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.

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 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.

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.

An LC-TOF /MS and LC-MS /MS method was established for the identification the related substances in ambrisentan.HPLC separation was carried out on an XBridge C18 column(4.6 mm ×150 mm,3.5 μm)with linear gradient elution using a mobile phase consists of acetonitrile,water and 0.15% formic acid.The structures of the related substances were identified by electrospray positive ESI high resolution TOF /MS and MS /MS spec-tra,and verified further through reference substances.Ambrisentan and its related substances can be separated under the established HPLC conditions.Ten related substances were detected and identified.The established method is useful for the identification of related substances in ambrisentan.The results obtained are valuable for its manufacturing process optimization and quality control.

To study the related substances in nicergoline, electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of the related substances. Triple quadrupoles tandem MS/MS was employed for the determination of the fragmentations of the parent ions. 16 related substances were detected and identified to be eight synthetic by-products and eight degradation products, by using impurity references matching, product mass spectra fragmentations elucidation, and verified further according to synthetic processes and stress testing results. The results obtained are valuable for nicergoline manufacturing process control and quality assurance.

The study aims to investigate different pharmacokinetic profilesof anthraquinones after oral administration of Rhei Radix et Rhizoma and Niuhuang Jiedu Tablets(NHJDT)in rats, respectively. Rats were administrated with 96 mg/kg of Rhei Radix et Rhizoma(1. 83 mg/kg of total anthraquinone, equivalent to 0. 28 mg/kg of rhein, 0. 30 mg/kg of emodin, 0. 81 mg/kg of chrysophanol, 0. 23 mg/kg of aloe-emodin and 0. 20 mg/kg of physcion)or 250 mg/kg of NHJDT(equal dose of total anthraquinone as Rhei Radix et Rhizoma, equivalent to 0. 33 mg/kg of rhein, 0. 38 mg/kg of emodin, 0. 71 mg/kg of chrysophanol, 0. 24 mg/kg of aloe-emodin and 0. 17 mg/kg of physcion), respectively. Followed by protein precipitation with methanol, the anthraquinones in plasma samples were determined by LC-MS/MS. The pharmacokinetic parameters were calculated by WinNonlin 7. 0. The cmaxof rhein were(121±103)and(474±251)μg/L, and the AUC0-twere(275±176)and(406±194)μg ·h/L for Rhei Radix et Rhizoma and NHJDT, respectively. The cmaxof chrysophanol isomer were(2 325±1 390)and(3 580±2 169)μg/L, and the AUC0-twere(8 170±2 661)and(8 856±4 023)μg ·h/L, respectively. Emodin in very low levels was only detected in rat plasma samples after oral gavage of NHJDT. The cmax, AUC and t1/2 of rhein, as well as Vd and CL of chrysophanol isomer were observed with a much increased degree in comparison with Rhei Radix et Rhizoma counterparts. However, much shorter tmaxwas found in NHJDT group. Therefore, NHJDT with co-existing components enhanced the absorption and influenced the pharmacokinetic behaviors of active ingredients in Rhei Radix et Rhizoma.

A sensitive and selective method was developed for the separation and characterization of related substances (RSs) in EVT-401 by hyphenated LC–MS techniques. Complete separation of the RSs was achieved with an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) by linear gradient elution using a mobile phase consisting of 0.2% formic acid solution, methanol and acetonitrile. EVT-401 was found to be susceptible to acid, alkaline and oxidative stresses, while relatively stable under photolytic and thermal dry stress conditions. Fourteen RSs including six process-related substances and eight degradation products were detected and identified in EVT-401 with positive ESI high-resolution TOF-MS analysis of their parent ions and the corresponding product mass spectra elucidation, and some of them were further verified by chemical synthesis and NMR spectroscopy. The specific LC–MS method developed for separation, identification and characterization of RSs is valuable for EVT-401 manufacturing process optimization and quality control.

A suitable liquid chromatography quadrupole time-of-flight mass spectrometric (LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on LiChrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed.