Pan Deng

An ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants.

Abscisic acid (ABA) is the most important hormone for plants to resist drought and other abiotic stresses. ABA binds directly to the PYR/PYL family of ABA receptors, resulting in inhibition of type 2C phosphatases (PP2C) and activation of downstream ABA signaling. It is envisioned that intervention of ABA signaling by small molecules could help plants to overcome abiotic stresses such as drought, cold and soil salinity. However, chemical instability and rapid catabolism by plant enzymes limit the practical application of ABA itself. Here we report the identification of a small molecule ABA mimic (AM1) that acts as a potent activator of multiple members of the family of ABA receptors. In Arabidopsis, AM1 activates a gene network that is highly similar to that induced by ABA. Treatments with AM1 inhibit seed germination, prevent leaf water loss, and promote drought resistance. We solved the crystal structure of AM1 in complex with the PYL2 ABA receptor and the HAB1 PP2C, which revealed that AM1 mediates a gate-latch-lock interacting network, a structural feature that is conserved in the ABA-bound receptor/PP2C complex. Together, these results demonstrate that a single small molecule ABA mimic can activate multiple ABA receptors and protect plants from water loss and drought stress. Moreover, the AM1 complex crystal structure provides a structural basis for designing the next generation of ABA-mimicking small molecules.

Metabolism and Pharmacokinetics of 3-n-Butylphthalide (NBP) in Humans: The Role of Cytochrome P450s and Alcohol Dehydrogenase in Biotransformation

3-n-Butylphthalide (NBP) is a cardiovascular drug currently used for the treatment of cerebral ischemia. The present study aims to investigate the metabolism, pharmacokinetics, and excretion of NBP in humans and identify the enzymes responsible for the formation of major metabolites. NBP underwent extensive metabolism after an oral administration of 200 mg NBP and 23 metabolites were identified in human plasma and urine. Principal metabolic pathways included hydroxylation on alkyl side chain, particularly at 3-, ω-1-, and ω-carbons, and further oxidation and conjugation. Approximately 81.6% of the dose was recovered in urine, mainly as NBP-11-oic acid (M5-2) and glucuronide conjugates of M5-2 and mono-hydroxylated products. 10-Keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-hydroxy-NBP (M3-2), and M5-2 were the major circulating metabolites, wherein the areas under the curve values were 1.6-, 2.9-, 10.3-, and 4.1-fold higher than that of NBP. Reference standards of these four metabolites were obtained through microbial biotransformation by Cunninghamella blakesleana. In vitro phenotyping studies demonstrated that multiple cytochrome P450 (P450) isoforms, especially CYP3A4, 2E1, and 1A2, were involved in the formation of M3-1, M3-2, and 11-hydroxy-NBP. Using M3-2 and 11-hydroxy-NBP as substrates, human subcellular fractions experiments revealed that P450, alcohol dehydrogenase, and aldehyde dehydrogenase catalyzed the generation of M2 and M5-2. Formation of M5-2 was much faster than that of M2, and M5-2 can undergo β-oxidation to yield phthalide-3-acetic acid in rat liver homogenate. Overall, our study demonstrated that NBP was well absorbed and extensively metabolized by multiple enzymes to various metabolites prior to urinary excretion.

Identification of Amiodarone Metabolites in Human Bile by Ultraperformance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

