, the Agilent 1200 Infinity with a diode array detector was used (Agilent Technologies, Inc. Santa Clara, CA, United States).

The following additional instrumental equipment was used: analytical balance Mettler Toledo XPE-205 (Mettler-Toledo International Inc. Greifensee, Switzerland), pH meter Mettler Toledo Seven Easy (Mettler-Toledo International Inc. Greifensee, Switzerland) and US bath Elmasonic P (Elma Schmidbauer GmbH, Singen, Germany). The regenerated nylon (NY) 0.2 µm syringe filters were purchased from Agilent Technologies.

Reagents and standards

Combined powder paracetamol, phenylephrine hydrochloride, pheniramine maleate and ascorbic acid was purchased from a local pharmacy. The standards for the active ingredients paracetamol (purity ≤ 99% (HPLC)), phenylephrine hydrochloride (purity ≤ 99% (HPLC)), pheniramine maleate (purity ≤ 99% (HPLC)) and its specified impurity of paracetamol, 4-aminophenol (purity ≤ 99% (HPLC)) was supplied by EDQM and USP.

The used chemicals, methanol, sodium octanesulfonate and phosphoric acid, were gradient grade, purchased from Merck, Darmstadt, Germany. The demineralized water that was used for analyses was an in-house product of Stilman with a conductivity of less than 0.5 µS/cm.

Sample preparation and chromatographic conditions

Sample preparation and chromatographic conditions for determination of impurity of paracetamol (4-aminophenol) in powder

For mobile phase A, the 1.1 g of sodium octanesulfonate was dissolved in 900 ml of water for chromatography R, adjusted to pH 3.2 with phosphoric acid R and dilute to 1000 ml in the water for chromatography R.

Mobile phase B—Methanol R.

Solvent mixture— mix of equal values of methanol R and water for chromatography R, the pH which has been adjusted to pH 3.5 with phosphoric acid R 1:1 (v/v).

Preparation of test solution

A solution was prepared with a concentration of 800 µg/mL of paracetamol, in a solvent mixture.

Preparation of reference solution

A solution was prepared with 16.0 mg of reference standard paracetamol and 16.0 mg of reference standard 4-aminophenol, add 10.0 mL of methanol R. This solution was diluted to 50.0 mL with the methanol R. Make 1.0 mL of the resulting solution up to 50.0 ml with methanol R and mix. Make 2.5 ml of the resulting solution up to 20.0 ml with water R, the pH of which has been adjusted to 3.5 with phosphoric acid R, and mix (0.8 µg/mL of paracetamol, 0.8 µg/mL of 4-aminophenol).

Chromatography was carried out on a liquid chromatograph with a spectrophotometric detector under the following conditions: a 50 mm × 4.6 mm octadecylsilyl column, 5 µm (Zorbax SB-Aq); a flow rate of 1.0 mL/min; detection at 225 nm; a column temperature of (40 ± 1) °C; injection with 10 μL; and elution mode: gradient according to the following program (Table 1).

Table 1.

Gradient mode for the HPLC method for analysis of 4-aminophenol in powder

Time, min	Mobile Phase A, %	Mobile Phase B, %
0	90	10
7	90	10
12	40	60
15	40	60
17	90	10
20	90	10

The system suitability for reference solution:

Symmetry factor: maximum 1.5 for the peak due to 4-aminophenol;

Resolution factor: minimum 5 between the peaks due to 4-aminophenol and paracetamol.

Sample preparation and chromatographic conditions for quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder

Mobile phase A, mobile phase B and the solvent mixture were the same as those in Sect. "Sample Preparation and Chromatographic Conditions for Determination of Impurity of Paracetamol (4-aminophenol) in Powder.".

Preparation of test solution

A solution was prepared with a concentration of 260 µg/mL of paracetamol, 8 µg/mL phenylephrine hydrochloride and 16 µg/mL of pheniramine maleate in a solvent mixture.

Preparation of reference solution (a)

A solution was prepared with a concentration of 400 µg/mL of reference standard phenylephrine hydrochloride in the solvent mixture.

Preparation of reference solution (b)

A solution was prepared with a concentration of 800 µg/mL of reference standard pheniramine maleate in the solvent mixture.

Preparation of reference solution (c)

A solution was prepared with 26.0 mg of reference standard paracetamol, added 2.0 mL of reference solution (a), 2.0 mL of reference solution (b) and 5.0 ml of methanol R. This solution was diluted to 100.0 mL with the solvent mixture (260 µg/mL of paracetamol, 8 µg/mL phenylephrine hydrochloride and 16 µg/mL of pheniramine maleate).

Chromatography as described in the test Sect. "Sample Preparation and Chromatographic Conditions for Determination of Impurity of Paracetamol (4-aminophenol) in Powder." with the following modification: detection at 273 nm and elution mode: gradient according to the following program (Table 2).

Table 2.

