Quantitative Method for Analysis of Hydrocodone, Hydromorphone and Norhydrocodone in Human Plasma by Liquid Chromatography-tandem Mass Spectrometry
ARMY INST OF SURGICAL RESEARCH FORT SAM HOUSTON TX
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A selective, sensitive and accurate high-performance liquid chromatography-tandem mass spectrometry LC MS MS method for the quantitation of hydrocodone, hydromorphone and norhydrocodone in human plasma was developed. The internal standard stock solution comprised of hydrocodoned6, hydromorphone-d6 and norhydrocodone-d3 was added to 0.5 mL plasma samples. Samples were extracted using a copolymeric sorbent mixed mode solid phase extraction SPE column. Chromatographic separation was carried out using a reversed-phase C18 analytical column with a gradient mobile phase consisting of solvent A 5 acetonitrile with 0.1 formic acid and solvent B 100 acetonitrile. MS analysis was performed using positive electrospray ionization ESI in multiple reaction monitoring MRM mode. Linearity was established over the range 1 100 ngmL with correlation coefficients 0.998 for all three analytes. The coefficient of variation CV of intra-day samples was 5.6 at 10 ngmL. The precision of inter-day 6 days samples resulted in CVs 8.1 at concentrations tested at 2.5, 10 and 25 ngmL for all three analytes. The lower limit of quantification LOQ was 1.0 ngmL with signal-tonoise SN ratio 10, the limit of detection LOD was 0.25 ngmL with SN ratio 3 for the drug and its metabolites. Dilution effects, extraction recovery, stability, interference, carryover and ion suppression were also evaluated. This method was successfully applied to human subject plasma samples in support of a hydrocodone pharmacokinetic study.
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