Use of Surrogate Reference Standards in Quantitative HPLC

In quantitative HPLC, pure reference standards of analytes are used either to draw calibration curves or for one point assays. These reference standards are sometimes very scarce to come by and if available may be very expensive. The use of readily available drugs to replace the reference standards of the analytes was therefore investigated. A simple and sensitive HPLC method was developed for the assay of Indometacin and Diazepam. The assay method made use of surrogate reference standards being run together with the pure sample of the analyte to obtain a constant of proportionality, „K‟ that relates the concentration and peak areas of the two. The K values obtained were then used to assay the analyte. The assay of Indometacin employed a microbore column packed with a C18 reversed-phase material (5μm HICHROM ODS column) with an isocratic mixture of methanol and phosphate buffer, pH 5.8±0.02 (60:40, v/v) as the mobile phase. The chromatographic separation was monitored by a UV detector at a wavelength of 254 nm. When Naproxen was used as surrogate standard the K value obtained was 1.6735±0.021 with percentage content of 99.20%±1.98, 95.09%±1.85 and 96.98%±1.70 for the three brands of Indometacin capsule used. With Benzoic acid as surrogate standard, the K value obtained was 3.426±0.073 with percentage content of 99.02%±1.81, 94.89±1.47 and 98.52%±1.63 for the three brands. When Diazepam was used as surrogate reference standard the K value was 3.0955±0.19 with assays of 99.65%±0.59, 92.99%±1.28 and 98.14%±0.81 respectively for the three brands. Comparatively, the standard method (BP method) of assay showed percentage content of 98.04%±0.21, 92.51%±0.77 and 99.15%±0.77 respectively for the three brands. The methods were statistically comparable at the 99% confidence interval. The assay of Diazepam also employed a microbore column packed with a C18 reversed-phase material (5μm HICHROM ODS column) with an isocratic mixture of methanol and phosphate buffer, pH 5.8±0.02 (75:25, v/v) as the mobile phase. The chromatographic separation was monitored by a UV detector at a wavelength of 300 nm. When Piroxicam was v used as surrogate, the K value obtained was 0.20418±0.0015 with percentage content of 95.00%±1.98, 103.01%±1.01 and 95.67%±1.05 for the three brands of Diazepam tablet used. With Metronidazole as surrogate standard, the K value obtained was 0.135271±0.009 with percentage content of 94.84%±0.55, 103.94±1.44 and 98.10%±0.82 for the three brands. When Indometacin was used as surrogate reference standard the K value was 0.3230±0.018 with assays of 95.10%±1.25, 97.57%±1.27 and 93.11%±1.30 respectively for the three brands of Diazepam tablet. The standard method from the BP also showed results of 94.38%±0.74, 98.07%±0.48 and 99.05%±0.38 respectively for the three brands of Diazepam tablets. The methods were statistically comparable to the Standard BP method at the 99% confidence interval except for two brands which showed slight systematic errors with Piroxicam as surrogate reference standard. All the assays for the Diazepam and Indometacin with the developed methods however fell within the permissible range of the British Pharmacopoeia with detection limit of 0.37μg/ml for Indometacin and 0.67μg/ml for Diazepam. Naproxen, Benzoic acid and Diazepam can be used as surrogate reference standards for the assay of Indometacin using the K values obtained. Piroxicam, Metronidazole and Indometacin can be used as surrogate reference standard for the assay of Diazepam.

Source: KNUST