Xingjie Guo

Simultaneous determination of trantinterol and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid-liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1mm×100mm, 3μm), with the mobile phase consisted of methanol-0.2% formic acid (30:70, v/v) at the flow rate of 0.2mL/min. Each sample was chromatographed within 5min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1mg/kg trantinterol to rats.

Rapid separation and identification of 31 major saponins in Shizhu ginseng by ultra-high performance liquid chromatography–electron spray ionization–MS/MS

Background Among the various ginseng strains, Shizhu ginseng is endemic to China, mainly distributed in Kuandian Manchu Autonomous County (Liaoning Province, China); however, not much is known about the compounds (especially saponins) in Shizhu ginseng. Methods A rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with MS/MS (UHPLC–MS/MS) method was developed to separate and identify saponins in Shizhu ginseng. Results The separation was carried out on a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phase under a gradient elution at 40°C. The detection was performed on a Micromass Quattro Micro API mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 31 saponins were identified or tentatively characterized by comparing retention time and MS data with related literatures and reference substances. Conclusion The developed UHPLC–MS/MS method was suitable for identifying and characterizing the chemical constituents in Shizhu ginseng, which provided a helpful chemical basis for further research on Shizhu ginseng.

Simultaneous determination of icariin, naringin and osthole in rat plasma by UPLC-MS/MS and its application for pharmacokinetic study after oral administration of Gushudan capsules.

A rapid, sensitive and selective ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of icariin, naringin and osthole in rat plasma. Plasma samples pretreatment involved a one-step liquid-liquid extraction with a mixture of ethyl acetate-methyl tert-butyl ether (3:1, ν/ν). The separation was performed on an ACQUITY UPLC™ BEH C18 column with a gradient mobile phase system of methanol and water. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM), with the transitions at m/z 513.3→366.8 (icariin), m/z 579.3→150.9 (naringin), m/z 245.1→189.0 (osthole) and m/z 237.1→194.1 (IS), respectively. A good linear response was observed over the concentration ranges of 1.06-424ng/ml, 2.10-525ng/ml and 1.05-1.05×10(3)ng/ml with lower limit of quantification (LLOQ) of 1.06, 2.10 and 1.05ng/ml for icariin, naringin and osthole, respectively. The intra- and inter-day precisions (R.S.D.) were within 14.3%, and the accuracy (R.E.) ranged from -4.1% to 4.6% at three quality control levels. The sensitive and selective method was applied to a pharmacokinetic study of icarrin, naringin and osthole in rats after oral administration of Gushudan capsule.

Simultaneous quantification of Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid from Herba Siegesbeckiae in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

A rapid and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16β,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1mm×100mm, 3.5μm) with isocratic elution using methanol-5mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0-25,000ng/mL for Kirenol, and 25.0-12,500ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from -10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.

Development of an UPLC–MS/MS method for simultaneous quantitation of 11 d-amino acids in different regions of rat brain: Application to a study on the associations of d-amino acid concentration changes and Alzheimer’s disease

There are significant differences in d-amino acid concentrations between healthy people and Alzheimers disease patients. In order to investigate the potential correlation between d-amino acids and Alzheimers disease, a simple and sensitive ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed. The method was applied to simultaneous determination of 11 d-amino acids in different regions of rat brain. Rat brain homogenates were firstly pretreated with protein precipitation procedure and then derivatized with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE]. Baseline separation of the derivatives was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm×50mm, 1.7μm). The mobile phase consisted of acetonitrile and water (containing 8mM ammonium hydrogen carbonate) and the flow rate was 0.6mLmin-1. The derived analytes were sensitively detected by multiple reaction monitoring in the positive ion mode. The lower limits of quantitation ranged from 0.06 to 10ngmL-1 with excellent linearity (r≥0.9909). The intra- and inter-day RSD were in the range of 3.6-12% and 5.7-12%, respectively. The recovery rate was 82.5%-95.3%. With this UPLC-MS/MS method, the 11 d-amino acids in hippocampus, cerebral cortex, olfactory bulb and cerebellum from Alzheimers disease rats and age-matched controls could be simultaneously determined. Compared with the normal controls, the concentrations of d-serine, d-alanine, d-leucine, and d-proline in hippocampus and cerebral cortex of Alzheimers disease rat brain were significantly decreased, while no differences in olfactory bulb and cerebellum of all the d-amino acids were observed. The different amounts and distribution of d-amino acids in brain between the two groups, which regulated by particular pathological changes of Alzheimers disease, would give new insights into further study in neuropathogenesis and provide novel therapeutic targets of Alzheimers disease.

Multi-components determination by single reference standard and HPLC fingerprint analysis for Lamiophlomis rotata Pill.

Abstract A validated HPLC method was developed to evaluate the quality of Lamiophlomis rotata Pill combining the multi-components analysis by single reference standard with HPLC fingerprint analysis. Five bioactive components (shanzhiside methyl ester, loganin, 8-O-acetylshanzhiside methyl ester, forsythoside B and luteolin-7-O-β-D-glucopyranoside) were selected as markers to control the quality of L. rotata Pill. The results revealed that the chromatographic fingerprint method coupled with multi-components analysis provides an effective and feasible way to determine the components in L. rotata Pill.

