The extensive metabolism and administration of low doses of ethinylestradiol (EE) in preclinical animal species necessitates a sensitive analytical method to quantify the drug at low picogram-per-milliliter concentrations in biological matrixes. A highly sensitive and accurate method based on the derivatization of EE with dansyl chloride coupled with liquid chromatography/tandem mass spectrometry is described. The dansyl derivatization of EE introduced a basic secondary nitrogen into the molecule that was readily ionized in commonly used acidic HPLC mobile phases. The derivative showed an intense protonated molecular ion at m/z 530 under positive turbo ion spray ionization. The collision-induced dissociation of this ion formed a distinctive product at m/z 171, corresponding to the protonated 5-(dimethylamino)naphthalene moiety. The selected reaction monitoring, based on the m/z 530 --> 171 transition, was highly specific for EE, since no background signal was observed from blank plasma obtained from rhesus monkeys. The limit of detection, at a signal-to-noise ratio of 5, was 0.2 fg/mL EE spiked into blank plasma. This allowed for a lower limit of quantitation of 5 pg/mL using a 50-microL plasma sample and 10-microL injection of dansylated derivative into the CTC-PAL Leap autosampler coupled to a Sciex API 4000 mass spectrometer. Using fast-gradient liquid chromatography, the analyte peak eluted at 1.6 min. The validation results showed high accuracy (% bias < 4) and precision (% CV < 7.5) at broad linear dynamic ranges (0.005-20 ng/mL), using deuterated EE as internal standard. Therefore, the facile dansyl derivatization coupled with tandem mass spectral analysis allowed the development of a highly sensitive and specific method for quantitation of trace levels of EE in the plasma of rhesus monkeys dosed orally and intravenously with EE.
Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [M + H]+ m/z values of expected metabolites allows for the construction of user-defined MS(n) protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional "survey scans" (e.g., precursor ion and constant neutral loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/MS(n) data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 "hits", pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/MS(n) analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/MS(n) run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.
A new type of quadrupole linear ion trap mass spectrometer, Q TRAP TM LC/MS/MS system (Q TRAP TM ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma.With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS 2 ) and MS 3 experiments to obtain structural information of drug metabolites 'on-thefly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage. Copyright # 2003 John Wiley & Sons, Ltd.Liquid chromatography/tandem mass spectrometry (LC/ MS/MS) has been widely applied in both qualitative and quantitative analyses of drug candidates and their metabolites in biological matrices, in support of drug discovery and development. 1-3 Commonly used tandem mass spectrometers include triple stage quadrupole (tandem-in-space), three-dimensional (3D) ion trap (tandem-in-time), and quadrupole time-of-flight (Q-TOF) mass spectrometers. Threedimensional ion trap mass spectrometers are widely used for conducting consecutive product ion scans (MS n ) in a data-dependent fashion that provides useful information for structural elucidation. Triple quadrupole mass spectrometers, on the other hand, can be used to conduct tandem mass scan experiments in various modes including product ion scan (MS 2 ), precursor ion scan, constant neutral loss scan, and multiple reaction monitoring (MRM). The constant neutral loss and precursor ion scan experiments on a triple quadrupole serve as a convenient tool for detecting unknown metabolites in complex biological matrices, especially for phase II drug metabolites, such as acyl glucuronide (loss of 176 Da) and sulfate (loss of 80 Da) conjugates. Thus far, MRM is the most sensitive and selective detection method for confirming trace levels of circulating metabolites in plasma, 4 for example, as in human ADME studies, as well as in animal organs, and it is the method of choice for quantifying drugs and their metabolites in biological fluids. Nevertheless, both instruments have...
ABSTRACT:The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 M). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (V max /K m ) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoformspecific monoclonal antibodies, in which the inhibition of formation was ϳ54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.Oral contraceptives (OCs) have been used widely by 60 to 70 million women worldwide since the 1970s. Ethinylestradiol (EE) and norethindrone are two components in OCs. Interactions with OCs in clinical studies have been reported for several compounds, including rifampicin (Bolt et al., 1977), rifabutin (LeBel et al., 1998), and ritonavir (Ouellet et al., 1998), and the subsequent failure to inhibit contraception was attributed to the increased metabolism of EE.The metabolism of EE has been studied extensively. It undergoes hydroxylation at the 2 (major), 4, 6, and 16␣ positions of the steroid nucleus (Fig. 1). Metabolism can occur by way of glucuronidation at 17 and 3 positions, methylation, or sulfation at the 3 position. Hydroxylation of EE is catalyzed primarily by CYP3A4 (Guengerich, 1988), 3-O-glucuronidation by uridine diphosphate glucuronosyltransferase 1A1 (Ebner et al., 1993), and 3-O-sulfation by sulfotransferase 1E1 (Forbes-Bamforth and Coughtrie, 1994). Changes in the activities of these metabolizing enzymes have been implicated in the observed OC interactions (Guengerich, 1990a(Guengerich, ,b, 1997. More recently, estradiol and estrone have been shown to be oxidized at several positions by several P450 isoforms, namely, CYP1A2, CYP3A4, CYP1B1, and CYP2C9 (Hayes et al., 1996;Shou et al., 1997;Yamazaki et al., 1998;Badawi et al., 2001;Lee et al., 2001Lee et al., , 2002. However, the involvement of P450 isoforms, other than CYP3A4, in the metabolism of EE has not been established clearly, although a previous report suggested that isoforms from the CYP2C and CYP2E families also may be involved (Ball et al., 1990). The objective of this present study was to investigate systematically the human P450 isoforms involved in the ox...
ABSTRACT:Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1-hydroxymidazolam, which is equipotent to midazolam. 1-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1-hydroxymidazolam in human liver microsomes enriched with uridine 5-diphosphoglucuronic acid (UDPGA). The structure of the Nglucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n ؍ 1.2-1.5). The apparent S 50 values for the formation of O-and N-glucuronides were 43 and 18 M, respectively, and the corresponding apparent V max values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1-hydroxymidazolam in human liver microsomes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.