A UPLC-MS/MS method for therapeutic drug monitoring of etonogestrel.

INTRODUCTION: Etonogestrel (ENG) is a progestin used in the contraceptive vaginal ring NuvaRing and the subdermal implant Implanon. A sensitive method for measuring ENG is useful for further investigating the progestin's pharmacokinetics with these alternative contraceptive formulations and generating important information about possible continued efficacy or potential failure to remove the subdermal implant. METHODS: Standards and serum samples were spiked with D8-progesterone (internal standard) and subsequently extracted with dichloromethane, dried, and reconstituted in 25% methanol with formic acid. ENG was analyzed by positive electrospray ionization in multiple reaction monitoring mode with a run time of 5.5 minutes using a C18 BEH column. The mobile phase was a gradient of water:acetonitrile, with 0.1% formic acid. The method was applied successfully to study the pharmacokinetics of ENG during vaginal ring use. The method was also used in routine patient care to assess ENG levels. RESULTS: The method is linear from 50 to 2000 pg/mL. The limits of detection and quantification are 25 and 50 pg/mL, respectively. There was no observed ionization suppression within the linear range of the assay, and the average recovery was 87%. Serum ENG levels of n = 3 subjects were all within the linear range of the assay for a total study period of 42 days after insertion of the ring. Of n = 20 patients with nonpalpable subdermal implants, n = 13 had ENG levels >25 pg/mL, whereas n = 7 had levels <25 pg/mL. CONCLUSIONS: We developed a rapid, sensitive, and robust ultra performance liquid chromatography-tandem mass spectometry (UPLC-MS/MS) method for the quantification of ENG in serum that is useful to study the progestin's pharmacokinetics and inform physicians about successful implantation or potential failure to remove a subdermal device.

High performance liquid chromatography-mass spectrometry assay for polymyxin B1 and B2 in human plasma.

BACKGROUND: Polymyxin B is an old antibiotic with increasing clinical relevance in the treatment of multidrug-resistant Gram-negative bacterial infections. However, current dosing regimens are largely empiric as clinical pharmacological characterization of the drug has been hindered by the lack of assays to measure polymyxin B in human plasma. METHODS: A high-performance liquid chromatography-mass spectrometry assay was developed to quantify polymyxin B1 and B2 in human plasma using pure calibrators. After purification with a solid-phase extraction column, polymyxin B1 and B2 were separated on a C18 column by gradient chromatography with an overall runtime of 12 minutes. Polymyxin B1 and B2 were ionized by positive electrospray ionization, and the resulting ions specific to polymyxin B1 and B2 were monitored (selected ion recording). RESULTS: The dominant ions produced were (M + 2H) at m/z 602.6 and 595.5 for polymyxin B1 and polymyxin B2, respectively. The assay was linear between concentrations of 100 and 2500 ng/mL, with interday precision of 5.9% and 3.4% at 100 ng/mL and 5.3% and 4.0% at 2000 ng/mL for polymyxin B1 and polymyxin B2, respectively. Accuracy was 80.2% and 82.2% at 100 ng/mL and 99.9% and 109.6% at 2000 ng/mL for polymyxin B1 and polymyxin B2, respectively. No interference from other drugs commonly administered with polymyxin B was detected. The performance of the assay is affected by gross hemolysis and hyperlipemia. The method was successfully applied to patient samples. Interestingly, in a single patient the ratio of B1 and B2 did not change over a period of 12 hours after administration of the drug. CONCLUSIONS: A simple method for the simultaneous measurement of polymyxin B1 and polymyxin B2 in human plasma is described, which has the potential to optimize clinical use of this valuable antibiotic by permitting pharmacokinetic studies and therapeutic drug monitoring.

Placental transfer and fetal elimination of morphine-3-beta-glucuronide in the pregnant baboon.

The glucuronide metabolites of several widely used drugs are detected in fetal plasma after maternal drug administration. However, the disposition of these metabolites is poorly understood and clinical concerns have been raised about accumulation of active metabolites in the fetus. For this reason, morphine-3-beta-glucuronide (M3G), an active metabolite of morphine, was studied to provide quantitative data on disposition. Maternal, fetal, and bidirectional placental clearances of M3G were measured in three pregnant baboons. During maternal infusion of M3G to steady-state, the glucuronide metabolite readily appeared in fetal plasma achieving a mean +/- S.D. fetal-to-maternal concentration ratio of 0.79 +/- 0.04. In paired maternal and fetal infusions, steady-state clearances were 53 +/- 3 (maternal), 1.5 +/- 0.5 (maternal-to-fetal), 2.6 +/- 0.1 (fetal-to-maternal), and -0.70 +/- 0.6 ml x min(-1) (fetal). These clearance values support bidirectional transfer of M3G across the placenta and indicate negligible direct clearance from the fetus. The clearance of M3G across the placenta is more than 20-fold less than that of morphine. Despite this low index of permeability, placental transfer contributes significantly to the glucuronide pool in the fetus. Placental transfer emerges as the major clearance pathway for the glucuronide from the fetus and suggests a component of active efflux. What is more, the results do not support the concept of sequestration in the fetal intestine as a significant route of clearance. Together these results clarify the distribution and clearance of glucuronides in the pregnant primate and facilitate prediction of fetal exposure to active metabolites.

