The use of surrogate reference standards in quantitative HPLC; a case study of the analysis of Amlodipine and Lisinopril tablets

A simple and rapid isocratic RP-HPLC method was developed and validated in accordance with ICH guidelines for the quantitation of Lisinopril and Amlodipine using Diclofenac sodium; Metronidazole and Ibuprofen in place of their own chemical reference standards as surrogate reference standards. The appropriate surrogate constants for each surrogate was determined in relation to each analyte and with these constants the assays of the analytes in commercial formulations were deduced. The HPLC system used for the determinations comprised of a Schimadzu-CTO-20A pump, Prominence programmable UV/Vis absorbance detector SPD-20A, LC-Real time analysis integrator and LUMX Iu C18 5μm, 3.9 x 150mm, Å~100 column. The analysis of Amlodipine and Lisinopril using Diclofenac sodium, Metronidazole and Ibuprofen in each case was achieved using an ODS column (LUMX Iu C18 5μm, 3.9 x 150mm, Å~100) and a mobile phase system comprising 0.1M orthophosphoric acid: methanol in a ratio 15:85 under isocratic elution mode using a 1.2ml/min flow rate and with UV detection at 219nm and 230nm for lisinopril and amlodipine respectively. The mean retention times obtained for analysis of Lisinopril using the surrogates were 1.2298 ± 0.0395, 3.0987 ± 0.031, 1.6301 ± 0.0026, 3.2828 ± 0.0029 for Lisinopril, Diclofenac sodium, Metronidazole and Ibuprofen respectively. For the assay of Amlodipine using the surrogate constants. Mean retention times of 1.1761 ± 0.0243, 2.8349 ±0.0001, 1.4987 ± 0.0018, 2.9699 ± 0.0051 were obtained respectively for Amlodipine, Diclofenac sodium, Metronidazole and Ibuprofen. The surrogate constants obtained for the analysis of Lisinopril were 0.2221 ± 0.008, 0.5916 ± 0.2815, 0.3390 ± 0.018 using Diclofenac sodium, Metronidazole and Ibuprofen respectively. For the analysis of Amlodipine, the constants obtained were 0.7460 ± 0.074, 1.2226 ± 0.737, 1.5203 ± 0.971 using Diclofenac sodium, Metronidazole and Ibuprofen respectively. The assays obtained for commercial samples of Lisinopril tablets using Diclofenac sodium as surrogate were 97.50 ± 0.850, 98.43 ± 0.954, 101.53 ± 0.793 respectively for brands L-AA, L-BB, and L-CC. When Metronidazole was used as surrogate, the assay values calculated were 97.35 ± 0.189, 98.29 ± 0.045, 101.78 ± 0.469 respectively for brands L-AA, L-BB, and L-CC while the assay values; 97.82 ± 0.008, 98.81 ± 0.002, 101.17 ± 0.203 respectively for brands L-AA, L-BB, and L-CC were obtained when Ibuprofen was employed as surrogate. The assay obtained for commercial samples of Amlodipine tablets using Diclofenac sodium as surrogate were 94.70 ± 0.086, 97.40 ± 0.084, 96.15± 0.112 respectively for brands A-AA, A-BB, and A-CC. When Metronidazole was used as surrogate, the assay values calculated were 95.83 ± 0.214, 98.27 ± 0.151, 96.50 ± 0.440 respectively for brands A-AA, A-BB, and A-CC while the assay values; 95.14 ± 0.521, 97.96 ± 0.703, 96.80 ± 0.295 respectively for brands A-AA, A-BB, and A-CC were obtained when Ibuprofen was employed as surrogate The methods developed on comparison with official methods employing the statistical tool; T-test showed no significant statistical difference between the method developed and the official methods. This demonstrates the accuracy of the method and its suitability for routine analysis of the analytes. The methods developed were validated in accordance with the guidelines of the International Conference on Harmonization (ICH). The results of the validation showed the methods developed were accurate, precise, had good linearity of the working concentration range, robust, sensitive, and