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The deproteinized supernatant was then removed and transferred to a fresh tube and immediately analysed for 4-OH CP as described above. The remainder of the plasma sample was http://www.selleckchem.com/products/pifithrin-alpha.html stored at ?80��C until analysis for CP. Briefly, plasma (0.4?ml) was spiked with ifosfamide as internal standard and diluted to 2?ml with Milli Q water. Following solid phase extraction (C18 cartridges, Alltech associates, USA) the eluate was evaporated to dryness and the residue reconstituted in 100??l mobile phase prior to LC/MS analysis as previously described [22]. The limit of quantification was 0.1??m with a coefficient of variation of http://www.selleckchem.com/screening/pfizer-licensed-library.html of total drug and metabolite exposure (AUC) [14]. A further blood sample (8.5?ml) was collected into PAXgene? blood tubes (Qiagen, Hilden, Germany) for CYP2C19 and CYP2B6 genotyping. Genomic DNA was extracted from human liver tissue using a DNeasy tissue kit or from whole blood using a PAXgene? blood DNA kit according to manufacturer's instructions (Qiagen, Hilden, Germany). The CYP2C19 genotype of individual human livers was determined by PCR-RFLP analysis of the two major LoF allelic variants (CYP2C19*2 and CYP2C19*3) and the gain of function variant (CYP2C19*17) using previously published methods [9, 11]. CYP2B6 genotype was determined by PCR-RFLP analysis of the G516T, A785G and C1459T SNPs which allows determination of the, *4 (A785G), *5 (C1459T), *6 (G516T, A785G), http://en.wikipedia.org/wiki/Temsirolimus *7 (G516T, A785G and C1459T) and *9 (G516T) alleles as described previously [23]. Expression of CYP2C19 and CYP2B6 protein in human liver microsomes was determined by immunoblotting using standard techniques and commercially available primary antibodies (BD Biosciences, New Jersey, USA and Abcam Inc., Cambridge, UK, respectively). Immunoreactive proteins were visualized by enhanced chemiluminescence detection (Pierce Biotechnology, Illinois, USA) and the relative intensity of the signal analysed with the Scion imaging analysis program (Scion Corporation, Maryland, USA). The relationship between CYP protein expression and cyclophosphamide bioactivation was analysed using Spearman rank order correlation. Student's t-test (two-tailed) was used to determine the statistical difference between groups using GraphPad Prism version 5.02. Values of P