ABSTRACT
A simplified protein precipitation method in combination with gas chromatography-triple quadrupole mass spectrometry (GC-MS-MS) analysis was developed and validated for the simultaneous determination of codeine, morphine, 6-acetylmorphine (6-MAM), hydrocodone and hydromorphone in human blood samples. A protein precipitation with 10% trichloroacetic acid followed by solid-phase extraction using a mixed-mode cartridge was used to separate the analyte from the blood samples. A BSTFA + 1% TMCS was used for derivatization of opiates prior to the analysis. Codeine-D3, morphine-D3, 6-acetylmorphine-D3 and hydrocodone-D6 were used as internal standards. The GC-MS-MS was operated under multiple-reaction monitoring mode using electron ionization technique. The transition ions used for quantitation were 371 â 234 for codeine, 429 â 146 for morphine, 399 â 287 for 6-MAM, 299 â 228 for hydrocodone and 357 â 314 for hydromorphone. The method was linear over the concentration range 2.5-1000 ng/mL for all analytes, except hydrocodone which was linear over 5-1000 ng/mL with a correlation coefficient (r2) = 0.99. The limit of detection was 1.0 ng/mL for all compounds except hydrocodone which was 2.5 ng/mL. The limit of quantitation was 2.5 ng/mL for all compounds except hydrocodone which was 5.0 ng/mL. The precision (% RSD) was within 1.26-14.81 and the accuracy (% Bias) was within -6.29-10.93% for all compounds. The method successfully analyzed morphine (305 ng/mL) and 6-acetylmorphine (6-MAM) (2.3 ng/mL) in a human blood sample received from an opiate user.
