Daria I. Vernikovskaya

Simultaneous quantitative determination of bupropion and its three major metabolites in human umbilical cord plasma and placental tissue using high-performance liquid chromatography-tandem mass spectrometry

A liquid chromatography in tandem with electro-spray ionization mass spectrometry method has been developed and validated for the quantitative determination of bupropion and its major metabolites (hydroxybupropion, threo- and erythrohydrobupropion) in human umbilical cord plasma and placental tissue. The samples were acidified with trichloroacetic acid, and protein precipitated by adding acetonitrile. Chromatographic separation of drug and metabolites was achieved by using a Waters Symmetry C(18) column, with an isocratic elution of 31% methanol and 69% formic acid (0.04%, v/v) aqueous solution at a flow rate of 1.0 mL/min. Detection was carried out by mass spectrometry using positive electro-spray ionization mode, and the compounds were monitored using multiple reactions monitoring method. Deuterium-labeled isotopes of the compounds were used as internal standards. Calibration curves were linear (r(2)>0.99) in the tested ranges. The lower limit of quantification of analytes in umbilical cord plasma samples is <0.72 ng/mL and 0.92 ng/g in placental tissue samples. The relative deviation of this method was <15% for intra- and inter-day assays, and the accuracy ranged between 88% and 105%. The extraction recovery of the four analytes ranged between 89% and 96% in umbilical cord plasma, and 64% and 80% in placental tissue. No significant matrix effect was observed in the presented method.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

A liquid chromatography method with single quadrupole mass spectrometry for quantitative determination of indomethacin in maternal plasma and urine of pregnant patients

A liquid chromatography with single quadrupole mass spectrometry method was developed for the quantitative determination of indomethacin in the maternal plasma and urine of pregnant patients under treatment. A deuterium-labeled isotope of indomethacin (d4-indomethacin) was used as an internal standard. The maternal plasma and urine samples were acidified with 1.0M HCl then extracted with chloroform to achieve the extraction recovery range of 94-104% with variation less than 11%. Chromatographic separation was achieved by a Waters Symmetry C₁₈ column with isocratic elution of 0.05% (v/v) formic acid aqueous solution and acetonitrile (47:53, v/v). An in-source fragmentation was applied on the single quadrupole mass spectrometer equipped with an electrospray ionization source at positive mode. The LC-ESI-MS quantification was performed in the selected ion monitoring mode targeting ions at m/z 139 for indomethacin and m/z 143 for its internal standard. The calibration curves were linear in the concentration ranges between 14.8 and 2.97 × 10(3) ng/mL for plasma samples and between 10.5 and 4.21 × 10(3) ng/mL for urine samples. The relative standard deviation of this method was less than 8% for intra- and inter-day assays, and the accuracy ranged between 90% and 108%.

Metabolism and Disposition of Bupropion in Pregnant Baboons (Papio cynocephalus)

Recent in vitro data obtained in our laboratory revealed similarities between baboons and humans in the biotransformation of bupropion (BUP) by both hepatic and placental microsomes. These data supported the use of baboons to study BUP biotransformation during pregnancy. The aim of this investigation was to determine the pharmacokinetics of BUP in baboons during pregnancy and postpartum, as well as fetal exposure to the drug after intravenous administration. Pregnant baboons (n = 5) received a single intravenous bolus dose of bupropion hydrochloride (1 mg/kg) at gestational ages 94–108 days (midpregnancy), 142–156 days (late pregnancy), and 6 weeks postpartum. Blood and urine samples were collected for 12 and 24 hours, respectively. The concentrations of BUP, hydroxybupropion (OH-BUP), threohydrobupropion, and erythrohydrobupropion in plasma were determined by liquid chromatography–tandem mass spectrometry. Relative to the postpartum period, the average midpregnancy clearance of BUP trended higher (3.6 ± 0.15 versus 2.7 ± 0.28 l/h per kg) and the average Cmax (294 ± 91 versus 361 ± 64 ng/ml) and the area under the curve (AUC) of BUP values (288 ± 22 versus 382 ± 42 h·ng/ml) trended lower. AUCOH-BUP also tended to be lower midpregnancy compared with postpartum (194 ± 76 versus 353 ± 165 h·ng/ml). Whereas the observed trend toward increased clearance of BUP during baboon pregnancy could be associated with a pregnancy-induced increase in its biotransformation, the trend toward increased renal elimination of OH-BUP may overshadow any corresponding change in the hydroxylation activity of CYP2B.

Quantitative determination of pravastatin and its metabolite 3α-hydroxy pravastatin in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α-hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one-step sample preparation by liquid-liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α-hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α-hydroxy pravastatin. The relative deviation of this method was <10% for intra- and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia.

Characteristics of Trophoblasts in Long-Term Culture

We analyzed more than 40 cytotrophoblast cultures derived from cell islets that grew from trypsinized tissue fragments of placental microvilli. Phenotypic variability of trophoblasts was demonstrated. Changes in trophoblast morphology from epithelium-like or oval cells to bipolar and spindle-shaped or twisted and then to mesenchymal-like cells as well as intensive expression of cytokeratin-7 and vimentin attested to epithelial-mesenchymal transition of trophoblasts during in vitro culturing. Analysis of the expression of specific markers in long-term trophoblast culture (≥7 passages) revealed the possibility of culture contamination with other non-trophoblast cells including fibroblasts. High risk of trophoblast culture contamination with rapidly growing cells necessitates regular control of the cultures used in fundamental studies. Our experiments confirmed the possibility of long-term culturing of cells maintaining trophoblast properties. The identity and purity of 4 trophoblast cultures free from contamination and retaining the properties of pure culture during long-term (>10 passages) culturing in vitro were confirmed.