Amiodarone is recognized as an effective drug in the treatment of arrhythmias. Previous experiments demonstrated that mono-N-desethylamiodarone (MDEA) was the major circulating metabolite in humans. In addition, dealkylation, hydroxylation, and deamination were minor metabolic pathways. The purpose of this study was to identify the metabolites of amiodarone in the bile obtained from patients with T-tube drainage after oral drug administration. Amiodarone metabolism in vitro was also investigated using human liver microsomes (HLMs) and S9 fraction. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) revealed 33 metabolites in human bile, including 22 phase I and 11 phase II metabolites. The major metabolites were MDEA (M7) and ω-carboxylate amiodarone (M12). Metabolite M12 was isolated from human bile, and the chemical structure was confirmed using UPLC-Q/TOF MS and 1H NMR. Moreover, the authentic standards of two hydroxylated metabolites, 2-hydroxylamiodarone and 3′-hydroxylamiodarone, were obtained through microbial transformation. Several novel metabolic pathways of amiodarone in human were proposed, including ω-carboxylation, deiodination, and glucuronidation. The in vitro study demonstrated that incubation of HLMs with amiodarone did not give rise to any carboxyl metabolites. In contrast, M12 and its metabolites were detected in human liver S9 incubation samples, and the production of these metabolites were inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, suggesting the involvement of alcohol dehydrogenase in the ω-carboxylation of amiodarone. Overall, UPLC-Q/TOF MS analysis leads to the discovery of several novel amiodarone metabolites in human bile and underscores the importance of bile as an excretion pathway.

Derivatization methods for quantitative bioanalysis by LC–MS/MS

LC with atmospheric pressure ionization MS is essential to a large number of quantitative bioanalyses for a variety of compounds, especially nonvolatile or highly polar compounds. However, in many instances, weak ionization, poor LC retention and instability of certain analytes hinder the development of the LC-MS/MS method. Chemical derivatization has been used for different classes of analytes to improve their ionization efficiency, chromatographic separation and chemical stability. This work presents an overview of chemical derivatization methods that have been applied to the quantitative LC-MS/MS analyses of nine classes of molecules, including aldehydes, amino acids, bisphosphonate drugs, carbohydrates, carboxylic acids, nucleosides and their associated analogs, steroids, thiol-containing compounds and vitamin D metabolites, in biological matrices.

Evidence for the Bioactivation of 4-Nonylphenol to Quinone Methide and ortho-Benzoquinone Metabolites in Human Liver Microsomes

4-Nonylphenol (4-NP) is a well-known toxic environmental contaminant. The major objective of the present study was to identify reactive metabolites of 4-NP. Following incubations of 4-NP with NADPH- and GSH-supplemented human liver microsomes, 6 GSH conjugates, along with 19 oxidized metabolites, were detected by UPLC/Q-TOF mass spectrometry utilizing the mass defect filter method. Several authentic key metabolite standards were chemically synthesized for structural identification. Three GSH conjugates were found to derive from quinone methide intermediates, and the other three resulted from ortho-benzoquinone intermediates. Conjugation of the quinone methides with GSH produced benzylic-orientated GSH conjugates by 1,6-addition, while the reaction of the ortho-benzoquinone intermediates offered aromatic-orientated GSH conjugates. The conversion of 4-NP to the quinone methides and ortho-hydroquinones required cytochromes P450, specifically CYPs1A2, 2C19, 2D6, 2E1, and 3A4, while the oxidation of ortho-benzohydroquinones to the corresponding benzoquinones was apparently independent of microsomal enzymes. The ortho-benzoquinone derived from 4-NP was isomerized to the corresponding hydroxyquinone methide, and the former dominated the latter at a rate of approximately 20:1. The findings of the quinone methide and benzoquinone metabolites intensified the concern on the impact of 4-NP exposure on human health.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of febuxostat in human plasma.

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d(7) -febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C(18) column (4.6 × 100 mm, 5 µm) using acetonitrile-5 mm ammonium acetate-formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor-to-product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d(7) -febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0-5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra- and inter-day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at -20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers.