Gradient mode for the HPLC method for analysis of Paracetamol, Phenylephrine Hydrochloride, Pheniramine Maleate in Powder

Time, min	Mobile Phase A, %	Mobile Phase B, %
0	90	10
4	40	60
6	40	60
8	90	10
10	90	10

The system suitability for reference solution (c):

Symmetry factor: maximum 1.5 for the peak due to paracetamol, phenylephrine hydrochloride and pheniramine maleate;

Relative standard deviation: maximum 1.0% for 3 injections;

The content of paracetamol in a powder should be from 308.8 to 341.3 mg, phenylephrine hydrochloride 9.5 mg to 10.5 mg, and pheniramine maleate 19.0 mg to 21.0 mg based on the average weight of one sachet powder. These ranges are derived from the nominal values of 325 mg, 10 mg, and 20 mg per sachet, with an allowable variation of ± 5%.

Sample preparation and chromatographic conditions for quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in «Uniformity of dosage units»

Mobile phase A, mobile phase B and the solvent mixture were the same as those in Sect. "Sample Preparation and Chromatographic Conditions for Determination of Impurity of Paracetamol (4-aminophenol) in Powder.".

Preparation of test solution

One sachet of powder was dissolved of solvent mixture and diluted to 200.0 mL with the same solvent. This solution was filtered through a membrane filter with a pore size of 0.2 μm, discarding the first portions of the filtrate.

Preparation of reference solution

Preparation of reference solution (c) was the same as those in Sect. "Sample Preparation and Chromatographic Conditions for Determination of Impurity of Paracetamol (4-aminophenol) in Powder.".

Chromatography was carried out on a liquid chromatograph with a spectrophotometric detector under the conditions described in Sect. "Sample preparation and chromatographic conditions for quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder".

The test requirements are considered fulfilled if the acceptance value is less than or equal to 15.0. If the acceptance value is greater than 15.0, the tests specified in Eur.Ph., 2.9.40 [3] should be performed.

Validation of HPLC method

All validation was carried out in accordance with the requirements of the ICH Validation of Analytical Procedures: Text and Methodology, Q2 (R2) [6].

Validation of HPLC method for the determination of 4-aminophenol in powder

To study the specificity, the absence of the interference of the peaks of 4-aminophenol with the peaks of the solvent mixture and components of the drug was checked.

To study the linearity, 7 solutions containing 4-aminophenol at a concentration of 0.160–1.200 μg/mL (equivalent to an impurity content of 0.020–0.148%).

To check the accuracy, 12 model solutions were prepared (4 concentration levels, 3 solutions for each level) containing a known addition of 4-aminophenol to the test solution, along with 3 parallel test solutions without the additive.

Precision was studied at the level of repeatability and intermediate precision. Under the conditions of repeatability, 6 parallels of the tested solution were prepared. Under the conditions of intermediate precision, 12 parallels of the tested solution were prepared.

Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder

To study the specificity, the absence of the interference of the peaks of paracetamol, phenylephrine hydrochloride and pheniramine maleate with the peaks of the solvent mixture and components of the drug was checked.

Linearity studies were conducted within 5 solutions in the following ranges:

Paracetamol from 157.447 µg/mL to 359.737 µg/mL, corresponding to 60.56%–138.36% of the nominal API content in the test solution;

Phenylephrine hydrochloride: from 4.843 µg/mL to 11.301 µg/mL, corresponding to 60.54%–141.26% of the nominal API content in the test solution;

Pheniramine maleate from 9.632 µg/mL to 22.474 µg/mL, corresponding to 60.20%–140.46% of the nominal API content in the test solution.

The accuracy of the methodology was evaluated using 9 model solutions containing placebo and the target analytes within the following concentration ranges:

Paracetamol from 168.101 µg/mL to 349.993 µg/mL, corresponding to 64.65%–131.61% of the nominal content in the test solution;

Phenylephrine hydrochloride: from 5.231 µg/mL to 11.170 µg/mL, corresponding to 65.39%–139.62% of the nominal content in the test solution;

Pheniramine maleate from 10.505 µg/mL to 21.847 µg/mL, corresponding to 65.66%–136.55% of the nominal content in the test solution.

The precision study was conducted at the levels of repeatability and intermediate precision. For the precision assessment, two analysts prepared 6 parallel test solutions for the quality parameter "Quantitative Determination" over two analytical days.

Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in «Uniformity of dosage units»

The specificity was evaluated by verifying the absence of interference between the peaks of paracetamol, phenylephrine hydrochloride, and pheniramine maleate and the peaks of the solvent mixture or other components of the drug.

The linearity was assessed by preparing 5 solutions with analytes in the solvent mixture, covering a range of concentrations, as described in Sect. "Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder".

To evaluate accuracy, 9 model solutions were prepared at three concentration levels (three solutions per level) according to Sect. "Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder".

The precision was studied at the levels of repeatability and intermediate precision. For this, two analysts prepared 10 parallel test solutions for the quality parameter "Uniformity of Dosage Units" over two analytical days.

Results and discussion

HPLC method development

During the development of our methods, we focused on optimizing analytical conditions to ensure selectivity, simplicity, and efficiency, which are critical for routine pharmaceutical quality control. Particular attention was given to the selection of chromatographic parameters, including the column and mobile phase, as well as instrumental settings, to achieve reproducible results and minimize analysis time. The choice of the Zorbax SB-Aq column (50 mm × 4.6 mm, 5 µm) was guided by its superior performance in separating polar and moderately lipophilic compounds under high aqueous conditions. Unlike conventional C18 columns, which may suffer from phase dewetting in highly aqueous mobile phases, the SB-Aq stationary phase is designed to remain fully hydrated, ensuring consistent retention and peak shape for hydrophilic analytes. This property was particularly beneficial for the simultaneous determination of paracetamol, phenylephrine hydrochloride, and pheniramine maleate, as well as the impurity 4-aminophenol, all of which exhibit varying degrees of polarity.