specific and can be employed for routine analysis A simple and rapid isocratic RP-HPLC method was developed and validated in accordance with ICH guidelines for the quantitation of Lisinopril and Amlodipine using Diclofenac sodium; Metronidazole and Ibuprofen in place of their own chemical reference standards as surrogate reference standards. The appropriate surrogate constants for each surrogate was determined in relation to each analyte and with these constants the assays of the analytes in commercial formulations were deduced. The HPLC system used for the determinations comprised of a Schimadzu-CTO-20A pump, Prominence programmable UV/Vis absorbance detector SPD-20A, LC-Real time analysis integrator and LUMX Iu C18 5μm, 3.9 x 150mm, Å~100 column. The analysis of Amlodipine and Lisinopril using Diclofenac sodium, Metronidazole and Ibuprofen in each case was achieved using an ODS column (LUMX Iu C18 5μm, 3.9 x 150mm, Å~100) and a mobile phase system comprising 0.1M orthophosphoric acid: methanol in a ratio 15:85 under isocratic elution mode using a 1.2ml/min flow rate and with UV detection at 219nm and 230nm for lisinopril and amlodipine respectively. The mean retention times obtained for analysis of Lisinopril using the surrogates were 1.2298 ± 0.0395, 3.0987 ± 0.031, 1.6301 ± 0.0026, 3.2828 ± 0.0029 for Lisinopril, Diclofenac sodium, Metronidazole and Ibuprofen respectively. For the assay of Amlodipine using the surrogate constants. Mean retention times of 1.1761 ± 0.0243, 2.8349 ±0.0001, 1.4987 ± 0.0018, 2.9699 ± 0.0051 were obtained respectively for Amlodipine, Diclofenac sodium, Metronidazole and Ibuprofen. The surrogate constants obtained for the analysis of Lisinopril were 0.2221 ± 0.008, 0.5916 ± 0.2815, 0.3390 ± 0.018 using Diclofenac sodium, Metronidazole and Ibuprofen respectively. For the analysis of Amlodipine, the constants obtained were 0.7460 ± 0.074, 1.2226 ± 0.737, 1.5203 ± 0.971 using Diclofenac sodium, Metronidazole and Ibuprofen respectively. The assays obtained for commercial samples of Lisinopril tablets using Diclofenac sodium as surrogate were 97.50 ± 0.850, 98.43 ± 0.954, 101.53 ± 0.793 respectively for brands L-AA, L-BB, and L-CC. When Metronidazole was used as surrogate, the assay values calculated were 97.35 ± 0.189, 98.29 ± 0.045, 101.78 ± 0.469 respectively for brands L-AA, L-BB, and L-CC while the assay values; 97.82 ± 0.008, 98.81 ± 0.002, 101.17 ± 0.203 respectively for brands L-AA, L-BB, and L-CC were obtained when Ibuprofen was employed as surrogate. The assay obtained for commercial samples of Amlodipine tablets using Diclofenac sodium as surrogate were 94.70 ± 0.086, 97.40 ± 0.084, 96.15± 0.112 respectively for brands A-AA, A-BB, and A-CC. When Metronidazole was used as surrogate, the assay values calculated were 95.83 ± 0.214, 98.27 ± 0.151, 96.50 ± 0.440 respectively for brands A-AA, A-BB, and A-CC while the assay values; 95.14 ± 0.521, 97.96 ± 0.703, 96.80 ± 0.295 respectively for brands A-AA, A-BB, and A-CC were obtained when Ibuprofen was employed as surrogate The methods developed on comparison with official methods employing the statistical tool; T-test showed no significant statistical difference between the method developed and the official methods. This demonstrates the accuracy of the method and its suitability for routine analysis of the analytes. The methods developed were validated in accordance with the guidelines of the International Conference on Harmonization (ICH). The results of the validation showed the methods developed were accurate, precise, had good linearity of the working concentration range, robust, sensitive, and specific and can be employed for routine analysis

Source: KNUST