Association of 4p14 and 6q27 variation with Graves disease: a case-control study and a meta-analysis of available evidence.

BACKGROUND: The etiology of the Graves' disease (GD) is largely unknown. However, genetic factors are believed to play a major role. A recent genome-wide association study in a Han Chinese sample collection revealed two new Graves' disease (GD) risk loci within chromosome band 4p14 and 6q27. In this study, we aimed to investigate these associations with Weifang Han Chinese population of Shandong province and perform a meta-analysis of associations with GD. METHODS: A case-control study was conducted to investigate association of variation within 4p14 and 6q27 to GD susceptibility in Weifang Han Chinese population of Shandong province. SNP rs6832151 at chromosome 4p14 and SNP rs9355610 at chromosome 6q27 was selected for genotyping in 2,382 GD patients and 3,092 unrelated controls. SNP genotyping was performed using TaqMan Real-time PCR technique assays on ABI7900 platform. A meta-analysis was performed with the data obtained in the current sample-set and those available from prior studies. RESULTS: Association analysis revealed both rs6832151 located in 4p14 (odds ratio (OR) = 1.27, P Allelic = 1.48 × 10-9) and rs9355610 located in 6q27 (OR = 1.10, P Allelic = 1.04 × 10-2) was associated with GD susceptibility. By model of inheritance analysis, we found the recessive model should be preferred (P Recessive = 2.75 × 10-11) for rs6832151. The dominant model should be preferred (P Dominant = 7.15 × 10-3) for rs9355610, whereas analysis of recessive model showed no significant association (P Recessive = 0.13). Meta-analysis with the data of 10,781 cases and 16,304 controls obtained from present sample-set and those available from prior studies confirmed association of rs6832151 at 4p14 with GD susceptibility using a fixed model (OR = 1.27, 95% CI: 1.22 to 1.32; I2 = 0%). Meta-analysis with the data of 11,306 cases and 12,756 controls confirmed association of rs9355610 at 6q27 with GD susceptibility using a fixed model (OR = 1.18, 95% CI: 1.13 to 1.22; I2 = 41.2%). CONCLUSIONS: Our findings showed that chromosome 4p14 and 6q27 variants were associated with Graves' disease in Weifang Han Chinese population of Shandong province.

Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention.

Determination of trantinterol enantiomers in human plasma by high-performance liquid chromatography - tandem mass spectrometry using vancomycin chiral stationary phase and solid phase extraction and stereoselective pharmacokinetic application.

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human.

Simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study after administration of Reduning injection.

A simple, specific and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established and validated for simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma using puerarin as an internal standard (IS). Plasma samples were pretreated by a one-step direct protein precipitation procedure with acetonitrile after acidified using as little as 50 µL plasma. Chromatographic separation was performed on an Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm) at a flow rate of 0.2 mL/min by a gradient elution, using 0.2% acetic acid-methanol as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with negative ion mode. Calibration curves showed good linearity (r > 0.995) over wide concentration ranges. The intra- and inter-day precisions were <15%, and the accuracy was within ±8.0%. The validated method was successfully applied to a pharmacokinetic study of the four bioactive components in rats after intravenous administration of Reduning injection.

Comparative pharmacokinetic study of four major components after oral administration of pure compounds, herbs and Si-Ni-San to rats.