Additionally, this column demonstrated optimal peak resolution and symmetry while maintaining high efficiency, allowing for rapid analysis without sacrificing chromatographic performance. Compared to other reversed-phase options, the Zorbax SB-Aq column provided a robust and reproducible separation profile, reducing analysis time and improving method reliability. These characteristics made it the most suitable choice for achieving precise, efficient, and high-throughput quality control of the multicomponent pharmaceutical formulation under study.

All proposed HPLC methods—the determination of 4-aminophenol as a critical impurity, the quantitative determination of active pharmaceutical ingredients, and the “Uniformity of Dosage Units” test—were designed to use the same mobile phase, consisting of a 1.1 g/L solution of sodium octanesulfonate adjusted to pH 3.2 with phosphoric acid. This unified approach not only simplified the analytical process but also reduced the need for additional solvents and reagents, aligning with modern principles of cost-effectiveness and sustainability in analytical chemistry. The selected concentration of ion-pairing reagent allowed for improved peak shape while maintaining a suitable balance between retention time and resolution. This pH level (3.2) was chosen because it provided ideal peak symmetry for phenylephrine hydrochloride and pheniramine maleate, preventing tailing.

To further enhance the methods, we systematically optimized the gradient elution program to balance separation efficiency and run time. Particular emphasis was placed on reducing the overall analysis duration without compromising accuracy and precision. The resulting methods allowed for the determination of 4-aminophenol within 20 min and the quantitative determination of active ingredients within just 10 min, achieving a twofold reduction in analysis time compared to official methods.

Validation of the methods was carried out following ICH guidelines, encompassing parameters such as accuracy, precision, linearity, specificity, robustness, and system suitability. The methods demonstrated high robustness, ensuring consistent performance even under slight variations in experimental conditions, which is essential for routine pharmaceutical applications. Acceptability criteria were defined and met for all validation characteristics, confirming the reliability and suitability of these methods for the quality control of multicomponent pharmaceutical products.

The implementation of these optimized methodologies offers numerous advantages, including enhanced analytical efficiency, reduced resource consumption, and improved sustainability. By minimizing analysis time and simplifying procedures, the methods facilitate seamless integration into routine quality control processes, particularly in high-throughput manufacturing environments. Furthermore, the use of a single mobile phase across multiple tests reduces operational complexity, lowers costs, and minimizes potential errors during method execution.

In addition to their efficiency, these methods also reflect modern pharmaceutical industry trends, focusing on the environmental impact of analytical procedures. The reduced consumption of reagents and solvents contributes to sustainability efforts, making these methods not only practical but also environmentally responsible.

The full details of the optimized methods, including step-by-step procedures for the determination of “4-aminophenol,” “Quantitative Determination,” and “Uniformity of Dosage Units,” are provided in Appendices A–C. These methods are highly adaptable and can be widely applied in pharmaceutical analysis, particularly for products with similar multicomponent formulations.

HPLC method development for the determination of 4-aminophenol in powder

A reliable and accurate HPLC method was established for the determination of 4-aminophenol in pharmaceutical dosage forms. The analysis was conducted using a gradient mobile phase comprising a 1.1 g/L sodium octanesulfonate solution (pH 3.2) and methanol, with a flow rate of 1.0 mL/min at 40 °C. Separation was performed on a Zorbax SB-Aq column (50 × 4.6 mm, 5 µm) (Fig. 1), which was identified as the most suitable column for achieving optimal resolution and peak separation during experimental trials. This column features an endcapped octadecyl stationary phase with an active surface area of 180 m²/g, 80 Å pore size, and 10% carbon load. The total run time for the analysis was approximately 20 min, with an injection volume of 10 µL. The optimal detection wavelength was determined to be 225 nm, providing the highest sensitivity and a robust response. A flow rate of 1.0 mL/min ensured efficient separation within a reasonable time frame.

Fig. 1.

Chromatogram obtained using gradient mobile phase consisting of 1.1 g/L solution of sodium octanesulfonate (pH 3.2), methanol, at a flow rate of 1.0 mL/min and temperature 40 °C, used for the analysis on an Zorbax SB-Aq (50 × 4.6 mm, 5 µm) column

Building on the prior experience of our research group, we opted to use sodium octanesulfonate as an ion-pairing agent. The application of gradient elution enabled the effective separation of paracetamol and 4-aminophenol. This approach provided high specificity and resolution for both analytes. The results of peak identification, along with the calculated LOQ values, are presented in Table 3. Additionally, the use of sodium octanesulfonate contributed to improved peak symmetry and reduced interference from matrix components, ensuring reliable quantification.

Table 3.

Identification of peaks (reference solution)

Peak	RT	RRT	LOQ, %
Paracetamol	3.0	1.00	–
4-aminophenol	7.4	2.47	0.02%

Rationing at the time of release: 4-aminophenol: no more than 0.1%.