Simultaneous determination of shanzhiside methyl ester, 8-O-acetylshan- zhiside methyl ester and luteolin-7-O-β-d-glucopyranoside in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study after oral administration of Lamiophlomis rotata Pill

A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of shanzhiside methyl ester, 8-O-acetylshanzhiside methyl ester and luteolin-7-O-β-D-glucopyranoside of Lamiophlomis rotata Pill in rat plasma was developed and validated. After liquid-liquid extraction with n-butyl alcohol/ethyl acetate (70:30, v/v), analytes and paeoniflorin (internal standard, IS) were separated on an Acquity BEH UPLC C18 column (100 × 2.1 mm, 1.7 μm) with gradient elution at a flow rate of 0.2 mL/min. All calibration curves had good linearity (r>0.9929) over the concentration ranges of 1-1000 ng/mL for shanzhiside methyl ester and 8-O-acetylshanzhiside methyl ester, 0.3-150 ng/mL for luteolin-7-O-β-D-glucopyranoside. The intra- and inter-day precisions were all within 11.1% and the accuracy (relative error, RE%) all ranged from -13.6% to 5.3%. The method also guaranteed an acceptable selectivity, recovery and stability, which was successfully applied to a pharmacokinetic study of the three analytes in rats after oral administration of Lamiophlomis rotata Pill.

Simultaneous quantification of 10 saponins in Chinese Shizhu Panax by UPLC-ESI-MS.

A simple and rapid method was established and validated for the simultaneous quantification of 10 saponins, namely ginsenosides-Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Re, Rf and Notoginsenside R1, in Chinese Shizhu Panax by ultra performance liquid chromatography coupled with an electrospray mass spectrometry (UPLC-ESI-MS). In addition, the contents of the analytes in different parts of Chinese Shizhu Panax were also analysed. The results showed that the concentration of saponins had a reference to the different parts of Chinese Shizhu Panax. The established method could be used as a new analytical approach for assessment of the quantity of Chinese Shizhu Panax.

Osteoblastic Proliferative Activity of Extracts of Qing’e pill and its Disassembled Formulae

Qing’e pill is one of the famous traditional Chinese compound prescriptions used to treat bone diseases. It contains four botanical drugs, i.e., the cortex of Eucommia ulmoides Oliv., the fruit of Psoralea corylifolia L., the seed of Juglans regia L. and the rhizome of Allium sativum L. In this study, osteoblastic proliferation activity of extracts from Qing’e pill and its disassembled formulae was investigated with the osteoblast-like UMR106 cell line as a model. The extract of Qing’e pill, both alcohol and aqueous, stimulated cell proliferation in a dose-responsive manner. The proliferative activity of alcohol extract was more potent than that of the aqueous one with a maximal growth stimulation ratio (GSR) of 58.5% versus 38.8%. Eucommia ulmoides and Psoralea corylifolia produced a maximum proliferative promotion of 38.7% and 34.0%, respectively, when co-cultured with UMR106 cells. Neither Juglans regia nor Allium sativum showed a significant effect on osteoblastic proliferation. When Eucommia ulmoides and Psoralea corylifolia were combined, the stimulating action (GSR = 47.2%) became stronger than that of either individual drug. The enhancement effect was more marked when Juglans regia or both Juglans regia and Allium sativum were added. These results demonstrated the synergy of the four botanical drugs and the rationality of Qing’e pill prescription in a modern scientific way. This is the first time to study the stimulating osteroblastic proliferation effect of a traditional Chinese compound prescription on the basis of disassembled formulae.

Identification of the cytochrome P450 enzymes involved in the oxidative metabolism of trantinterol using ultra high-performance liquid chromatography coupled with tandem mass spectrometry

Trantinterol is a novel β2-adrenoceptor agonist used for the treatment of asthma. This study aimed to identify the cytochrome P450 enzymes responsible for the metabolism of trantinterol to form 4-hydroxylamine trantinterol (M1) and tert-butyl hydroxylated trantinterol (M2), which was achieved using the chemical inhibition study, followed by the metabolism study of trantinterol in a panel of recombinant CYPs, as well as the kinetic study with the appropriate cDNA-expressed P450 enzymes. A highly selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of M1 and M2. The inhibition study suggested that CYP2C19 and CYP3A4/5 were involved in the formation of M1 and M2, and CYP2D6 only contributed to the formation of M1. Assays with cDNA-expressed CYP enzymes further showed that the relative contributions of P450 isoforms were 2C19 > 3A4 > 2D6 > 2E1 for the formation of M1, and 3A4 > 2C19 > 2D6 for the formation of M2. The enzyme kinetic analysis was then performed in CYP2C19, CYP2D6 and CYP3A4. The kinetic parameters were determined and normalized with respect to the human hepatic microsomal P450 isoform concentrations. All the results support the conclusion that CYP3A4 and CYP2C19 are the major enzymes responsible for formation of M1 and M2, while CYP2D6 and CYP2E1 also engaged to a lesser degree. The results imply that potential drug–drug interactions may be noticed when trantinterol is used with CYP2C19 and CYP3A4 inducers or inhibitors, and we should pay attention to this phenomenon in clinical study.