Journal of Chromatography B (v.878, #7-8)
Editorial Board (i).
Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry by Robert S. Jansen; Hilde Rosing; Wiete Kromdijk; Rob ter Heine; Jan HM Schellens; Jos H. Beijnen (621-627).
Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within −10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within −14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.
Keywords: Emtricitabine; Tenofovir; Nucleotides; LC–MS; Weak anion-exchange;
Simultaneous determination of glucuronic acid and sulfuric acid conjugated metabolites of daidzein and genistein in human plasma by high-performance liquid chromatography by Kaori Hosoda; Takashi Furuta; Kazuo Ishii (628-636).
Isoflavone aglycones daidzein (Dein) and genistein (Gein) are mainly present as glucuronides and sulfates in human plasma, and small amounts of the intact aglycones are also detected. In the present study, we have developed a high-performance liquid chromatography (HPLC)-UV-diode-array detector (DAD) method for the determination of intact 16 metabolites of Dein and Gein in plasma, especially focusing on highly polar conjugated metabolites at both 4′ and 7 positions on the isoflavone ring with glucuronic acid and/or sulfuric acid (7-glucuronide-4′-sulfates and 4′,7-diglucuronides). Luteolin-3′,7-di-O-glucoside was used as an internal standard. Solid-phase extraction was performed on an Oasis® HLB cartridge (60 mg, 3 cm3) with a recovery of >ca. 80%. The HPLC assay was performed on a Hydrosphere C18 column (100 mm × 4.6 mm I.D., particle size 3 μm). The mobile phase consisted of a mixture of 10 mM ammonium acetate solution and acetonitrile run under gradient mode at a flow rate of 1.5 ml/min. The UV detection wavelength was set at 250 nm. For UV spectral analysis, the diode-array detection wavelength was set at 220–360 nm. All HPLC analyses were performed at 45 °C. Each calibration for the determination of 16 metabolites gave a linear signal (r > 0.997) over a concentration range of 5–5000 ng/ml. The lower limits of quantification of these metabolites were 21.1–23.4 ng/ml and the lower limits of detection were 7.9–9.4 ng/ml. This method was used in a preliminary experiment to determine the plasma concentration of intact 16 metabolites after oral administration of kinako (baked soybean powder) to a healthy volunteer. The present HPLC-UV-DAD method should be useful for the metabolic and pharmacokinetic investigations of isoflavones in humans.
Keywords: Isoflavone; Sulfoglucuronide; Diglucuronide; Glucuronide; Sulfate; HPLC;
Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma by M. Wagner; E. Bourgogne; E. Varesio; G. Hopfgartner (637-644).
We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.
Keywords: Mass spectrometry; Quantitative analysis; Opioid receptor agonist; Plasma; Column-switching;
Quantification of topotecan by liquid chromatography–mass spectrometry (LC–MS). Application to intestinal transport using rat everted gut sacs by Cécile Arellano; Peggy Gandia; Laure Bettuing; John Woodley; Etienne Chatelut (645-652).
Topetecan is an important anti-cancer drug that has recently become available as an oral formulation. In order to study its intestinal absorption in vitro and a potential drug–drug interaction with the anti-emetic ondansetron, a sensitive and accurate method for the analysis of topotecan in biological media was required. We developed a liquid–liquid extraction method at pH 7.0–7.5 with a recovery of 85% and which took into account the complex chemical behaviour of topotecan related to the lactone opening and the keto-enol tautomerism. This enabled us to validate a new specific and sensitive LC–MS method of analysis, with satisfactory inter- and intra-day repeatability and accuracy. The method was applied to a study of topotecan uptake in rat everted gut sacs that showed that, despite being a P-glycoprotein substrate like topotecan, ondansetron did not interfere with topotecan uptake.
Keywords: Topotecan; Ondansetron; LC–MS; Everted gut sacs; Keto-enol tautomerism;
Liquid chromatography–tandem mass spectrometry for the determination of low-molecular mass aldehydes in human urine by Clara Eugenia Baños; Manuel Silva (653-658).
A simple and sensitive method is proposed for the determination of seven low-molecular mass aldehydes in human urine samples using liquid chromatography with tandem mass spectrometric detection. Urine samples diluted twofold with 0.3 M hydrochloric acid are aspirated into a LiChrolut EN solid-phase extraction column impregnated with 2,4-dinitrophenylhydrazine for cleanup, derivatization and preconcentration of the aldehydes. After elution of the hydrazones with acetonitrile, an aliquot is injected directly into the chromatograph. Identification and quantification of aldehydes was performed with electrospray in negative ion mode by selected reaction monitoring. By using synthetic urine samples, linearity is established over the concentration range 0.1–30 μg/l and limits of detection from 15 to 65 ng/l. The intra- and inter-day precision (RSD, %) of the aldehydes ranged from 2.9% to 6.4% and 3.6% to 9.3%, respectively, and specific uncertainties were ca. 5.0 ± 0.3 ng for all aldehydes. Average recoveries performed on two levels by enriching synthetic urine samples ranged between 92% and 100%. The method was also validated in terms of study sample stability including long-term and short-term analyte stability, freeze–thaw and extract stability. In summary, the method proposed surpasses other recent chromatographic alternatives in terms of the limit of detection and sample requirements for analysis.
Keywords: Aldehydes; Solid-phase extraction; In situ derivatization; 2,4-Dinitrophenylhydrazine; LC–ESI-MS/MS; Urine samples;
Identification and quantitative determination of a major circulating metabolite of gambogic acid in human by Jing Yang; Li Ding; Shaohong Jin; Xiaoxue Liu; Wenyuan Liu; Zhenzhong Wang (659-666).
Gambogic acid (GA), a promising anticancer candidate, is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi. The major circulating metabolite of GA in human, 10-hydroxygambogic acid (10-OHGA), was identified by comparison of the retention time and mass spectra with those of reference standard using liquid chromatography–tandem mass spectrometry. The reference standard of 10-OHGA was isolated from bile samples of rats after intravenous injection of GA injection, and its structure was confirmed by NMR. Then, a selective and sensitive method was developed for the quantitative determination of this metabolite in human plasma. After liquid–liquid extraction by ethyl acetate, the analyte and the internal standard were separated on a Sepax HPC18 column (100 mm × 2.1 mm i.d., 3.0 μm) with a mobile phase of 10 mM ammonium acetate water solution containing 0.1% formic acid–acetonitrile (20:80, v/v). The detection was performed on a single quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. The calibration curve was linear over the range of 3–2000 ng/mL for 10-OHGA. The developed quantification method can now be used for the pharmacokinetic and pharmacological studies of 10-OHGA after intravenous infusion of GA injection in human.
Keywords: Gambogic acid; 10-Hydroxygambogic acid; Metabolite; Liquid chromatography; Mass spectrometry;
Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR by Jon S.B. de Vlieger; Ard J. Kolkman; Kirsten A.M. Ampt; Jan N.M. Commandeur; Nico P.E. Vermeulen; Jeroen Kool; Sybren S. Wijmenga; Wilfried M.A. Niessen; Hubertus Irth; Maarten Honing (667-674).
This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatography–high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-1H, 2D-COSY, 2D-NOESY, and 1H-13C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.
Keywords: Bioaffinity; High resolution screening; Biosynthetic enzymes; Nuclear magnetic resonance spectroscopy; High resolution mass spectrometry; Human estrogen receptor; Liquid chromatography–mass spectrometry; Hyphenation;
Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry by Maren I. Blonk; Bart C. van der Nagel; Liesbeth S. Smit; Ron A.A. Mathot (675-681).
A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C18 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.
Keywords: Ultra performance liquid chromatography; Mass spectrometry; Levetiracetam; Plasma; Neonates; Pharmacokinetics;
Simultaneous determination of paracetamol, pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry by Hong-gang Lou; Hong Yuan; Zou-rong Ruan; Bo Jiang (682-688).
For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C18 Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of −8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.
Keywords: Paracetamol; Pseudoephedrine; Dextrophan; Chlorpheniramine; LC–MS/MS; Bioequivalence;
Simultaneous quantification of venlafaxine and O-desmethylvenlafaxine in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study by Feng Qin; Ning Li; Ting Qin; Yi Zhang; Famei Li (689-694).
A rapid, selective and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine venlafaxine (VEN) and O-desmethylvenlafaxine (ODV) in human plasma. Sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with 10 mmol/L ammonium acetate and methanol as the mobile phase at a flow rate of 0.30 mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for VEN and ODV were both obtained in the concentration range of 0.200–200 ng/mL (r 2 ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.200 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were less than 13% and the accuracy (relative error, R.E.) was within ±5.3% and ±3.6% for VEN and ODV. The method herein described was superior to previous methods in sensitivity and sample throughput and successfully applied to clinical pharmacokinetic study of venlafaxine sustained-release capsule in healthy male volunteers after oral administration.
Keywords: Venlafaxine; O-desmethylvenlafaxine; UPLC-MS/MS; Human plasma; Pharmacokinetics;
Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology by Adrian G. Miller; Heather Brown; Tim Degg; Keith Allen; Brian G. Keevil (695-699).
In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard™ column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters® Quattro Premier™ XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L (r 2 = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.
Keywords: 5-Hydroxyindole acetic acid; Carcinoid; Liquid chromatography; Tandem mass spectrometry;
Targeted quantitative analysis of superoxide dismutase 1 in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells by Jong Won Kim; Bei Nie; Heather Sahm; Dawn P.G. Brown; Tony Tegeler; Jin-Sam You; Mu Wang (700-704).
Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies.
Keywords: Superoxide dismutase 1; Ovarian cancer; Cisplatin drug resistance; Mass spectrometry; Selected-reaction-monitoring;
Analysis of lidocaine interactions with serum proteins using high-performance affinity chromatography by Sony Soman; Michelle J. Yoo; Yoon Jeong Jang; David S. Hage (705-708).
High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1-acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (K a) of 1.1–1.7 × 105 M−1 at 37 °C and pH 7.4. Lidocaine had weak to moderate binding to HSA, with a K a in the range of 103 to 104 M−1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes.
Keywords: Lidocaine; Human serum albumin; α1-Acid glycoprotein; High-performance affinity chromatography;
The release kinetics of drug eluting stents containing sirolimus as coated drug: Role of release media by C.P. Naseerali; P.R. Hari; K. Sreenivasan (709-712).
Prior understanding on the in vitro release profile of the drug from drug eluting devices such as stent (DES) is crucial in designing and optimizing the drug embedded coating or matrices. In fact, assessing in vitro release profile is a mandatory requirement prior to the clinical evaluation of DES. The in vitro release is also employed to estimate parameters such as T1/2. The release profile largely depends on the release medium selected for the studies. Normally PBS with a pH of 7.4 is used for assessing the release kinetics of the drug. Often drug undergoes irreversible changes such as hydrolysis in PBS leading to erroneous assessment of the release profile. This is particularly true in the case of sirolimus, one of the widely used drugs in various applications. We studied the influence of various media on the release profile of sirolimus from DES. The data generated suggested that a release medium consisting of 9:1 (v/v) of normal saline and isopropanol is a most suitable one for assessing in vitro the release kinetics of sirolimus from DES.
Keywords: Sirolimus; Drug eluting stent; Release kinetics; Release medium;
A convenient method for saponin isolation in tumour therapy by Alexander Weng; Kristina Jenett-Siems; Peter Schmieder; Diana Bachran; Christopher Bachran; Cornelia Görick; Mayank Thakur; Hendrik Fuchs; Matthias F. Melzig (713-718).
Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.
Keywords: Gypsophila saponins; Isolation; Dialysis; Tumour therapy;
Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry by Bo Jiang; Zourong Ruan; Honggang Lou; Hong Yuan (719-723).
A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C18 column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H]+ m/z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation (r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.
Keywords: Cymipristone; ESI; LC–MS/MS; Pharmacokinetics;