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The incidence of drug-induced liver injury (DILI) is second only to viral hepatitis and steatohepatitis in China, and DILI has become a serious public health problem that cannot be ignored. Guri Gumu-13 pill (GRGM) is a traditional Chinese medicine (TCM), which has a protective effect on liver diseases. However, the underlying therapeutic mechanisms of GRGM for DILI are still vague. In this study, the protective effect of GRGM on acetaminophen (APAP)-induced DILI was investigated based on the proteomics clues. The effects of GRGM on APAP-induced DILI in rats were studied using tandem mass tag (TMT)-based quantitative proteomics technology. Besides, western blotting was exerted to verify related proteins. Using the TMT-based quantitative proteomics approach, 237 proteins were identified as regulated in APAP-induced DILI and 58 proteins were regulated by GRGM. The 17 overlapping differentially expressed proteins (DEPs) were identified, and 7 proteins were inversely regulated. Enrichment analysis of KEGG indicated that metabolic pathways, linoleic acid metabolism, and retinol metabolism might be affected in DILI. Next, Cyp2c11, Aldh1a1, and Fads2 were verified with molecular biotechnology. GRGM exerts therapeutic effects through multi-pathways regulation in the treatment of DILI. This work may provide proteomics clues for the continuation of research on DILI treatment with GRGM.

MP3950 is being developed as a gastroprokinetic candidate compound. To illustrate the pharmacokinetic profiles, absolute bioavailability after intravenous administration and oral administration with MP3950 as well as tissue distribution in vivo, an UPLC-MS/MS approach which was rapid and selective was developed to determine MP3950 in plasma and tissue of rat. Sample pre-treatment of plasma sample was one-step protein precipitation. 0.1% formic acid containing 5 mmol/L ammonium acetate-methanol(55/45,v/v) was used for isocratic elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm, 1.7 μm) to achieve the separation. The analysis was performed in MRM mode via positive ESI mode. LLOQ of the method was 10 ng/mL, and the linearity up to 10,000 ng/mL. The intra-day precision (relative standard deviation, RSD) was 4.0–9.0% and the inter-day precision was 4.2–10.6%. The accuracy (relative error, RE) was −1.2–2.4%. Tissue samples were collected from the brain, heart, liver, spleen, lung, kidney, stomach, duodenum, small intestine, large intestine, appendix and skeletal muscle. The same liquid chromatographic and mass spectrometric conditions were used, and it's proven that this method was feasible to analyze the MP3950 in tissues with good precision and accuracy over the range from 10 to 5000 ng·mL−1. It was found that the concentration of MP3950 is higher in digestive system. The tissue distribution, pharmacokinetic and bioavailability of MP3950 in rats were carried out by the method for the first time, which can provide enough information for the further development and investigation of MP3950.

Coenzyme Q10 (CoQ10) solid dispersion was prepared to improve its oral bioavailability due to the poor solubility of CoQ10. To evaluate the pharmacokinetic behaviors of CoQ10 solid dispersion, a simple, rapid, sensitive and environment friendly method for the determination of CoQ10 in rat plasma was developed. In this study, samples were prepared by one-step protein precipitation with acetone and then the supercritical fluid chromatography-electrospray ionization tandem mass spectrometry (SFC-ESI-MS/MS) method was used. The separation was achieved by an ACQUITY UPC(2)™ BEH 2-EP column (100mm×3mm, 1.7μm) maintained at 35°C, using carbon dioxide (≥99.99%) and methanol (85:15, v/v) as the mobile phase at a flow rate of 1.0ml/min. To improve the response and sensitivity, the compensation solvent of methanol with 2mM ammonium acetate at a flow rate of 0.2ml/min was used and the total analysis time was only 1.5min for each sample. The detection was carried out on a tandem mass spectrometer with electrospray ionization (ESI) source and the mass transition ion pair was m/z 881.0→197.0 and 285.1→193.0 for CoQ10 and diazepam, internal standard (IS), respectively. Calibration curve was linear over the concentration range of 2.00-500.00ng/ml (r(2)≥0.998) with a lower limit of quantification of 2.00ng/ml. The intra- and inter-day accuracy and precision were below 15% for all quality control samples. The proposed method was rapid, accurate and reproducible, which was suitable to compare the pharmacokinetic behaviors in rats after a single oral dose of 100mg/kg CoQ10 solid dispersion or tablets.