The pharmacokinetic differences of paeoniflorin, naringin, naringenin and glycyrrhetinic acid (GA) following oral administration of pure compounds, single herbs and Si-Ni-San (SNS) decoction to rats were studied. Blood samples were analyzed with a validated UPLC-MS/MS method. Student's t-test was used for the statistical comparison. The Cmax and AUC0-∞ were 1470±434 ng/mL and 4663±916 ng h/mL for paeoniflorin, 64.29±59.21 ng/mL and 311.8±131.8 ng h/mL for naringin, 244.2±138.8 ng/mL and 4761±3167 ng h/mL for naringenin, and 1183±294 ng/mL and 38 994±14 377 ng h/mL for GA after oral administration of paeoniflorin, naringin and glycyrrhizic acid. The Cmax and AUC0-∞ were 812.6±259.6 ng/mL and 2489±817 ng h/mL for paeoniflorin, 344.3±234.9 ng/mL and 1479±531 ng h/mL for naringin, 981.9±465.4 ng/mL and 12 284±6378 ng h/mL for naringenin, and 3164±742 ng/mL and 78 817±16 707 ng h/mL for GA after oral administration of SNS decoction. There were significant differences between the pharmacokinetic behavior after oral administration of SNS decoction compared with pure components or herbs. The results indicated that some components in the other herbs of SNS had a pharmacokinetic interaction with paeoniflorin, naringin, naringenin and GA.

Structure identification and elucidation of mosapride metabolites in human urine, feces and plasma by ultra performance liquid chromatography-tandem mass spectrometry method.

1. Mosapride citrate (mosapride) is a potent gastroprokinetic agent. The only previous study on mosapride metabolism in human reported one phase I oxidative metabolite, des-p-fluorobenzyl mosapride, in human plasma and urine using HPLC method. Our aim was to identify mosapride phase I and phase II metabolites in human urine, feces and plasma using UPLC-ESI-MS/MS. 2. A total of 16 metabolites were detected. To the best of our knowledge, 15 metabolites have not been reported previously in human. 3. Two new metabolites, morpholine ring-opened mosapride (M15) and mosapride N-oxide (M16), alone with one known major metabolite, des-p-fluorobenzyl mosapride (M3), were identified by comparison with the reference standards prepared by our group. The chemical structures of seven phase I and six phase II metabolites of mosapride were elucidated based on UPLC-MS/MS analyses. 4. There were two major phase I reactions, dealkylation and morpholine ring cleavage. Phase II reactions included glucuronide, glucose and sulfate conjugation. The comprehensive metabolic pathway of mosapride in human was proposed for the first time. 5. The metabolites in humans were compared with those in rats reported previously. In addition to M10, the other 15 metabolites in humans were also found in rats. This result suggested that there was little qualitative species difference in the metabolism of mosapride between rats and humans. 6. In all, 16 mosapride metabolites including 15 new metabolites were reported. These results allow a better understanding of mosapride disposition in human.

Kidney tissue targeted metabolic profiling of glucocorticoid-induced osteoporosis and the proposed therapeutic effects of Rhizoma Drynariae studied using UHPLC/MS/MS.

Traditional Chinese medicine and modern science have indicated that there is a close relationship between bone and kidney. In light of this, this project was designed to study the metabolic profiling by UHPLC/MS/MS of glucocorticoid-induced osteoporosis in kidney tissue and the possible therapeutic effects of Rhizoma Drynariae (RD), a classic traditional Chinese medicine, in improving the kidney function and strengthening bone. Twenty-one Wistar rats were divided into three groups: control group (rats before prednisolone inducing), a model group (prednisolone-induced group) and a treatment group (prednisolone-induced rats that were then administered RD ethanol extracts). By using pattern recognition analysis, a significant change in the metabolic profile of kidney tissue samples was observed in the model group and restoration of the profile was observed after the administration of RD ethanol extracts. Some significantly changed biomarkers related to osteoporosis such as sphingolipids (C16 dihydrosphingosine, C18 dihydrosphingosine, C18 phytosphingosine, C20 phytosphingosine), lysophosphatidycholines (C16:0 LPC, C18:0 LPC) and phenylalanine were identified. As a complement to the metabolic profiling of RD in plasma, these biomarkers suggest that kidney damage, cell cytotoxicity and apoptosis exist in osteoporosis rats, which is helpful in further understanding the underlying process of glucocorticoid-induced osetoporosis and the suggested therapeutic effects of RD. The method shows that tissue target metabonomics might provide a powerful tool to further understand the process of disease and the mechanism of therapeutic effect of Chinese medicines.

A dispersive liquid-liquid microextraction method based on the solidification of a floating organic drop combined with HPLC for the determination of lovastatin and simvastatin in rat urine.

A dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with HPLC was developed for the determination of lovastatin and simvastatin in rat urine for the first time. 1-Dodecanol and methanol were used as the extraction and disperser solvents, respectively. Several important parameters influencing the micro-extraction efficiency were studied and systematically optimized, including the type and volume of extraction solvent and disperser solvent, extraction time, pH and salt concentration. The analytes were separated on a Kromasil C18 column at 30°C with a mobile phase of methanol and 0.2% acetic acid in water (83:17, v/v) and detected at 238 nm. Under the optimal conditions, the maximum number of enrichment factors for both analytes was 27. The linear ranges were 20.08-1004 and 20.00-1000 µg/L with the correlation coefficients ranging from 0.9990 to 0.9994 for lovastatin and simvastatin, respectively. The volume of organic solvent consumed in extraction was <0.3 mL, and the extraction time was 10 min. The newly developed environment-friendly sample pretreatment method will be a good alternative to conventional techniques, such as solid-phase extraction, liquid-liquid extraction and protein precipitation, for the HPLC determination of lovastatin and simvastatin in biological samples.

In vivo and in vitro metabolism of a novel ß2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR.

Trantinterol is a novel ß(2)-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.

Bidirectional chiral inversion of trantinterol enantiomers after separate doses to rats.

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new ß2 agonist, were studied in rats dosed (+)- or (-)-trantinterol separately. Plasma concentrations of (+)- and (-)-trantinterol were measured by chiral stationary phase liquid chromatography tandem mass spectroscopy (LC-MS/MS). The apparent inversion ratio was calculated as the ratio of AUC0-t of (-)-trantinterol or (+)-trantinterol inverted from their antipodes to the sum of the AUC0-t of (-)- and (+)-trantinterol. Following single intravenous administration, both given enantiomers declined in similar plasma concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics by the route of intravenous administration. However, after single oral administration, plasma concentrations of uninverted (-)-trantinterol at many timepoints were significantly higher than those of uninverted (+)-trantinterol, suggesting that the two enantiomers undergo apparently different absorption or metabolism after oral administration. Significant bidirectional chiral inversion occurred after intravenous and oral administration of (+)- or (-)-trantinterol. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated in vivo. The AUC0-36 of (+)-trantinterol after intravenous and oral dosing of (-)-trantinterol were 16.6 ± 5.2 and 33.3 ± 16%, respectively of those of total [(+) + (-)] trantinterol. The AUC0-36 of (-)-trantinterol after intravenous and oral dosing of (+)-trantinterol were 19.6 ± 8.8 and 37.9 ± 4.5%, respectively, of those of total [(-) + (+)] trantinterol. After intravenous administration of (+)- and (-)-trantinterol the chiral inversion ratios of the two enantiomers were not significantly different and similar results were found for oral administration. The extent of chiral inversion after intravenous administration was apparently lower, indicating that the bidirectional chiral inversion was not only systemic but also presystemic.

Development and validation of UPLC-MS/MS method for determination of domperidone in human plasma and its pharmacokinetic application.

A sensitive, rapid and selective ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination and pharmacokinetic study of domperidone in human plasma. Diphenhydramine was used as the internal standard. Plasma sample pretreatment involved a one-step liquid-liquid extraction with a mixture of diethyl ether-dichloromethane (3:2, v/v). The analysis was carried out on an Acquity UPLC(TM) BEH C(18) column. The mobile phase consisted of methanol-water containing 10 mmol/L ammonium acetate and 0.5% (v/v) formic acid (60:40, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionizationsource with positive mode. Each plasma sample was chromatographed within 2.1 min. The standard curves for domperidone were linear (r(2) ≥ 0.99) over the concentration range of 0.030-31.5 ng/mL with a lower limit of quantification of 0.030 ng/mL. The intra- and inter-day precision (relative standard deviation) values were not higher than 13% and accuracy (relative error) was from -7.6 to 1.2% at three quality control levels. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration.

