The use of surrogate reference standards in quantitative high performance liquid chromatography; a case study of the analysis of Chlorpheniramine Maleate tablets and Metformin Hydrochloride tablets

The study sought to investigate the use of compounds that were physico-chemically related to the analytes; (Chlorpheniramine Maleate and Metformin Hydrochloride) as surrogate reference standards for the assay of the analytes using High Performance Liquid Chromatography. The surrogate reference standards for Chlorpheniramine Maleate were Piroxicam, Ascorbic Acid and Caffeine while those for Metformin Hydrochloride were Metronidazole and Paracetamol. A reversed-phase isocratic HPLC method with UV detection was developed and validated. Phosphate buffer and Acetate buffer were used to effectively control the pH of the mobile phase. Phosphate buffer (0.025M) and Methanol in a ratio of 50:50 and detection at 266nm eluted well resolved peaks of Chlorpheniramine Maleate and its surrogate reference standards; Piroxicam, Ascorbic Acid and Caffeine within the pH range of 6.37 ± 0.02, while Acetate buffer and Methanol in a ratio of 70:30 within the pH range of 5.46 ± 0.02 was used for Metformin Hydrochloride and its surrogate reference standards; Metronidazole and Paracetamol and detection at 254nm. Both were carried out on a C18 Phenomenex, 250x4.6mm, 5μ column. The mean retention times obtained were as follows; Chlorpheniramine Maleate 2.6 ± 0.09min, Metformin Hydrochloride 3.4 ± 0.03min, Ascorbic acid 3.2 ± 0.02min, Piroxicam 6.5 ± 0.02min, Caffeine 5.9 ± 0.02min, Metronidazole 5.3 ± 0.20min and Paracetamol 4.7 ± 0.02min. The peak areas obtained from the chromatograms and the specific concentrations of the solutions analysed were used to find the surrogate constant K for Chlorpheniramine Maleate and the values obtained as follows: Piroxicam 0.8095 ± 0.003, Caffeine 0.2224 ± 0.006, and Ascorbic acid 0.1560 ± 0.002 and that of Metformin Hydrochloride are Metronidazole 1.3262 ± 0.02 and Paracetamol 0.8623 ± 0.02. The K values were found to be influenced by the molecular weight ratio of analyte to surrogate where the lower the ratio, the higher the K value. However, the K values were not affected by changes in concentration of both analyte and surrogate. The K values were then inserted into the hypothetical formular and the percentage content of Chlorpheniramine Maleate in four different brands of Chlorpheniramine Maleate tablets and Metformin Hydrochloride in four different brands of Metformin Hydrochloride tablets was found. Although the percentage contents were within the pharmacopoeial limits, statistical tests i.e. t-Test and F-test were carried out to compare the results obtained from the new method to the results obtained from the standard method in the British Pharmacopoeia and it was found out that there was no significant difference between the two methods, though some brands deviated. Similarity in physico-chemical parameters between analyte and surrogate is found to be favourable as observed in all the brands of Metformin Hydrochloride analyzed when Paracetamol and Metronidazole are used as surrogate reference standards, the two methods did not differ significantly in their precision because of the similarity in their solubility. Similar trend was observed in the analyses of Chlorpheniramine Maleate where the two methods did not differ significantly in their precision for all the brands when Piroxicam was used as the surrogate reference standard due to closeness of the wavelength of maximum absorption. With the experimental conditions maintained, Chlorpheniramine Maleate and Metformin Hydrochloride can therefore be analyzed using their respective surrogate reference standards in place of the their pure reference standards.

Source: KNUST