HPLC method development for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder

A reliable and environmentally friendly HPLC method was successfully developed for the simultaneous determination of paracetamol, phenylephrine hydrochloride, and pheniramine maleate in pharmaceutical dosage forms. The method utilized a gradient elution system with mobile phase A, consisting of a 1.1 g/L sodium octanesulfonate solution (pH 3.2), and mobile phase B, comprising methanol. The analysis was performed on a Zorbax SB-Aq column (50 × 4.6 mm, 5 μm) (Fig. 2) at a flow rate of 1.0 mL/min.

Fig. 2.

Chromatogram obtained using gradient mobile phase consisting of 1.1 g/L solution of sodium octanesulfonate (pH 3.2), methanol, at a flow rate of 1.0 mL/min and temperature 40 °C, used for the analysis on an Zorbax SB-Aq (50 × 4.6 mm, 5 µm) column

The retention times for paracetamol, phenylephrine hydrochloride, and pheniramine maleate were 2.620 min, 4.268 min, and 5.644 min, respectively, demonstrating excellent resolution between the analytes. An injection volume of 10 μL was used, and the optimal detection wavelength of 273 nm provided the highest sensitivity with no interference from the solvent mixture peak. The flow rate of 1.0 mL/min was confirmed to deliver efficient separation within a reasonable runtime, ensuring the method’s practicality for routine analysis.

HPLC method development for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in «Uniformity of dosage units»

An accurate and environmentally friendly HPLC method was developed for the determination of paracetamol, phenylephrine hydrochloride, and pheniramine maleate in the "Uniformity of Dosage Units" test. The chromatographic conditions were identical to those optimized for the quantitative determination of these analytes in pharmaceutical dosage forms.

The method utilized a gradient elution system comprising mobile phase A (a 1.1 g/L sodium octanesulfonate solution at pH 3.2) and mobile phase B (methanol). The analysis was carried out on a Zorbax SB-Aq column (50 × 4.6 mm, 5 μm) (Fig. 2) at a flow rate of 1.0 mL/min and ambient temperature. Retention times were 2.20 min for paracetamol, 3.88 min for phenylephrine hydrochloride, and 5.34 min for pheniramine maleate, ensuring good resolution between the analytes. The injection volume was 10 μL, providing reliable and reproducible results.

Method validation

The procedure was validated in compliance with the standards in accordance with the International Conference on Harmonization (ICH) [6].

Validation of HPLC method for the determination of 4-aminophenol in powder

To confirm the efficiency of the method, the following parameters were studied: specificity; linearity in the range of application; accuracy; precision; and limit of quantification (LOQ) [6]. The formulas that were used for the calculations are given in Appendix A. The summary of the validation results is given in Appendix B, Table A1.

The separation of impurities and other components in the spiking sample was appropriate. No interference was obtained among the 4-aminophenol peak and the matrix peaks.

The linear regression parameters were calculated in accordance with the recommendations of the ICH Validation of Analytical Procedures: Text and Methodology, Q2 (R2) [6]. Excellent linearity of the method for the analysis of impurities of 4-aminophenol was confirmed by the obtained correlation coefficient was almost ideal (Table 4, Appendix B, Table A1).

Table 4.

Linear regression parameters

Parameter	a	|a|, %	b	r	S0	Sa
Result	− 0,120	0688	21,900	0,99,949	0305	0238

The parameters accuracy and precision were evaluated using the spiking method for 4-aminophenol with the test solution in 12 replicates. Recovery and RSD were assessed for six replicates analyzed by Analyst 1, six replicates analyzed by Analyst 2, and the difference in results between the two analysts. The results are presented in Table 5.

Table 5.

Calculations for the parameter “accuracy”and “precision”

Sample	Xi, %	Xaverage, %	RSD, %
M1	86,202–102,928	95,861	6706
M2-M4	93,689–104,465	99,712	3261

The LOQ of the method for the determination of 4-aminophenol was determined to be ~ 0.020%, (acceptability criteria was LOQ ≤ 0.05%).

To calculate the content of 4-aminophenol, the USP reference standard of 4-aminophenol was used.

Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in powder

To evaluate the method's efficiency, several parameters were examined, including specificity, linearity within the application range, accuracy, and precision [6]. The formulas used for calculations are provided in Appendix A, while a summary of the validation results is presented in Appendix C, Table A2.

Linear regression parameters were determined following the guidelines outlined in the ICH "Validation of Analytical Procedures: Text and Methodology, Q2 (R2)" [6]. The regression analysis for the concentration ranges of paracetamol, phenylephrine hydrochloride, and pheniramine maleate demonstrated a high determination coefficient. The correlation coefficients, ranging from 0.9998 to 0.9999, indicate nearly perfect linear relationships for the analysis of these analytes (Table 6, Appendix C, Table A2).

Table 6.

