High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS

Ghassabian, Sussan, Moosavi, Seyed Mojtaba, Valero, Yarmarly Guerra, Shekar, Kiran, Fraser, John F. and Smith, Maree T. (2012) High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS. Journal of Chromatography B, 903 126-133.


Author Ghassabian, Sussan
Moosavi, Seyed Mojtaba
Valero, Yarmarly Guerra
Shekar, Kiran
Fraser, John F.
Smith, Maree T.
Total Author Count Override 6
Title High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS
Journal name Journal of Chromatography B   Check publisher's open access policy
ISSN 1570-0232
1873-376X
Publication date 2012-08-15
Sub-type Article (original research)
DOI 10.1016/j.jchromb.2012.07.005
Volume 903
Start page 126
End page 133
Total pages 8
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Collection year 2013
Language eng
Abstract A rapid LC–MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3-β-d-glucuronide (M3G), morphine-6-β-d-glucuronide (M6G), norfentanyl, 1′-hydroxymidazolam (1-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) in samples of human plasma has been developed and validated. Robotic on-line solid phase extraction (SPE) instrumentation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150 μL) of human plasma and aliquots (150 μL) of a mixture of two internal standards, viz. morphine-d3 (200 ng/mL) and 1′-hydroxymidazolam-d5 (50 ng/mL) in 50 mM ammonium acetate buffer (pH 9.25). Cartridges were washed using 10% methanol in ammonium acetate buffer, pH 9.25 (1 mL, 2 mL/min) before elution with mobile phase comprising 0.1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6 mL/min using an 11.5 min run time. The analytes were separated on a C18 X-Terra® analytical column. The linear concentration ranges were 0.5–100 ng/mL for fentanyl, norfentanyl and midazolam; 1–200 ng/mL for 4-hydroxymidazolam, 2.5–500 ng/mL for 1′-hydroxymidazolam and 3.5–700 ng/mL for morphine, M3G, and M6G. The method showed acceptable within-run and between-run precision (relative standard deviation (RSD) and accuracy <20%) for quality control (QC) samples spiked at concentrations of 80% and 50% of the ULOQ, 3 times higher than the LLOQ, and also at the LLOQ. Furthermore, analytes were stable in samples (after mixing with internal standard) for at least 48 h in the autosampler (except for 4-hydroxymidazolam which decreased by 22% after 24 h), 5 h at room temperature and after three cycles of freeze and thaw. No autosampler carry-over was observed and the absolute recovery (the area ratio of analyte in plasma relative to that in ammonium acetate buffer 50 mM, pH 9.25) was in the range 40% (midazolam) to 110% (morphine). The assay was applied successfully to the measurement of the analytes of interest in plasma samples from patients on extracorporeal membrane oxygenation (ECMO).
Keyword Morphine
M3G
M6G
Fentanyl
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Sun, 16 Sep 2012, 07:53:56 EST by Professor Maree Smith on behalf of School of Pharmacy