Simultaneous quantification of metformin and glipizide in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A selective, sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one-step protein precipitation of 200 µL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol-10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10-656 ng/mL for metformin and 2.55-408 ng/mL for glipizide. The relative standard deviation of intra-day and inter-day precision was below 10% and the relative error of accuracy was between -7.0 and 4.6%. The presented HPLC-MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration.

Determination of L-trantinterol in rat plasma by using chiral liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid method for determination of L-trantinterol in rat plasma was developed for the first time by using LC coupled to MS/MS based on chiral stationary phase. A baseline separation of the enantiomers of trantinterol was achieved on a Chirobiotic V column, using a mixture of acetonitrile-methanol-ammonia-acetic acid (80:20:0.01:0.02, v/v/v/v) as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via ESI. The calibration curve was linear in concentration range from 0.270 to 108 ng/mL in plasma with the lower limit of quantification of 0.270 ng/mL. The intra- and interday precision (relative standard deviation) values were within 10.9% and the accuracy (relative error) was from 2.6 to 9.2% at all quality control levels. The method has been successfully applied to a study of L-trantinterol pharmacokinetics in rats.

Quantitative determination of lisinopril in human plasma by high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine lisinopril in human plasma. Sample pretreatment involved a one-step protein precipitation with methanol of 0.1 mL plasma. Analysis was performed on an Inertsil ODS-3 column (2.1 × 50 mm i.d., 3 µm) with mobile phase consisting of methanol-water (containing 0.2% formic acid; 55:45, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via an electrospray ionization source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for lisinopril were obtained in the concentration range of 1.03-206 ng/mL (r(2) ≥ 0.99) with a lower limit of quantification of 1.03 ng/mL. The intra- and inter-day precisions (relative standard deviation) were not higher than 11%, and accuracy (relative error) was within ±6.8%, determined from quality control samples for lisinopril, which corresponded to the guidance of the Food and Drug Administration. The method described herein was fully validated and successfully applied to the pharmacokinetic study of lisinopril tablets in healthy male volunteers after oral administration.

[Effect of phenformin hydrochloride on pharmacokinetics of puerarin in rats].

OBJECTIVE: To study the effect of phenformin hydrochloride that may be illegally added in traditional Chinese medicine preparations on the pharmacokinetics of puerarin in rats. METHOD: Rats were randomly divided into the single pueraria group and the phenformin hydrochloride combined with pueraria group. After oral administration in the two groups, their bloods were sampled at different time points to determine the drug concentration of puerarin in rat blood and calculate pharmacokinetic parameters. RESULT: After oral administration with pueraria extracts and phenformin hydrochloride combined with pueraria extracts, the two groups showed main pharmacokinetic parameters as follows: Cmax were (2.39 +/- 1.01), (1.03 +/- 0.35) mg x L(-1), respectively; Tmax were (0.50 +/- 0.09), (1.5 +/- 0.5) h, respectively; Ke were (0.153 +/- 0.028), (0.172 +/- 0.042) h(-1), respectively; t(1/2) were (4.65 +/- 0.86), (4.20 +/- 0.81) h, respectively; AUC(0-t), were (5.73 +/- 2.60), (5.45 +/- 1.81) mg x h x L(-1), respectively; AUC(0-infinity) were (6.72 +/- 2.89), (6.26 +/- 1.88) mg x h x L(-1), respectively. Compared with the single puerarin group, the Cmax was significantly decreased (P < 0.05) and the Tmax was markedly longer (P < 0.01) than the hydrochloride combined with pueraria group. CONCLUSION: Phenformin hydrochloride can slow down the absorption process of puerarin and change the pharmacokinetic process of puerarin to some extent.

[Study on in vitro and in vivo material base of Sini San by UPLC-PDA-MS/MS].