Results of the calculation of the linear regression parameters and accuracy and precision of the method according to the calibration curve

Parameter	a	|a|, %	b	r
Paracetamol	− 6823	0259	10,205	0,9999
Phenylephrine hydrochloride	− 0155	0353	5531	0,9998
Pheniramine maleate	− 0374	0659	3584	0,9999

The proposed method demonstrated excellent accuracy for the analysis of paracetamol, phenylephrine hydrochloride, and pheniramine maleate, as evidenced by the recovery rates at each concentration level, which ranged between 99.0% and 101.0% (99.5%, 99.1%, 100.1%). These results confirm the method's reliability in accurately quantifying the target analytes.

Furthermore, the method exhibited outstanding precision, with the relative standard deviation (RSD) for the measured content across samples being less than 2.2% (1.136%, 0.807%, 1.233%) (Appendix C, Table A2). The obtained results demonstrate good accuracy and precision of the proposed HPLC method (Tables 7 and 8).

Table 7.

Calculations for the parameter “accuracy” for the Paracetamol, Phenylephrine Hydrochloride, Pheniramine Maleate in Powder

Parameter	∆c_i, %	∆c_Average, %	RSD, %
Paracetamol	98.5–100.4	99.5	0.02–0.72
Phenylephrine hydrochloride	98.5–100.0	99.1	0.02–0.70
Pheniramine maleate	98.3–101.2	100.1	0.06–0.68

Table 8.

Calculations for the parameter “precision”

Parameter	Xi, mg/Sachet	Xaverage, mg/Sachet	RSD, %
Paracetamol	327.730–327.093	327.411	1.136
Phenylephrine hydrochloride	9.887–9.907	9.897	0.807
Pheniramine maleate	20.576–20.879	20.727	1.233

Validation of HPLC method for the quantitative determination of paracetamol, phenylephrine hydrochloride, pheniramine maleate in «Uniformity of dosage units»

To confirm the efficiency of the method, the following parameters were studied: specificity; linearity in the range of application; accuracy; and precision [6]. The formulas that were used for the calculations are given in Appendix A. The summary of validation results is given in Appendix C, Table A2. The linear regression parameters were calculated in accordance with the recommendations of the ICH Validation of Analytical Procedures: Text and Methodology, Q2 (R2) [6]. Excellent linearity of the method for the analysis of paracetamol, phenylephrine hydrochloride, pheniramine maleate was confirmed by the obtained correlation coefficient was almost ideal (0.9998–0.9999) (Table 6).

The obtained results demonstrate good accuracy and precision of the proposed HPLC method (Tables 7).

Excellent precision for the method for analysis of the paracetamol, phenylephrine hydrochloride, pheniramine maleate was confirmed by the RSD of the found content between samples, which was 1.885%, 4.058% and 2.462%. (Appendix C, Table A2). The obtained results demonstrate good accuracy and precision for the proposed HPLC method (Tables 9).

Table 9.

Calculations for the parameter “precision”

Parameter	Xi, %	Xaverage, %	RSD, %
Paracetamol	101.128–99.403	100.265	1.885
Phenylephrine hydrochloride	100.538–97.018	98.778	4.058
Pheniramine maleate	103.483–103.444	103.467	2.462

Efficiency improvement through method optimization

The presented data demonstrate a significant reduction in the analysis time following the optimization of methods for various analytical procedures.

Previous research, as detailed in the qualification scientific work “The Theoretical and Experimental Substantiation of Creation and Research of Combined Solid Dosage Forms” [5], proposed HPLC methods for the assay of paracetamol, phenylephrine hydrochloride, and pheniramine maleate, as well as for the impurity 4-aminophenol in combined powder formulations. Building upon these foundations, we optimized these methodologies and achieved a reduction in analysis time by more than 52%.

Quantitative Determination: The initial analysis time of 352 min was reduced to 160 min, resulting in a time savings of 192 min. Uniformity of Dosage Units: The method optimization reduced the analysis time from 550 to 250 min, achieving a notable time savings of 300 min. 4-Aminophenol: The optimization process decreased the analysis time from 304 to 160 min, saving 144 min. Total Analysis Time: The cumulative analysis time for all methods was significantly reduced from 1206 to 570 min, yielding an overall time savings of 636 min (Table 10).

Table 10.

Time Savings Achieved Through Optimization of Analytical Methods

Method	Analysis time before optimization (min)	Analysis time after optimization (min)	Time savings (min)
Quantitative Determination	352	160	192
«Uniformity of dosage units»	550	250	300
4-aminophenol	304	160	144
Total analysis time	1206	570	636

These results underscore the effectiveness of the optimized methodologies in enhancing efficiency by reducing the total analysis time by over half. Such advancements are critical for improving the throughput and practicality of analytical procedures in pharmaceutical research and quality control.

Overview of existing RP-HPLC methods for the simultaneous determination of API with MOGAPI score is presented in Table 11. The main problem with the existing methods is that they performed on columns 250 mm. Then the peaks of phenylephrine hydrochloride and pheniramine maleate are too stretched along the chromatogram and poor RSD, due to their very low content (10 and 20 mg, respectively, per 13.6 g packet). A short column solves this problem, the peaks become sharp and the accuracy of the method increases.

Table 11.

