Quantitation of Over the Counter Cold Medicine Formulations using UPLC Technology

Pharmaceutical formulations used to treat the common cold often contain multiple active ingredients to treat different symptoms. These actives can include combinations of decongestants, antihistamines, pain relievers, cough suppressants, and expectorants in addition to numerous excipients, all of which exhibit different chemical properties, including polarity. It is this range of analyte polarity that often makes chromatographic methods development difficult. 

In this study, we used Waters UltraPerformance LC (UPLC) technology to develop a single chromatographic method for the analysis of the most common pharmaceutical formulations targeted to relieve symptoms associated with the common cold. Utilizing UPLC for this analysis improves productivity by enabling the separation of such complex mixtures while maintaining a high level of chromatographic resolution in a rapid analysis time.

We used a new High Strength Silica (HSS) UPLC stationary phase to develop a single chromatographic method for the analysis of a number of possible formulation compositions. This stationary phase was selected due to its ability to enhance the retention of polar analytes while also having good chromatographic selectivity of hydrophobic species.

This methodology was then applied to several over-the-counter (OTC) formulations to quantitate the accuracy of reported concentrations of the active ingredients. 

A mixture of standards of 20 common components of cold medicine formulations including active ingredients, impurities and counter ions was separated on a 2.1 x 100 mm, ACQUITY UPLC HSS T3, 1.8 μm Column, as depicted in Figure 1. 

This methodology was then applied to two OTC liquid formulations. A four-point calibration curve from 1 to 1,000 μg/mL was used to quantitate the accuracy of the reported values of the active ingredients respective to each OTC formulation. 

Sample Preparation

Reference standards for the mixture depicted in Figure 1 were prepared in a solution of 75:25 (v/v) water:methanol containing 0.2% formic acid at a concentration of 25 μg/mL for each component except acetaminophen, which was 12.5 μg/mL and its respective impurities, 4-aminophenol, 4-nitrophenol and 4-chloroacetanilide, were prepared at a concentration of 2.5 μg/mL. 

Individual stock solutions of the active ingredients of OTC formulation 1 (pseudoephedrine and diphenhydramine) were prepared at 2,000 μg/mL in a solution of 75:25 (v/v) water:methanol containing 0.2% formic acid. A mixture of the two actives was prepared at 1,000 μg/mL followed by serial dilutions of each subsequent concentration to make calibration standards at the concentrations of 100, 10 and 1 μg/mL. 

A similar process was followed for the stock solution preparation of the active ingredients of OTC formulation 2 (acetaminophen, doxylamine and dextromethorphan). Individual stock solutions were prepared at a concentration of 3,000 μg/mL in a solution of 75:25 (v/v) water:methanol containing 0.2% formic acid. A mixture of the three actives was prepared at 1,000 μg/mL followed by serial dilutions of each subsequent concentration to make calibration standards at the concentrations of 100, 10 and 1 μg/mL. 

The OTC liquid formulations were obtained from a local pharmacy and diluted with a 75:25 (v/v) methanol:water solution and directly injected on column without any further sample treatment or filtration. OTC formulation 1 was diluted 1:9 for the quantitation of pseudoephedrine and diphenhydramine. OTC formulation 2 was diluted 1:39 for the quantitation of doxylamine and dextromethorphan and diluted 1:199 for the quantitation of acetaminophen. 

Linearity

Serial dilutions from stock solutions were made to obtain four concentrations per calibration curve ranging from 1 to 1,000 μg/mL. Each concentration was injected in triplicate. A linear regression was used for doxylamine and dextromethorphan. A linear regression with weighting (1/X) was used for acetaminophen, pseudoephedrine and diphenhydramine. Table 1 lists the correlation coefficient and line equation for each analyte. Accepted value is greater than 0.995 for all sample components. 

Quantitation of OTC Formulation 1

OTC formulation 1 was diluted 1:9 with a 75:25 methanol:water solution and directly injected on column. As depicted in Figure 2, the active ingredients, pseudoephedrine and diphenhydramine, are well resolved from the excipients. As listed in Table 2, when quantitated against the calibration curve, percent recoveries for pseudoephedrine and diphenhydramine were 82% and 102%, respectively.

Quantitation of OTC Formulation 2

OTC formulation 2 was diluted 1:39 with a 75:25 methanol:water solution for the quantitation of doxylamine and dextromethorphan and diluted 1:199 for the quantitation of acetaminophen. These solutions were directly injected on column. As depicted in Figures 3 and 4, active ingredients acetaminophen, doxylamine and dextromethorphan, are well resolved from the excipients. As listed in Table 3, when quantitated against the calibration curve, percent recoveries for acetaminophen, doxylamine and dextromethorphan were 79%, 77% and 80%, respectively. 

A fast, high-resolution UPLC chromatographic method was developed for pharmaceutical formulations targeted to relieve symptoms of the common cold by utilizing a new High Strength Silica (HSS) UPLC stationary phase. This stationary phase was selected due to its ability to enhance the retention of polar analytes while also having good chromatographic selectivity of hydrophobic species. This single chromatographic method can be used to analyze a number of possible formulation compositions containing different active ingredients. 

By utilizing UPLC technology, improvements in productivity were realized by enabling the separation of complex mixtures while maintaining a high level of chromatographic resolution in a rapid analysis time. This results in a higher degree of confidence in results due to the ease of analyst familiarization with a single methodology, reducing the need for repeat analysis. 

When we applied this methodology to over-the-counter (OTC) formulations, adequate resolution for the active ingredients from the excipients was observed. Analyte recoveries ranged from 77 to 102% of their reported value.