Molecular cloning of the baboon UDP-glucuronosyltransferase 1A gene family: evolution of the primate UGT1 locus and relevance for models of human drug metabolism.

BACKGROUND: Glucuronidation by the UDP glucuronosyltransferase 1A enzymes (UGT1As) is a major pathway for elimination of drugs and endogenous substances, such as bilirubin. OBJECTIVE: To identify the baboon UGT1A gene family, compare it with that of the human, and evaluate the baboon as a model for human glucuronidation. METHODS AND RESULTS: Aligning the human and baboon UGT1 loci identified rearrangements occurring since the divergence of baboons and humans. The baboon UGT1A cDNAs were cloned and shown to have an orthologous relationship with several genes in the human UGT1A family. This indicates that most protein encoding UGT1A first exons were duplicated before the divergence of baboons and humans. Gene conversions interfered with the phylogenetic signal for exons 1A4, 1A5, and 1A10, and led to concerted evolution of exon groups 1A2-1A5 and 1A7-1A13. The activity of the baboon UGT1As resembled those of their human counterparts in glucuronidating endobiotics, such as serotonin, bilirubin, and various xenobiotics. CONCLUSION: These insights demonstrate that the baboon has significant clinical relevance as a model for examining toxicological metabolism in humans.

Fetal morphine metabolism and clearance are constant during late gestation.

Fetal metabolism significantly contributes to the clearance of drugs from the fetus. To understand how the changes in fetal metabolism expected in late gestation alter fetal drug clearance, serial measurements of morphine metabolism were made in the fetal baboon over the latter third of gestation. Clearance and metabolism were evaluated in the context of fetal growth, onset of labor, and the administration of classical enzyme induction agents. Morphine, a probe substrate for the enzyme uridine diphosphate glucuronosyltransferase 2B7 (UGT2B7), was continuously infused to chronically catheterized fetal baboons while measuring morphine, morphine-3-beta-glucuronide, and morphine-6-beta-glucuronide concentrations. In some animals, intermittent infusions of the metabolites provided estimates of metabolite clearance and, hence, the rate of formation of metabolites and metabolic clearance. Overall, metabolic clearance of morphine from the fetus was 27 +/- 9.0 ml x min(-1) or 32% of total clearance. This is similar to the overall clearance in the adult baboon when standardized to weight. No change in any measure of metabolism or clearance of morphine or its glucuronide metabolites was found with gestational age, the presence of labor, or administration of UGT enzyme induction agents. Interpreting these findings using a physiologically based approach suggests that the intrinsic clearance of the fetal liver toward morphine is of sufficient magnitude that fetal hepatic clearance is flow-limited. The implication of a high intrinsic clearance is for significant placento-hepatic first-pass metabolism when drugs are administered to the mother. The previously held view of the "inadequacy of perinatal glucuronidation" needs to be reconsidered.

The contribution of fetal metabolism to the disposition of morphine.

The contribution of fetal metabolism to drug disposition in pregnancy is poorly understood. With maternal administration of morphine, like many drugs, steady-state concentrations in fetal plasma are less than in maternal plasma. The contribution of fetal metabolism to this difference is unknown. Morphine was used as a model drug to test the hypothesis that fetal metabolism contributes significantly to drug clearance by the fetus. Infusions of morphine, morphine-3-beta-glucuronide (M3G), and morphine-6-beta-glucuronide (M6G) were administered to the fetal baboon. Plasma concentrations of drug and metabolite obtained near steady state were measured by high-performance liquid chromatography. During morphine infusion, morphine, M3G, and M6G concentrations rose linearly with dose. M3G concentrations exceeded M6G by 20-fold. Mean +/- S.D. clearances of morphine, M3G, and M6G from the fetus were 69 +/- 17, 2.3 +/- 0.60, and 1.6 +/- 0.24 ml x min(-1), respectively. Clearances seemed to be dose-independent. The mean +/- S.D. fraction of morphine dose metabolized was 32 +/- 5.5%. This converts to a fetal metabolic clearance of 22 +/- 6.5 ml x min(-1). In conclusion, one third of the elimination of morphine from the fetal baboon is attributable to metabolism, one third to passive placental transfer, and one third undefined. Furthermore, there is no evidence for saturation of metabolism. Fetal metabolism is surprisingly high compared with in vitro estimates of metabolism and morphine clearance in human infants. For morphine, fetal drug metabolism accounts for half the difference between fetal and maternal plasma concentrations.