Development and validation of HPLC/UV-procedures for quantification of metronidazole in the blood and urine
Keywords:metronidazole, high-performance liquid chromatography, blood, urine, sample preparation, validation, method of calibration curve, method of standard, method of additions
Metronidazole belongs to the group of antiprotozoal medicines and widely used for the treatment of infectious diseases; the medicine has a number of side effects manifested by usual symptoms of acute intoxication, especially when interacting with other drugs and alcohol.
Aim. To apply the system of MiLiChrome® A-02 HPLC-analyzer widely used in the Ukrainian laboratories of forensic toxicology for the metronidazole quantitative determination in biological fluids and carry out validation of the procedures developed.
Materials and methods. The sample preparation of blood and urine was carried out by extraction with acetonitrile and 2-propanol followed by separation of the organic layer under the conditions of the aqueous phase saturation with ammonium sulfate. Previously blood and urine were treated with acids. Isolation was carried out in the strong acid, neutral and weak alkaline medium.
Results and discussion. To find the optimal conditions of the sample preparation such validation parameters as specificity/selectivity and recovery were determined. The results of the blank samples analysis were acceptable for all variants of the sample preparation procedures. Recovery values were reproducible for all procedures of analysis studied, but efficacy of metronidazole isolation was variable – from 85% to 97 %. The results of verification of metronidazole stability showed the necessity to carry out all measurements within 12 hours after obtaining the solutions to be analyzed. The results of determination of linearity, accuracy and precision were the evidence
of acceptable systematic and random errors of the HPLC/UV-procedures studied in the variant of the method of calibration curve, method of standard and method of additions.
Conclusions. The set of HPLC/UV-procedures for the metronidazole quantitative determination in blood and urine has been developed. Validation of the procedures developed has been carried out.
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