OBJECTIVE: To analyze chemical constituents of Sini San its migrating components in rat plasma and study its in vitro and in vivo material base using ultra-performance liquid chromatography coupled with photo-diode-array detector and tandem mass spetrometry (UPLC-PDA-MS/MS). METHOD: ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 microm) was adopted, with gradient elution system of water containing 2 mmol x L(-1) ammonium acetate and acetonitrile at flow rate of 0.2 mL x min(-1). The column temperature was maintained at 35 degrees C. The mass spectra were obtained by electrospray ionization source operating in both positive and negative ion mode. Ions were scanned from the m/z 100 to 1 000, and the characteristic ions were schizolysised twice to obtain the secondary MS data. RESULT: Twenty chemical constituents were detected, including paeoniflorin, glycyrrhizic acid, saikosaponins a and naringin. In vivo, there were 8 ingredients directly absorbed into blood after the administration of Sini San decoction, such as paeoniflorin, naringin and hesperidin. Besides, 6 metabolites were also detected, involving glucuronides, sulfate and sulfoglucuronides. CONCLUSION: In vitro and in vivo chemical materials of Sini San decoction is analyzed by UPLC-PDA-MS/MS to reflect in vitro and in vivo material base of Sini San decoction in a comprehensive and rapid manner and provide basis for further study on efficacious material basis of Sini San decoction.

[Metabonomic study on protective effect of ethanol extracts of drynariae rhizoma on osteoporosis in rats urine by using UPLC-MS/MS].

This paper was designed to study metabonomic characters of the osteoporosis induced by high dose of hydrocortisone and the protective effects of Drynariae Rhizoma, which can replenish the kidney and strengthen the bones. A urinary metabonomics method based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) was developed. Clear separation of healthy control group, model group and treatment group was achieved by using the principal components analysis (PCA) and 9 significantly changed metabolites were identified as potential biomarkers of osteoporosis. Compared with the health control group, the model group rats showed lower levels of creatinine, citric acid, azelaic acid, hippurate, tryptophan and indoxyl sulfate together with higher levels of phenylalanine, cresol sulfate and phenaceturic acid. These changes in urinary metabolites suggest that the disorders of amino acid metabolism, energy metabolism, gut microflora and anti-oxidative damage are related to osteoporosis induced by high dose of hydrocortisone and the potential effect of Drynariae Rhizoma on all the four metabolic pathways.

Antibacterial polyketides from the jellyfish-derived fungus Paecilomyces variotii.

Four new polyketides (1-4) were isolated from the fungus Paecilomyces variotii, which was derived from the jellyfish Nemopilema nomurai. The planar structures and relative configurations of these polyketides were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. The compounds showed inhibitory activity against pathogenic bacteria including methicillin-resistant Staphylococcus aureus 3089 and multi-drug-resistant Vibrio parahemolyticus 7001 with MIC values in the range 5-40 µg/mL.

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution.

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl(3)-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl(3)-MeOH-0.2 M HCl (4:2:2, v/v) and CHCl(3)-MeOH-0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of (1)H-NMR, (13)C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

Development and validation of a reliable high-performance liquid chromatographic method for determination of nodakenin in rat plasma and its application to pharmacokinetic study.

A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed for the determination of nodakenin in rat plasma. The concentration of nodakenin was determined in plasma samples after deproteinization with methanol using hesperidin as internal standard. HPLC analysis was performed on a Diamonsil C(18) analytical column using acetonitrile-water (25:75, v/v) as the mobile phase and a UV detection at 330 nm. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day variation). The extraction recoveries were 91.3 ± 10, 87.8 ± 4.8 and 92.6 ± 5.1 at concentrations of 0.500, 5.00 and 40.0 µg/mL, respectively. The standard curve for nodakenin was linear (r(2) ≥ 0.99) over the concentration range 0.250-50.0 µg/mL with a lower limit of quantification of 0.250 µg/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were not higher than 12% and the accuracy (relative error, RE) was within ± 5.8% at three quality control levels. The validated method was successfully applied for the evaluation of the pharmacokinetics of nodakenin in rats after oral administration of Rhizoma et Radix Notopterygii decoction and nodakenin solution.