The study aims to identify the related substances in fasudil hydrochloride by hyphenated techniques. A WondaSil C18 (250 mm x 4.6 mm, 5 microm) column was used for the separation of the related substances with a mixture of methanol and ammonium acetate buffer solution as the mobile phase by gradient elution. The structures of the related substances were speculated by electrospray positive ionization LC-TOF/MS accurate ion mass and MS/MS determination and elucidation, and verified further through synthesis and spectroscopic analysis. Fasudil hydrochloride and the related substances were separated under the established HPLC condition. Three related substances in fasudil hydrochloride were characterized by hyphenated techniques. The hyphenated LC-MS method is useful for the identification of related substances in fasudil hydrochloride and the results obtained are valuable for its manufacturing process and quality control.

Objective To isolate and elucidate the structures of alkaloids in Huangyangning. Methods Alkaloids of Huangyangning were separated with preparative HPLC. The molecular structures were elucidated on the basis of chemical evidences and spectral analyses (UV, IR, MS, 1H-NMR, 13C-NMR, COSY, DEPT, HMQC, and HMBC). Results Cyclovirobuxine D is the major component in Huangyangning and cyclobuxine D and cyclovirobuxine C are the two related alkaloids. Conclusion It is demonstrated that all the Huangyangning alkaloids have the same structural frame with only minor differences in substitution through chromatographic and spectral analyses. Therefore, it is not easy to purify cyclovirobuxine D by using usual column, re-crystallization, or chemical approaches for the existence of the related alkaloids.

Object To develop a new method for the determination of curcumol in essential oil from rhizoma Curcumae L.. Methods The contents of curcumol were determined by high performance capillary gas chromatography with sequential increase of temperature on a HEWLETT PACKARD 5890A gas chromatograph. Results The method can be used to determine curcumol with accuracy at a recovery of 101.4% and RSD of 0.40%. Conclusion The present study provided a satisfactory method for the determination of curcumol, and it was found that its contents in four different species (C. wenyujin, C. longa, C. aeruginose, and C. kwangsiensis) were markedly different.

Object To establish the standardization and digit ization methods for gas chromatographic fingerprint chromatograms of the essenti al oil of Curcuma longa L. Methods A polynomial regression analysis technique was estab lished for the calculation and prediction of the gas chromatographic retention i ndices by using a series of normal aliphatic hydrocarbons as the reference stand ards. And it was used for the characterization of the features of the gas chroma tographic fingerprint spectra of the essential oil of C. longa. Results It was approved that retention indices of the gas chrom atographic fingerprint spectra obtained at a variety of conditions were stable and reliable with excellent reproducibility, and fairly good ruggedness. It was also much better than the relative retention time indices. Conclusion The fingerprint spectra standard established on t he multiple references basis are much more reasonable and useful for the practic al quality assurance and validation of Chinese herbals.

Objective To establish a stable and reliable HPLC method and fingerprint reference substance for the measurement of the fingerprint, the practical quality control, and assay of the water-soluble components of Salvia miltiorrhiza. Methods The HPLC was run on C 18 columns with methanol-1.0 % glacial acetic acid solution as mobile phase in gradient elution mode at a flow rate of 1.0 mL/min. The chromatographic system suitability, the gradient elution mode, mobile phase acidity, and the effect of column type on fingerprint repeatability were tested. Results The HPLC fingerprints of the water-soluble extract of reference S. miltiorrhiza were obtained with very good resolution under the established chromatographic system. Fifteen peaks in the chromatograms were selected for the fingerprint identification and quality control of S. miltiorrhiza. The quality of ten batches of S. miltiorrhiza samples from different hibitats were assessed by comparing their chromatographic fingerprints with the reference fingerprints obtained at the same time, and the similarity showed no difference, eventhough the column filler was changed. Conclusion Because the inherent complexity of medicinal material components has been reflected by chromatographic fingerprints, there are many factors affecting the fingerprint repeatability for the changes of column type. The results of the quality assessment of S. miltiorrhiza, using fingerprints from different columns, are not all coincidence. In order to obtain the comparable and repeatable results in different laboratories, it is much practical with both a defined chromatographic system suitability and a fingerprint reference substance.