Overview of Existing RP-HPLC Methods for the Simultaneous Determination of Active Pharmaceutical Ingredients

RP-HPLC Method for Simultaneous estimation	Column	Mobile Phase	Flow rate, mL/min	Run time, min	Limitations of Existing Methods and Advantages of the Proposed Approach	MOGAPI
Chlorpheniramine Maleate, Paracetamol, Phenylephrine Hydrochloride [11]	CN, 250 × 4.6 mm, 5µ	Phosphate buffer (pH 6.2): ACN (70:30 v/v)	1.0	15	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy Red is operation 7 (acetonitrile usage) and 14 (waste 15 mL)	78
Paracetamol, Dextromethorphan Hydrobromide, Phenylephrine Hydrochloride [13]	LunaC18, 250 × 4.6 mm, 5µ	ACN and Buffer (1 mL of phosphoric acid in 1 L water) (20:80 V/V)	1.0	8	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy Red is operation 7 (acetonitrile usage)	80
Paracetamol, Phenylephrine Hydrochloride, Triprolidine Hydrochloride [14]	Prontosil C18, 250 × 4.6 mm, 5µ	ACN: 0.1 M potassium dihydrogen phosphate buffer (52:48)	1.0	10	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy Red is operation 7 (acetonitrile usage)	80
Paracetamol, phenylephrine, dextromethorphan, and chlorpheniramine [15]	C18, 250 × 4.6 mm, 5µ	Methanol: water (containing 6.0 g of ammonium acetate and 10 ml of triethylamine per liter, pH to 5.0 with orthophosphoric acid), 95:5 (v/v)	1.0	15	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy Red is operation 14 (waste 15 mL)	80
Paracetamol [19]	C18, 250 × 4.6 mm, 5µ	Acetonitrile: 0.1 M Acetic Acid 50:50(v/v)	1,2	10	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy. Determination of paracetamol alone requires a long run time, making it inefficient for routine analysis Red is operation 7 (acetonitrile usage) and 14 (waste 12 mL)	78
Paracetamol, Phenylephrine Hydrochloride, Pheniramine maleate [5]	C18, 250 × 4.6 mm, 5µ	Methanol: 1.18 g of sodium octane sulfonate pH = 3.2	1.0	22	250 mm columns are ineffective for phenylephrine HCl and pheniramine maleate due to high RSD, reducing method accuracy and have long run times for routine control Red is operation 14 (waste 22 mL)	80
Paracetamol, Phenylephrine Hydrochloride, Pheniramine maleate (developed method)	Zorbax SB-Aq C18, 50 × 4.6 mm, 5µ	Methanol: 1.1 g of sodium octane sulfonate pH = 3.2	1.0	10	High efficiency, low RSD, and an optimal run time make the proposed method suitable for routine quality control	82

Greenness assessment

To assess the environmental impact of the proposed methods, MOGAPI greenness metrics was applied [24]. For the HPLC method developed for the analysis of paracetamol, phenylephrine hydrochloride, and pheniramine maleate in powder, the total MOGAPI scores reached 82, and for determining paracetamol impurity 4-aminophenol in powder —80 (Fig. 3). In addition, AGREE greenness metrics was applied and AGREE score was 0.79 [25]. The obtained values indicate an excellent green analysis, which is a great advantage of the technique.

Fig. 3.

Comparison of the greenness profiles (MOGAPI score) between the proposed method for determining paracetamol, phenylephrine hydrochloride and pheniramine maleate in powder (A) and proposed method for determining paracetamol impurity 4-aminophenol in powder (B)

Analyzing Table 11 we can notice that the score MOGAPI was 78–82. The highest score is 82 in our developed method and there were no red operations. All the others had red operations. It was operation 7 (non-green solvents usage) and/or 14 (waste).

Conclusion

Precise and validated HPLC methods were successfully developed for the determination of 4-aminophenol, the quantitative determination of active ingredients, and the “Uniformity of Dosage Units” test in pharmaceutical formulations. These methods meet the stringent requirements of ICH guidelines, including specificity, linearity in the range of application, accuracy, precision, and LOQ.

A key achievement of the study was the significant optimization of analytical conditions. The use of the Zorbax SB-Aq column (50 mm × 4.6 mm, 5 µm) provided a perfect balance between separation efficiency, analytical speed, and reproducibility. The mobile phase, consisting of a 1.1 g/L solution of sodium octanesulfonate at pH 3.2, was consistent across all methods, ensuring simplicity and cost-effectiveness in routine applications.

The optimized methods offer substantial time savings compared to official methodologies. The analysis of 4-aminophenol was completed within 20 min, while the quantitative determination of active ingredients was achieved within 10 min—both representing a twofold reduction in analysis time. These improvements not only enhance operational efficiency but also align with sustainability principles by minimizing solvent and reagent consumption.

Validation results confirmed the robustness and reliability of the proposed methods under routine laboratory conditions. The methods maintained consistent performance even under slight variations in experimental parameters, making them highly adaptable for high-throughput quality control in pharmaceutical manufacturing.

The developed methodologies are not only applicable to the analyzed pharmaceutical formulation but can also be extended to similar multicomponent drugs. Their simplicity, speed, and reproducibility make them a valuable tool for ensuring the safety and efficacy of pharmaceutical products, particularly in industrial-scale production settings.

