van Maanen M J, Tijhof I M, Damen J M, Versluis C, van den Bosch J J, Heck A J, Rodenhuis S, Beijnen J H
Department of Pharmaceutical Analysis, University of Utrecht, The Netherlands.
Cancer Res. 1999 Sep 15;59(18):4720-4.
An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.
人们试图解析烷化剂N,N',N''-三乙烯硫代磷酰胺（硫替派）的代谢谱。通过带有选择性氮/磷检测的气相色谱法对接受治疗患者尿液中的硫替派及其代谢产物N,N',N-三乙烯磷酰胺（替派）进行定量。通过对硝基苄基吡啶反应性评估总烷化活性。总烷化活性超过了硫替派和替派的量，表明存在其他烷化代谢产物。固相萃取和液液萃取后进行气相色谱-质谱分析，结果显示替派的氮丙啶官能团转化为β-氯乙基部分。这种代谢产物，N,N'-二乙烯基-N''-2-氯乙基磷酰胺，通过带有选择性氮/磷检测的气相色谱法定量，仅占给药剂量的0.69%。大量尿液通过固相萃取进行浓缩，并用高效液相色谱法进行分馏。对每个2毫升馏分测定烷化活性，结果显示在8至12毫升之间洗脱的一种烷化化合物的存在。收集具有烷化活性的馏分，在室温下于氮气流下蒸发至干，用甲醇复溶，然后进行快原子轰击质谱和快原子轰击串联质谱分析。发现了一种分子量为352道尔顿的新代谢产物，与硫替派-巯基尿酸盐相同。硫替派-巯基尿酸盐可能是谷胱甘肽结合的结果，之后谷胱甘肽加合物在不同阶段失去两个氨基酸残基。这种新代谢产物的裂解模式和色谱性质与通过在pH 11和95℃下将硫替派与N-乙酰半胱氨酸孵育30分钟获得的参比物硫替派-巯基尿酸盐相同。通过液相色谱-质谱法对硫替派-巯基尿酸盐进行定量。尿液中的硫替派-巯基尿酸盐水平占给药剂量的12.3%，超过了之前被认为是硫替派主要代谢产物的替派的量。硫替派及其代谢产物的总排泄量占总烷化活性的54 - 100%，表明仍存在其他烷化代谢产物。