Validated LC-MS/MS method for quantitative determination of rasagiline in human plasma and its application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed to determine rasagiline in human plasma. The analytical method utilized liquid-liquid extraction of plasma with n-hexane-dichloromethane-isopropanol (20:10:1, v/v/v). Separation of analyte and the internal standard (IS) pseudoephedrine was performed on a Zorbax Extend C(18) column (150 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile-5 mM ammonium acetate-acetic acid (40:60:0.05, v/v/v) at a flow rate of 0.5 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transitions m/z 172.1-->m/z (117.1+115.1) for rasagiline, and m/z 166.0-->m/z 148.1 for the internal standard. The method was linear over the concentration range of 0.020-50.0 ng/mL. The intra- and inter-day precisions were less than 11.2% in terms of relative standard deviation (R.S.D.), and the accuracy was within +/-6.4% in terms of relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.020 ng/mL with acceptable precision and accuracy. The mean extraction-efficiency at three concentrations was 95.6+/-7.0%, 97.9+/-3.0% and 95.3+/-8.3%. The validated method offered increased sensitivity (10 times higher than those reported) and wide linear concentration range. This method was successfully applied for the evaluation of pharmacokinetics of rasagiline after single oral doses of 1, 2 and 5mg rasagiline to 12 Chinese healthy volunteers.

Determination of tropisetron in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the determination of tropisetron in human plasma was developed and validated over the concentration range of 0.100-100 ng/mL. Diphenhydramine was used as the internal standard (IS). The tropisetron and the IS were extracted from alkalized plasma samples into diethyl ether-dichloromethane (2:1, v/v) and the LC separation was performed by a Diamonsil C18 column (150 mm x 4.6 mm, i.d., 5 microm). The mobile phase was methanol:water (80:20, v/v) containing 0.2% formic acid delivered at a flow rate of 0.5 mL/min. The total chromatographic run time was 4.5 min. The MS data acquisition was accomplished by selected reaction monitoring (SRM) mode with positive atmospheric pressure chemical ionization (APCI) interface. The lower limit of quantification (LLOQ) achieved was 0.100 ng/mL with precision (RSD) of 3.1% and accuracy (RE) of -0.7%. For both inter-batch and intra-batch tests, the precision (RSD) for the entire validation was less than 6.0%, and the accuracy (RE) was within the -0.5% to 0.2% range. This validated LC-MS/MS method was later used to characterize the pharmacokinetics as well as the bioequivalence of tropisetron formulations.

Characterization of metabolites of GLS4 in humans using ultrahigh‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

RATIONALE GLS4 is a heteroaryldihydropyrimidine compound that inhibits hepatitis B virus (HBV) replication by drug-induced depletion of nucleocapsids. It is currently undergoing clinical trials in China to treat HBV infection. The aim of this study was to identify the metabolites of GLS4 in humans. METHODS A rapid and sensitive method based on ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry was used to identify GLS4 metabolites in human plasma, urine, and feces after an oral dose of 120 mg GLS4. RESULTS A total of 27 metabolites were detected and identified by comparing the accurate molecular masses, retention times and spectral patterns of the analytes with those of the parent drug. Nine metabolites were confirmed by comparison with reference substances. All of the metabolites had a bromine atom and displayed the isotope ion of [M + H](+)/[M + H + 2](+) at a ratio of 1:1. Fragmentation of the dihydropyrimidine structure was characterized by the loss of the m-bromofluorobenzene group to generate an ion at m/z 220.0175. The morpholine ring was characterized by an ion at m/z 100.0757. CONCLUSIONS The metabolites of GLS4 in humans were identified by the diagnostic ions of dihydropyrimidine and morpholine rings. GLS4 underwent extensive dealkylation, hydrolysis, dehydrogenation, oxidation, and glucuronidation reactions in humans.

Derivatization methods for LC–MS analysis of endogenous compounds

Sensitive and reliable analysis of endogenous compounds is critically important for many physiological and pathological studies. Methods based on LC-MS have progressed to become the method of choice for analyzing endogenous compounds. However, the analysis can be challenging due to various factors, including inherent low concentrations in biological samples, low ionization efficiency, undesirable chromatographic behavior and interferences of complex biological. The integration of chemical derivatization with LC-MS could enhance its capabilities in sensitivity and selectivity, and extend its application to a wider range of analytes. In this article, we will review the derivatization strategies in the LC-MS analysis of various endogenous compounds, and provide applications highlighting the impact of these important techniques in the evaluation of pathological events.