SPE: A: following 1800 ml Antimetabolites of water, 200 ml of methanol, 8.2 ml of formic acid. SPE B: methanol, HPLC A: 1760 ml water, 200 ml of methanol, 40 ml of 1 M ammonium acetate in methanol, 2 ml of acetic acid. HPLC B: 60 ml of water, 1900 ml of methanol, 40 ml of 1 M ammonium acetate in methanol, 2 ml of acetic acid. Sample preparation thoroughly mixed 150 l aliquots of calibrator samples contr And the patient were placed in 2 ml of R Hrchen round-bottom samples and 600 l of sample preparation L Was pipetted solution added. Min after mixing the samples for 20 proteins were By centrifugation for 20 min at 16,000 g and 4 executed Filled. Musterl Solutions were filled into HPLC-Fl Schchen and on the HPLC autosampler. An aliquot of 50 l were described in the online SPE-LC-MS / MS system for analysis in the following paragraph is injected. LC-MS / MS configuration The general layout of the on-line SPE instrumentation LC MS / MS was used for this test before pr Presents. In short, a tandem mass spectrometer equipped with a functioning Turbo V ion source in modewas electro-ionization detector as used. The HPLC system coupled to provided the instrument with an autosampler with a thermostated sample compartment, the syringe 100, a loop 100 and the sample, two pumps I Ren two modules degasser, and a tray column with a six-port switching valve fitted. The whole system was controlled by Analyst 1.4.2 software. A I Re pump was the autosampler and L Solvent delivery SPE SPE-S Column was connected stored at room temperature. The second I Re HPLC pump L Solvent delivery to the HPLC-S Column thermostatted at 70 and protected by a pilot Column. The output of the HPLC-S was Column is connected to the ion source of the mass spectrometer. SPE pump was programmed so that the lipophilic components were captured of the injected sample on the SPE-S Molecules. Rin after lacing hydrophilic matrix material with the L Solvent A PES, was the six-valve of the column oven is turned off and matrix components confinement, Lich lipophilic analyte purged back with HPLC mobile phase A on the HPLC-S Molecules. An L Solvent gradient from A to B HPLC HPLC was used to the analyte from the HPLC-S Molecules elute in the mass spectrometer. Meanwhile, the SPE-S Column rinsed with SPE B and compensated with SPE A. For details on configuring the individual volumetric Me or response times, see Table 1, Fig. Figure 2 shows the settings of the control valve. The mass spectrometer was in the mode weight Operated hlten reaction monitoring conditions. The ion source, which was in positive ESI mode with a sputtering voltage of 1600 V ions heated to 500. Gas ion parameters: gas source 1 to 70 psi, the ion source gas 2 to 90 psi, 10 psi gas curtain, gas collision cell at 6 psi. Two SRM Trnsfer Length were recorded for each analyte and each internal standard. Since no internal standard is isotopically labeled commercially Ltlich of cyproterone acetate, was used as internal standard etonogestrel d7 for this substance. To help optimize the residence time in a cycle time of 600 ms f Rderf compatibility available, the window of the retention time of the analyte was divided into three periods. Conditions for SRM and retention times of the analytes, see Table 2 The analyst quantization 1.4.2 was used to generate the quantization method.