Validated method for the determination of risperidone and 9-hydroxyrisperidone in human plasma by liquid chromatography–tandem mass spectrometry

Since the first entry of risperidone on to the market in the early 1990s, investigation of the pharmacokinetic behaviour of the compound for which the availability of a bioanalytical method was a conditio sine qua non, has received considerable attention. Most of the published methods, however, did not reach the level of sensitivity and selectivity which can be obtained today since the evolution of liquid chromatography–tandem mass spectrometry (LC–MS–MS) towards a routine technique in the bioanalytical laboratory. Therefore, we developed and validated a new LC–MS–MS method for the determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma. This paper describes in detail the bioanalytical procedure and summarizes the validation results obtained. In addition, it focuses on the pitfalls one might encounter when developing similar assays. Despite the particular physicochemical characteristics of risperidone and 9-hydroxyrisperidone, the LC–MS–MS method enabled the quantification of both compounds down to 0.1 ng/ml. The method uses a sample preparation step by solid-phase extraction at pH 6 using a mixed-mode phase. In a short chromatographic run, separation of 9-hydroxyrisperidone from the minor metabolite 7-hydroxyrisperidone is achieved. Detection takes place by (turbo)ionspray tandem mass spectrometry in the positive ion mode. The validated concentration range is from 0.100 to 250 ng/ml, using 500 μl of sample, with accuracy (bias) and precision (coefficient of variation) being below 15%. Although new developments in equipment will allow us to further improve and speed up the method, the assay reported can be used as a routine method to support a wide range of pharmacokinetic studies.