Quantification of 4-Methylaminoantipyrine, the Active Metabolite of Dipyrone, in Human Plasma

Abstract

Dipyrone (metamizole) is a nonsteroidal anti-inflammatory drug used as an analgesic and antipyretic in both pediatric and adult patients. Dipyrone hydrolyses to 4-methylaminoantipyrine (MAA) in the stomach before absorption. There are several HPLC methods available for analysis of MAA from human plasma but no method has yet been developed on liquid chromatography-mass spectrometry (LC-MS) or LC tandem MS (LC-MS/MS), which are much more specific and sensitive techniques. A high-performance LC-MS method for the quantification of 4-methylaminoantipyrine from human plasma has been developed, validated and applied to a pharmacokinetic study of 500 mg oral dose dipyrone. Following liquid-liquid extraction, the analyte was first separated on a reverse phase column using isocratic mobile phase and then analyzed by MS in selected ion monitoring mode using [M+H](+) ions, m/z 218.2 for 4-methylaminoantipyrine and 231.3 for 4-isopropylantipyrine (internal standard). The method exhibited a linear range from 0.2 to 10.0 µg/ml when only 100 µl human plasma sample was used. The lower limit of detection was 0.04 µg/ml (160 pg on column). The recovery was 80%. The accuracy and precision were obtained over the calibration curve range and were well within the limits specified under guidelines for bioanalytical method validation. The compound was stable under the experimental conditions. The method was demonstrated to be, simple, sensitive and rapid. It can be easily adopted in laboratories with access to LC-MS or MS/MS and applied to sample analysis in clinical settings where a large number of samples are generated.