The optimized methods provide a practical, efficient, and environmentally friendly approach to the quality control of multicomponent pharmaceutical products, meeting modern requirements for analytical accuracy and sustainability.

Appendix A

Formulas for calculations

The difference between the retention times for the peaks of 4-aminophenol, paracetamol, phenylephrine hydrochloride and pheniramine maleate on the chromatograms of the reference solution and the tested solution was calculated using the following formula:

$${\mathrm{\Delta }}_{\mathit{RT}}=\frac{\left|{\mathit{RT}}_{\mathit{RS}},-,{\mathit{RT}}_{\mathit{TS}}\right|}{{\mathit{RT}}_{\mathit{RS}}}·100\%$$

where.

${\mathit{RT}}_{\mathit{RS}}$ is the retention time of 4-aminophenol, paracetamol, phenylephrine hydrochloride and pheniramine maleate on the chromatogram of the reference solution;

${\mathit{RT}}_{\mathit{TS}}$ is the retention time of 4-aminophenol, paracetamol, phenylephrine hydrochloride and pheniramine maleate on the chromatogram of the test solution.

To verify the accuracy of solution preparation, a reference solution and a control solution were prepared. Response factors for each solution were calculated using the following formula:

$${\mathit{RF}}_i=\frac{\overline{S_{0i}}}{C_{0i}}$$

where.

$C_{0i}$ is the concentration of the analyte in the respective solution, µg/mL;

$\overline{S_{0i}}$ is the mean peak area of the analyte.

The relative response factor was calculated using the formula:

$$RRF=\frac{{\mathit{RF}}_2}{{\mathit{RF}}_1}·100\%$$

Concentration values in normalized coordinates were calculated using the following formula:

$$x_i=\frac{C_i}{C_{\mathit{ref}}}·100\text{\%}$$

The value of the response of the device in normalized coordinates was calculated according to the following formula:

$$y_i=\frac{S_i}{S_{\mathit{ref}}}·100\text{\%}$$

The “found/put” ratio was calculated using the following formula:

$$z_i=\frac{y_i}{x_i}·100\text{\%}$$

Coefficient b was calculated using the following formula:

$$b=\frac{m·{\sum }_{i=1}^m{x}_i{y}_i-{\sum }_{i=1}^m{x}_i{\sum }_{i=1}^m{y}_i}{m·{\sum }_{i=1}^m{x}_i^2-{\left({\sum }_{i=1}^m,x_i\right)}^2}$$

where.

m is the number of model solutions.

Coefficient a was calculated using the following formula:

$$a=\frac{{\sum }_{i=1}^m{y}_i-b·{\sum }_{i=1}^m{x}_i}{m}$$

Correlation coefficient r was calculated using the following formula:

$$r=\frac{m·{\sum }_{i=1}^m{x}_i{·y}_i-{\sum }_{i=1}^m{x}_i·{\sum }_{i=1}^m{y}_i}{\sqrt{\left(m,\bullet ,\sum _{i=1}^m,x_i^2,-,{\left(\sum _{i=1}^m,x_i\right)}^2\right)·\left(m,·,\sum _{i=1}^m,y_i^2,-,{\left(\sum _{i=1}^m,y_i\right)}^2\right)}}$$

The concentration of 4-aminophenol in the tested solutions with additives was calculated using the following formula (“found”):

$$C_{m\_i}=\frac{C_{01}\bullet \overline{S_i}}{\overline{S_{01}}}$$

where.C₀₁is the concentration of 4-aminophenol in the reference solution;

$\overline{S_i}$ Is the peak area of 4-aminophenol in the test solution;

S₀₁is the peak area of 4-aminophenol in the reference solution.

The “found”/”put” ratio (in percent) was calculated using the following formula:

$$\text{Recov}er{y}_i=\frac{C_{m\_i}}{C_{t\_i}}·100\%$$

The limit of quantification (LOQ) were calculated using the following formula:

$$LOQ=\frac{S_a·10}{b}$$

where.

$S_a$ is the standard deviation of the response;

$b$ is the slope of the calibration curve.

See Table 12

Table 12.

Summary of validation results of the quality control method of the drug “paracetamol, phenylephrine hydrochloride, pheniramine maleate powders” according to the indicator “4-aminophenol”

Investigation	Parameter		Eligibility Criteria	Evaluation
Specificity	The peak area in the chromatograms of the blank solution and the placebo solution, which could interfere with the 4-aminophenol peak		≤ 0005%	Not detected
System suitability	Tailing factor of 4-aminophenol		≤ 15	≤ 11
	Resolution factor between the peaks of paracetamol and 4-aminophenol		≥ 5	≥ 137
	Signal-to-noise ratio for the peaks of 4-aminophenol from the chromatogram of the sensitivity solution		≥ 10	≥ 11
	Relative Standard Deviation between parallel injections		≤ 100%	≤ 33%
Linearity	Intercept		≤|50%|	09%
	Correlation coefficient		≥ 0990	0999
Range of application	The range in which linearity, precision, and accuracy have been studied		005%—0,12% (041 mkg/ml – 0,78 mkg/ml)	004%—013% (0,33 mkg/ml – 1,08 mkg/ml)
Accuracy (spiked samples)	Individual recovery values 4-aminophenol	M1	800%—1200%	86,202%—102,928%
		M2-M4	900%—1100%	93,689%—104,465%
	Mean recovery 4-aminophenol	M1	900%—1100%	95,861%
		M2-M4	950%—10,0%	99,712%
Precision (spiked samples)	Standard deviation of the detected content between samples prepared by the same analyst	M1	≤ 150%	≤ 45%
		M2-M4	≤ 100%	≤ 24%
	Standard deviation of detected content between samples prepared by two analysts	M1	≤ 200%	≤ 67%
		M2-M4	≤ 150%	33%
	The difference between the content obtained in the results by two analysts	M1	≤ 200%	106%
		M2-M4	≤ 150%	54%
LOQ (0.02%)	Individual recovery values 4-aminophenol		800%—1200%	1024%
	Relative Standard Deviation between parallel injections		≤ 150%	40%
	Signal to noise ratio		≥ 10	13

