Quantification of N, N' N"-triethylenethiophosphoramide, N, N"-triethylenephosphoramide, cyclophosphamide, and 4-hydroxy-cyclophosphamide in microvolume human plasma to support neonatal and pediatric drug studies.

N, N' N"-triethylenethiophosphoramide (thiotepa) and cyclophosphamide (CP) are alkylating agents used for a variety of malignant and non-malignant disorders. Both drugs are metabolized by cytochrome P450 enzymes to form active metabolites. To support pharmacokinetic studies of thiotepa and CP in children, we sought to develop assays to determine parent drug and metabolite concentration in small volume plasma samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for assay development. CP metabolite 4-hydroxycyclophosphamide (4OHCP) was converted to the more stable semicarbazone derivative (4OHCP-SCZ) for quantitation. Samples (10 μL) were extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a pentafluorophenyl reverse phase column (2.1 × 50 mm, 2.7 μm). Electrospray ionization in positive mode was used for detection. Multiple reaction monitoring of the precursor-to-product ion transitions 190→147 for thiotepa, 174→131 for tepa, 261→233 for CP, and 334→221 for 4OHCP-SCZ was selected for quantification. The ion transitions 202→155 for thiotepa-d, 186→139 for tepa-d, 267→237 for CP-d, and 340→114 for 4OHCP-d-SCZ were selected for the internal standard (IS) corresponding to each analyte The less abundant IS ions from Cl were used for CP-d and 4OHCP-d-SCZ to overcome the cross-talk interference from the analytes. Under optimized conditions, retention times were 0.67 min for tepa and its IS, 2.50 min for thiotepa and its IS, 2.52 min for 4OHCP-SCZ and its IS, and 2.86 min for CP and its IS. Total run time was 5 min per sample. The calibration ranges were 2.5-2,000ng/mL for thiotepa and tepa, 20-10,000ng/mL for CP and 20-5,000 ng/mL for 4OHCP; Dilution integrity for samples above the calibration range was validated with 10-fold dilution for thiotepa/tepa and 20-fold dilution for CP/4OHCP. Recoveries ranged from 86.3-93.4% for thiotepa, 86.3-89.0% for tepa, 90.2-107% for CP, and 99.3-115% for 4OHCP-SCZ. The IS normalized matrix effect was within (100±7) % for all 4 analytes. Plasma samples at room temperature were stable for at least 60 hours for thiotepa, 6 days for tepa, and 24 hours for CP and 4OHCP-SCZ. Plasma samples for thiotepa/tepa were stable after 4 freeze-thaw cycles, and for CP/4OHCP-SCZ were stable after 3 freeze-thaw cycles. The assays were validated and applied to clinical studies requiring small sample volumes.