MP 3950 is an active metabolite of mosapride which exhibits high 5-HT4 receptor agonistic effect. The present paper describes an enantioselective chiral HPLC method for separation and quantification of MP 3950 enantiomers in rat plasma. The plasma samples were prepared by liquid-liquid extraction with ethyl acetate and the baseline chromatographic separation was achieved on a Chiralcel OJ-H column with a mobile phase consisting n-hexane/ethanol/methanol/diethylamine (85:5:10:0.4, v/v/v/v) at the flow rate of 1.0mL/min. The ultraviolet detection was performed at 276nm. The calibration curve was linear in the range from 0.100 to 5.00μg/mL with the lower limit of quantification of 0.100μg/mL for each enantiomer. The intra- and inter-day precisions were not more than 14.7%. The accuracy was from -6.4% to 14.0%. The validated method was successfully applied to chiral inversion and stereoselective pharmacokinetic study in rats after oral administration of MP 3950 racemate. The results indicated that no stereochemical inversion was occurred in rats. And the plasma concentrations and area under plasma concentration-time curve of (S)-MP 3950 were all significantly higher than those of (R)-MP 3950 in both male and female rats, which indicated that the disposition of MP 3950 in rats might be stereoselective.

Xiao-Chai-Hu-Tang (XCHT) has been proven to be effective for the clinical treatment of depression. However, the mechanisms of definite antidepressant-like effects and detailed metabolic biomarkers were still unclear in this prior study. Here, we have investigated the metabolic profiles and potential biomarkers in a chronic unpredictable mild stress model after treatment with XCHT. Metabonomics based on ultra-high performance liquid chromatography coupled with mass spectrometry was used to profile the metabolic fingerprints of serum obtained from a rat model with chronic unpredictable mild stress with and without XCHT treatment. The model rats showed a significant decrease in sucrose preference and food consumption, and these depression-like symptoms were significantly improved by XCHT. Through principal component analysis (PCA), nine potential biomarkers of tryptophan, uric acid, phenylalanine, cholic acid and lysophosphatidylcholine (C18:0 LPC, C16:0 LPC, C16:1 LPC, C18:1 LPC, C20:4 LPC) were characterized as potential biomarkers involved the pathogenesis of depression. The therapeutic effect of XCHT on depression may involve in amino acid metabolism, lipid metabolism, oxidative stress and inflammation response. The present investigation highlights that metabonomics is a valuable tool for studying the essence of depression as well as evaluating the efficacy of the corresponding drug treatment.

Gushudan, a Chinese compound formulation based on the theory of traditional Chinese medicine and desgined to treat osteoporosis. However, its intergated intervention effective mechanism in vivo is not well understood. In this study, an intergated serum and urinary metabonomic strategy based on UPLC-MS technique have been developed to increase the understanding of the metabolism characters of osteoporosis and to investigate the holistic therapeutic efficacy of Gushudan on prednisolone-induced osteoporosis rat model. Principle component analysis (PCA) was utilized to identify differences in metabolic profiles of rats in the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group and clear separation was achieved among three groups. Furthermore, 17 potential biomarkers from urine and 10 potential biomarkers from serum were identified, primiarily related to amino acid metabolism, energy metabolism, lipid metabolism, intestinal flora metabolism and kidney damage. Gushudan has therapeutic effects on rat with osteoporosis via the regulation of multiple metabolic pathways. It's worth mentioning that some new biomarkers associated with osteoporosis such as 3-methoxydopa, 2,8-digydroxyadenine have been discovered in this study for the first time. This study provides a useful approach to get insight into the intergated metabonomic mechanism of prednisolone-induced osteoporosis and to assess the efficacy of Gushudan on osteoporotic rats. The work also shows that the metabonomics method is a promising tool in the efficacy and mechanism research of traditional Chinese compound medicines.

Lovastatin (LOV) is an antihyperlipidemic agent which exhibits low bioavailability due to its poor solubility. Therefore, a nanosuspension (NS) was developed as an efficient strategy to improve its oral bioavailability. To evaluate the pharmacokinetics of LOV-NS, a novel, sensitive, and rapid UPLC-MS/MS method was developed and validated for the simultaneous determination of LOV and its metabolite lovastatin acid (LOVA) in rat plasma. Simvastatin (IS) was chosen as the internal standard, and a liquid-liquid extraction method was used to isolate LOV and LOVA from biological matrices. The analytes were analyzed on an Acquity UPLC BEH C18 column, and a gradient program was applied at a flow rate of 0.2mL/min. Then, a tandem quadrupole mass spectrometer coupled with a positive/negative ion-switching electrospray ionization interface was employed to detect the analytes. Quantitation of the analytes was performed in the multiple reaction monitoring mode to monitor the transitions of m/z 427.1→325.0 for LOV and m/z 441.1→325.0 for IS in the positive ion mode and m/z 421.0→101.0 for LOVA in the negative ion mode, respectively. The method was validated over the concentration range 0.25-500ng/mL (r(2)≥0.99) for both LOV and LOVA. The intra-day and inter-day precision (RSD%) of LOV and LOVA were less than 12.87% and the accuracy (RE%) was less than 5.22%. The average extraction recoveries were 90.1% and 91.9% for LOV and LOVA, and the matrix effects were found to be between 85% and 115%. The stability study showed that both analytes were stable during the experiment. Finally, this method has been successfully applied to a pharmacokinetic study in rats following a single oral dose of 10mg/kg LOV-NS.

