Enantioselective Determination of Methylphenidate and Ritalinic Acid in Whole Blood from Forensic Cases Using Automated Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

Abstract

A chiral liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed and validated for quantifying methylphenidate and its major metabolite ritalinic acid in blood from forensic cases. Blood samples were prepared in a fully automated system by protein precipitation followed by solid-phase extraction. The LC-MS-MS method was linear in the range of 0.5 to 500 ng/g for the enantiomers of both analytes. For concentrations above the limit of quantification, coefficients of variation were 15% or less, and the accuracy was 89 to 94%. For 12 postmortem samples in which methylphenidate was not determined to be related to the cause of death, the femoral blood concentration of d-methylphenidate ranged from 5 to 58 ng/g, and from undetected to 48 ng/g for l-methylphenidate (median d/l-ratio 5.9). Ritalinic acid was present at concentrations 10-20 times higher with roughly equal amounts of the d- and l-forms. In blood from 10 living subjects that were not suspected of being intoxicated by methylphenidate, the concentration ranges and patterns were similar to those of the postmortem cases. Thus, methylphenidate does not appear to undergo significant postmortem redistribution.

Pharmacokinetics of methylphenidate and ritalinic acid in plasma correlations with exhaled breath and oral fluid in healthy volunteers

“…Thomsen et al [29] suggested that a low ratio of dthreoto l-threo-methylphenidate plasma concentration might identify a subject as a poor metabolizer of methylphenidate via CES1. In the present single-dose study, the plasma concentrations of l-threo-methylphenidate were detectable in only five of the twelve subjects and even then in only a minority of the samples.…”

The impact of CES1 genotypes on the pharmacokinetics of methylphenidate in healthy Danish subjects

“…Plasma samples were prepared using solid‐phase extraction as described for whole blood elsewhere , but without the initial protein precipitation step. Lower limit of quantification was 0.205 ng ml −1 for methylphenidate isomers and 2.05 ng ml −1 for RA isomers.…”

“…Chromatographic separation was performed using the same system as described elsewhere . Briefly, a CHIRALPAK ® ‐AGP column (Sigma‐Aldrich, St. Louis, MO) using isocratic elution was employed to separate the enantiomers of both analytes.…”

Methylphenidate for Treating ADHD: A Naturalistic Clinical Study of Methylphenidate Blood Concentrations in Children and Adults With Optimized Dosage

“…MPH is rapidly and extensively metabolized in liver and other tissues by non-microsomal hydrolytic esterases, carboxylesterases (CES) [6–8] to its major and essentially inactive metabolite ritalinic acid (RA) [9]. The half-life of MPH ranges from 2 to 3 h both in adults and in children [10, 11].…”