Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in doping analysis.

Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography-mass spectrometry (GC-MS) in steroid analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation.

The degradation of N,N',N"-triethylenephosphoramide in aqueous solutions: a qualitative and kinetic study.

The degradation of N,N',N"-triethylenephosphoramide (TEPA) in aqueous solutions has been investigated over a pH range of 3-14. Samples were analyzed using a gas chromatographic system with nitrogen/phosphorus selective detection. The degradation kinetics were studied as function of pH, sodium chloride concentration and temperature. The degradation of TEPA in buffers follows pseudo first order kinetics. The logk(obs)8 the methoxy derivative of TEPA was formed, as a consequence of the applied procedure.

Determination of N,N',N"-triethylenethiophosphoramide and its active metabolite N,N',N"-triethylenephosphoramide in plasma and urine using capillary gas chromatography.

A sensitive assay for the determination of N,N',N"-triethylenethiophosphoramide (thioTEPA) and its metabolite N,N',N"-triethylenephosphoramide (TEPA) in micro-volumes human plasma and urine has been developed. ThioTEPA and TEPA were analysed using gas chromatography with selective nitrogen-phosphorus detection or mass spectrometry after extraction with a mixture of 1-propanol-chloroform from the biological matrix. Diphenylamine was used as internal standard. The limit of detection was 1.5 ng/ml for thioTEPA and 2.5 ng/ml for TEPA, using 100 microl of biological sample; recoveries ranged between 70 and 90% and both accuracy and precision were less than 10%. Linearity was accomplished in the range of 10-1000 ng/ml for plasma and 100-10000 ng/ml for urine using thermionic nitrogen-phosphorus detection. With mass spectrometry a linear range of 100-25000 ng/ml TEPA in plasma or urine was obtained. For thioTEPA a second-order polynomial function describes the relationship between the analyte concentration in the range of 500-25000 ng/ml and detection response. TEPA proved to be stable in plasma and urine for at least 10 weeks at -80 degrees C. ThioTEPA and TEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.

Simultaneous determination of N,N',N"-triethylenethiophosphoramide, cyclophosphamide and some of their metabolites in plasma using capillary gas chromatography.

A sensitive assay for the simultaneous determination of N,N',N"-triethylenethiophosphoramide (thioTEPA), its metabolite N,N',N"-triethylenephosphoramide (TEPA), cyclophosphamide (CP) and its metabolite 2-dechloroethylcyclophosphamide (2-DCE-CP) in plasma has been developed and validated. The analytes were determined using gas chromatography with nitrogen/phosphorus selective detection after liquid-liquid extraction with chloroform using 100 microl of plasma. Diphenylamine (for TEPA, thioTEPA and 2-DCE-CP) and imipramine (for CP) were used as internal standards. The limits of quantitation for thioTEPA, TEPA, CP and 2-DCE-CP were 5, 5, 50 and 250 ng/ml, respectively. Linear calibration curves were observed over two decades of concentration. Accuracy, within-day and between-day precision were less than 13% for all analytes. Stability of the analytes proved to be satisfactory for at least 1 month, stored at -70 degrees C. Analysis of samples obtained from patients receiving cyclophosphamide, thioTEPA and carboplatin in a high-dose regimen demonstrated the applicability of the assay.