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC ? MS/MS) method was established to determine 2-oxo-clopidogrel, a crucial intermediate metabolite in human plasma. A chromatographic separation was performed on a Sapphire C18 column following a liquid–liquid extraction sample preparation with methyl t-butyl ether. Detection was carried out on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) with an electrospray ionization (ESI) mode. The method was validated in terms of specificity, accuracy, precision and limit of quantification. The calibration curves ranged from 0.50 to 50.0 ng/mL with good linearity. The stability was fully validated with addition of 1,4-dithio-DL-threitol (DTT) into the plasma sample prior to and in the preparation procedure. The validated method was proved to be suitable for use in pharmacokinetic study after single oral administration of 75 mg clopidogrel tablets in human subjects, which could make contribution to intensive study of the clinical drug–drug interactions of clopidogrel and individual treatment.

A high performance liquid chromatography-electrospray ionization mass spectrometry- charged aerosol detection ( HPLC-MS-CAD) method was established for the simultaneous quantitative analysis of four Lignans in Magnoliae Flos extract. The components were separated on a YMC-Pack ODS-A column (250 mm× 4. 6 mm, 5 μm) by gradient elution with methanol and water as the mobile phase at aflow rate of 1. 0 mL/min. Then the elution solution was routed into MS equipment at a flow rate of 0. 3 mL/min and CAD detector at a flow rate of 0. 7 mL/min by a split ratio of 3:7 for the further detection. The column temperature was 25 ℃ and the detection wavelength was 278 nm. A method was developed for the quantitative analysis of muti-components by single maker ( QAMS) to determine pinoresinol dimethylether, magnoli, 1irioresinol B dimethylethe and epi-magnoli A . Magnoli was selected as internal standard and the relative correction factors ( RCF) of the four Lignans were calculated. The contents of the four Lignans in Magnoliae Flos extract were determined by both external standard method and QAMS. The QAMS method was evaluated by comparison of its assay result and that of external standard method. Under the selected chromatographic condition, the limits of detection of pinoresinol dimethylether, magnoli, lirioresinol B dimethylethe and epi-magnoli A were 0. 34, 0. 55, 0. 50 and 0. 58 mg/L, respectively, while the linear range were within 6. 8-270 mg/L, 11-546 mg/L, 2. 0-101 mg/L and 2. 3-116 mg/L. The recoveries ( n=9 ) were 98. 2%-99. 5%, and the correlation coefficient were 0 . 9995-0 . 9998 . No significant differences were found between the quantitative results of external standard method and QAMS method. The developed method is accurate, feasible, and can be used for quality evaluation of Magnoliae Flos .

After oral administration of Salvia miltiorrhiza (Danshen in Chinese), Panax notoginseng (Sanqi in Chinese) and Danshen Sanqi combination suspensions to Beagle dogs, the plasma concentration-time profiles of danshensu, tanshinone II(A), cryptotanshinone, notoginsenoside R1, ginsenoside Rg1 and Rb1 were analyzed by LC-MS/MS. Pharmacokinetic parameters were calculated and analyzed with BAPP 2.0 software. The results showed that the Cmax and AUC of danshensu, notoginsenoside R1, ginsenoside Rg1 and Rb1 in Danshen Sanqi combination group all decreased in comparison with those of Danshen or Sanqi given alone, while the CLz/F and Vz/F increased to some extent. No significant differences of the pharmacokinetics of tanshinone II(A) and cryptotanshinone were observed between groups.

A selective precolumn derivatization liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated.Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid.Chromatographic separation was performed on a Phenomenex ODS column (150 mm × 4.6mm,5μm) using linear gradient elution by a mobile phase consisting of methanol (A),and an aqueous solution containing 0.2％ ammonium acetate and 0.1％ formic acid (B) at a flow rate of 1 mL/min.Tolterodine tartrate was used as the internal standard (IS).With protein precipitation by acetonitrile and then the simple one-step derivatization,a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma.The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012-8.27 μg/mL in plasma and 1.80-84.1 μg/mL in urine.The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500,1000 and 1500 mg of glucosamine sulfate,as well as multiple oral doses of 500 mg t.i.d.for 7 consecutive days.