Enantioselective gas chromatography-negative ion chemical ionization mass spectrometry for methylphenidate in human plasma.
Therapeutic doses of Ritalin, a racemic mixture of d- and l-threo-methyphenidate, result in low plasma concentrations of methylphenidate. In order to assess the safety and efficacy of methylphenidate, a sensitive analytical method is needed. A gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) assay capable of measuring both d- and l-enantiomers in human plasma was developed and validated to support clinical studies involving administration of d,l-methylphenidate. d,l-Methylphenidate-d3 is added to 1-mL plasma samples. The plasma samples are made basic, mixed with isopropanol and extracted with hexane. The hexane extracts are then back-extracted into 0.1 N HCl. The acidified aqueous extract is made basic, cooled to ice temperature, and the methylphenidate derivatized with heptafluorobutyryl-l-prolyl chloride. The two diastereomeric derivatives are then extracted into hexane. The hexane extract is evaporated to dryness, reconstituted in ethyl acetate, and analyzed by GC-NCI-MS. This method can accurately (+/- 5% target) and precisely (< 11.1% coefficient of variation) quantitate enantiomers of threo-methylphenidate in human plasma and in the whole blood at concentrations ranging from 0.75 to 100 ng/mL. Plasma samples are stable for up to five freeze-thaw cycles when the duration of each cycle did not exceed 0.5 h. The drug degraded gradually when plasma samples were left at room temperature; a 6% loss at 3 h progressed to 17% at 12 h and to 35% at 24 h. Therefore, it is important that extraction of plasma samples begins within 0.5 h after samples are removed from the freezer. Whole blood stability results show that concentrations of methylphenidate in whole blood, with or without NaF, stored for up to 6 h at room temperature did not deviate from the target concentration by more than 13%. The derivatized methylphenidate in extract is stable at 4 degrees C for up to 10 days.
Lin
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Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112-9457, USA. sn.lin@m.cc.utah.edu
Andrenyak
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DM
Moody
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Foltz
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RL
D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D000697:Central Nervous System Stimulants / Q000097:blood*; D004355:Drug Stability; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D066298:In Vitro Techniques; D008774:Methylphenidate / Q000097:blood*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013997:Time Factors
D000697:Central Nervous System Stimulants; D008774:Methylphenidate