Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry.

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.

Determination of testosterone metabolites in human hepatocytes. I. Development of an on-line sample preparation liquid chromatography technique and mass spectroscopic detection of 6beta-hydroxytestosterone.

A rapid and sensitive RP-HPLC assay for determination of 6beta-hydroxytestosterone in human hepatocytes with corticosterone as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20x4 mm I.D.) extraction pre-column and subsequent gradient separation on a Prontosil 60-5 C(18)-H (250x2 mm I.D., 5 micrometer particle size) analytical column in the back-flush mode using a ternary eluent system composed of methanol, tetrahydrofuran and water. Signal monitoring was done by measurement of the responses from liquid chromatography coupled to mass spectroscopy (LC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source conducted in the selected reaction monitoring (SRM) mode. Mean recoveries of 6beta-hydroxytestosterone from an estimate of the biological matrix, i.e., Dulbecco's modified Eagle medium "High Glucose", ranged from 101.8-104.4% for samples containing the target analyte at the 250, 500 and 1000 ng/ml level. The limit of quantitation (LOQ) was 20 ng/ml at an injection volume of 100 microliter determined in the same matrix. Linearity of signal responses versus concentration for all three analytes was accomplished in the range of 100-4000 ng/ml. Mean values of the coefficients of variation (C.V.) for the target analyte obtained for the concentrations 250, 500 and 1000 ng/ml at 5 different days in quintuplicate ranged from 1.5-7.7% (within-day) and 4.8-7.3% (between-day). The corresponding values for the accuracy ranged from 87.7-106.1% for the within-day and from 98.8-102.5% for the between-day measurements. The target analyte was sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.

Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection.

A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20 x 4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-HT (100 x 3 mm I.D., 3.5 microm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC-MS-MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2 +/- 3.5, 96.7 +/- 2.2, and 100.9 +/- 3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 microl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10-600 ng/ml. The mean values of the coefficients of variation (CV) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46 +/- 1.15, 3.94 +/- 2.13 and 4.79 +/- 2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.