See Table 13

Table 13.

Summary of validation results of the quality control method of the drug “paracetamol, phenylephrine hydrochloride, pheniramine maleate powders” according to the indicator “Quantitative determination” and “Uniformity of Dosage Units”

Investigation	Parameter		Eligibility Criteria	Evaluation
Specificity	Peak area in the blank/placebo chromatogram, which may interfere with the peaks of paracetamol, phenylephrine or pheniramine		≤ 016%	Not detected
	The difference in the retention times of paracetamol, phenylephrine hydrochloride, pheniramine maleate on the chromatogram of the reference solution and the tested solution		≤ 20%	Not detected
System suitability	Tailing factor of the peak of paracetamol, phenylephrine, pheniramine calculated from chromatograms of the comparison solution (c)	Paracetamol	08—15	1,1—1,4
		Phenyleprine HCl		1,1—1,4
		Pheniramine maleate		1,2—1,5
	Relative standard deviation calculated for peak areas of paracetamol, phenylephrine, pheniramine from chromatograms of reference solution (c)	Paracetamol	≤ 10%	≤ 0,2%
		Phenyleprine HCl		≤ 0,5%
		Pheniramine maleate		≤ 1,0%
	Comparison solution and control solution response ratio	Paracetamol	980%—1020%	99,0%—100,0%
		Phenyleprine HCl		98,3%—100,0%
		Pheniramine maleate		99,6%—100,0%
Linearity	Intercept	Paracetamol	≤ 10%	0,3%
		Phenyleprine HCl		0,4%
		Pheniramine maleate		0,7%
	Correlation coefficient	Paracetamol	≥ 0998	1,000
		Phenyleprine HCl		1,000
		Pheniramine maleate		1,000
Range of application	The range in which linearity, precision, and accuracy have been studied	Paracetamol	1820 mkg/ml – 3432 mkg/ml	168,1 mkg/ml – 350,0 mkg/ml
		Phenyleprine HCl	56 mkg/ml – 10,6 mkg/ml	5,2 mkg/ml – 11,2 mkg/ml
		Pheniramine maleate	11,2 mkg/ml – 21,1 mkg/ml	10,5 mkg/ml – 21,8 mkg/ml
Accuracy	Individual recovery values 4-aminophenol	Paracetamol	980%—1020%	98,6%—100,3%
		Phenyleprine HCl		98,5%—100,0%
		Pheniramine maleate		98,3%—101,2%
	Mean recovery 4-aminophenol	Paracetamol	990%—1010%	99,5%
		Phenyleprine HCl		99,1%
		Pheniramine maleate		100,1%
Precision (“Quantitative determination”)	Relative standard deviation for 6 parallels	Paracetamol	≤ 16%	≤ 1,5%
		Phenyleprine HCl		≤ 1,1%
		Pheniramine maleate		≤ 1,2
	Relative standard deviation for 12 parallels	Paracetamol	≤ 22%	1,1%
		Phenyleprine HCl		0,8%
		Pheniramine maleate		1,2%
	Difference between mean results obtained within 2 days	Paracetamol	≤ 20%	0,2%
		Phenyleprine HCl		0,2%
		Pheniramine maleate		1,5%
Precision (“Quantitative determination”)	Relative standard deviation for 10 parallels	Paracetamol	≤ 48%	≤ 2,4%
		Phenyleprine HCl		≤ 4,2%
		Pheniramine maleate		≤ 3,3%
	Relative standard deviation for 20 parallels	Paracetamol	≤ 51%	1,9%
		Phenyleprine HCl		4,1%
		Pheniramine maleate		2,5%
	Difference between mean results obtained within 2 days	Paracetamol	≤ 53%	1,7%
		Phenyleprine HCl		3,5%
		Pheniramine maleate		0,05%
Stability of solutions (24 h)	Ratio of peak area in solution after storage to freshly prepared solution		980%—1020%	996%—1017%

Author contributions

Oleksandra Havrylenko, Yuliya Kondratova, Liliya Logoyda: Methodology and writing the original draft, Validation and reviewing, Lyudmyla Ivanets, Ihor Berdey: Formal analysis and reviewing, Liliya Logoyda: reviewing and publishing editing and supervision.

Funding

The research leading to these results has received funding from the JSC «Farmak», Ukraine.

Availability of data and materials

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.