Journal of Chromatography B, 903 (2012) 126–133 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb 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 Sussan Ghassabian a,∗ , Seyed Mojtaba Moosavi a , Yarmarly Guerra Valero a , Kiran Shekar b , John F. Fraser b , Maree T. Smith a,c a Centre for Integrated Preclinical Drug Development, The University of Queensland, Herston Campus, Brisbane, QLD 4029, Australia b Critical Care Research Group, The Prince Charles Hospital, University of Queensland, Brisbane, QLD, Australia c School of Pharmacy, The University of Queensland, Brisbane, QLD, Australia a r t i c l e i n f o Article history: Received 22 March 2012 Accepted 9 July 2012 Available online 20 July 2012 Keywords: Morphine M3G, M6G Fentanyl Norfentanyl Midazolam (MDZ), 1-OHMDZ 4-OHMDZ On-line SPE LC–MS/MS a b s t r a c t A rapid LC–MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3--d-glucuronide (M3G), morphine-6--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) instrumen- tation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150 L) of human plasma and aliquots (150 L) 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 tempera- ture 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). © 2012 Published by Elsevier B.V. 1. Introduction LC–MS/MS provides the opportunity for simultaneous quantifi- cation of tens of compounds with the current limiting step being that of adequate sample clean-up. Protein precipitation and solid phase extraction (SPE) in combination [1,2], or separately [3] have ∗ Corresponding author at: Centre for Integrated Preclinical Drug Development, The University of Queensland, Level 7, Block 6, Herston Campus, Brisbane, Queens- land 4029, Australia. Tel.: +61 07 3346 5194; fax: +61 07 3365 5444. E-mail addresses: s.ghassabian@uq.edu.au, susan.ghassabian@gmail.com (S. Ghassabian). been used for this purpose; however, SPE alone is the method of choice. Subramanian et al. [4] separated and quantified nine antiepileptic drugs using a single SPE. Low-speed centrifugation was used to force solutions and samples through the cartridges. Likewise, 21 benzodiazepines were separated from urine and quan- tified using a single SPE method by Quintela et al. [5]. Rate-limiting steps for these methods were manual analyte extraction as well as evaporation of sample eluants, and dried sample reconstitution. Ghassabian et al. [2] used an automated SPE instrument (off-line) to simultaneously extract 8 analytes from samples of human plasma following protein precipitation with acetonitrile. This was followed by an eluent evaporation step that limited the overall speed of the method. On-line SPE facilitates high-throughput analyte extraction 1570-0232/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2012.07.005