A novel pretreatment method involving microwave-assisted extraction and solid-phase purification combined with dispersive liquid-liquid microextraction (MAE-SPP-DLLME) followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established for the simultaneous determination of six antibacterial pharmaceuticals including metronidazole, tinidazole, chloramphenicol, thiamphenicol, malachite green and crystal violet. The conditions of MAE were optimized using an orthogonal design and the optimal conditions were found to be 8mL for acetonitrile, 50°C for 5min. Then, neutral alumina column was employed in the solid-phase purification. Finally, the critical parameters affecting DLLME, including selection of extraction and dispersive solvent, adjustment of pH, salt concentration, extraction time, were investigated by single factor study. Under optimum conditions, good linearities (r>0.9991) and satisfied recoveries (Recoveries>87.0%, relative standard deviation (RSD)

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.

Cortex Juglandis Mandshuricae is used as a folk remedy for treating cancer, diarrhea and dysentery in traditional Chinese medicine for many years. Six flavonoids (myricitrin, quercitrin, taxifolin, myricetin, quercetin and naringenin), gallic acid and 5,8-dihydroxy-1,4-naphthoquinone are major bioactive components in Cortex Juglandis Mandshuricae extract. In this study, an ultrahigh performance liquid chromatography and tandem mass spectrometry method was developed for simultaneous determination of eight ingredients in rat plasma using chloromycetin as an internal standard. Plasma samples added vitamin C (antioxygen) were acidified with hydrochloric acid and extracted by liquid–liquid extraction with ethyl acetate. Eight ingredients were separated on a Venusil ASB C 18 column and detected by multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9900. The validated lower limit of quantification was 20 ng/mL for gallic acid, 5 ng/mL for myricitrin, 3 ng/mL for quercitrin, 10 ng/mL for taxifolin, 6 ng/mL for myricetin, 3 ng/mL for quercetin, 2 ng/mL for naringenin and 1 μg/mL for 5,8-dihydroxy-1,4-naphthoquinone, respectively. Intra- and inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −6.9% to 6.9%. The validated method was successfully applied to investigate the pharmacokinetics of the eight analytes after oral administration of Cortex Juglandis Mandshuricae extract to rats.

A simple, novel, rapid and sensitive supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS) method was developed and validated for the determination of lacidipine in beagle dog plasma with nimodipine as internal standard. The method involved a simple liquid–liquid extraction method with tert-butyl methyl ether. The analytes were analyzed on an Acquity UPC 2 with a HSS C 18 SB column (3 mm × 100 mm, 1.8 μm) set at 50 °C. The mobile phase was carbon dioxide (≥99.99%) and methanol (92:8, v/v) at a flow rate of 2 ml/min, the compensation solvent was methanol with 2% formic acid at a flow rate of 0.2 ml/min and a total analysis time of 1.5 min for each sample. The multiple reaction-monitoring mode was used for quantification of ion transitions at m / z 473.32 → 354.10 and 419.00 → 343.10 for lacidipine and internal standard, respectively. The linearity range of proposed method was 0.10–100 ng/ml) ( r 2 ≥ 0.9990). The intra- and inter-day precision values were less than 15% and accuracy was from −0.83% to 3.27% at all quality control levels. The proposed method was successfully applied to a pharmacokinetic study of lacidipine in beagle dogs.

A liquid chromatography hybrid ion trap time-of-flight mass spectrometric (LC-IT-TOF-MS) method was developed and validated for identification and simultaneous determination of the potential bioactive components from green tea in rat plasma. The plasma samples were extracted by liquid-liquid extraction with ethyl acetate and separated on Shim-pack XR-ODS II column by a gradient elution within a runtime of 8.0 min. The mobile phase consisted of A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) at a flow rate of 0.4 ml/min. Two prototype components and one metabolite were successfully identified as caffeine, theobromine and theophylline according to their retention times, accurate molecule weight, and major fragment ions. Then they were determined with the addition of two internal standards, hypoxanthine and paracetamol. The linear range was 10-10,000 ng/ml for caffeine, 2.0-2000 ng/ml for theobromine and 1.0-1000 ng/ml for theophylline, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, and accuracy was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to investigate the dynamic change rules of caffeine, theobromine and theophylline in rat plasma after oral administration of caffeine, theobromine and green tea extract. The comparative analysis of the pharmacokinetic parameters indicated that there were obvious differences between green tea extract administration and single substances administration.