•Eleven impurities of tofacitinib were identified by LC-MS technology.•Two process-related substances and one degradation product were synthesized and confirmed by NMR.•Synthesis optimization was proposed and the process-related impurities were minimized or eliminated from the bulk drug.•The degradation pathway was studied and appropriate storage condition was suggested.A specific LC-MS method was developed for separation, identification and characterization of the process-related substances and degradation products in tofacitinib citrate. The separation was achieved on a LiChrospher C18 column (250 mm × 4.6 mm, 5 μm) by linear gradient elution of 0.1% ammonium acetate solution (pH adjusted to 4.0 by formic acid) and acetonitrile at a flow rate of 1.0 mL/min. Forced degradation studies were conducted under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions as described in ICH. It was found that tofacitinib was stable under photolytic condition, but degraded obviously in acidic, basic, thermal and oxidative conditions. The high resolution TOF-MS and MS/MS were used for determination and structural identification of the related substances. Eleven major related substances were detected and identified as five process-related substances and six degradation products, and three of them were further synthesized and characterized by NMR spectroscopy. The most plausible mechanisms involved in the formation of the related substances were also proposed. Since related substances have a significant impact on drug safety, quality and efficacy, the data obtained are valuable for process monitoring and quality assurance of tofacitinib citrate.

•7 related substances were identified, and five of them had not been reported.•LC-QTOF mass spectrometric method was developed for the identification.•Two related substances were synthesized and confirmed by NMR.•The manufacturing processes of alogliptin and its tablets were optimized.A highly specific and efficient LC-QTOF mass spectrometric method was developed for the separation and characterization of process related substances and the major degradation products in alogliptin benzoate and its tablets. The separation was performed on Phenomenex Gemini-NX C18 column (250 mm × 4.6 mm, 5 μm) using 0.2% formic acid-0.2% ammonium acetate in water as mobile phase A, acetonitrile and methanol (60:40, v/v) as mobile phase B in linear gradient elution mode. Forced degradation studies were also conducted under ICH prescribed stress conditions. Alogliptin benzoate and its tablets were tending to degrade under acid, alkaline, oxidative and thermal stresses, while relatively stable to photolytic stress. A total of seven related substances were detected and characterized through liquid chromatography-high resolution QTOF mass spectrometry techniques, including process related substances and degradation products, and two of them were further synthesized and characterized by NMR spectroscopy. Based on the related substances elucidation and the plausible formation mechanisms, efficient approaches were proposed to reduce or eliminate related substances, and in consequence the quality of alogliptin benzoate and its tablets have been promoted obviously. Therefore, the impurity profiles obtained are critical to the quality control and manufacturing processes optimization and monitoring of alogliptin benzoate and its tablets.

A high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC–HG–AFS) method was developed for the simultaneous determination of four arsenic species (As(III), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate As(V)) in dog plasma. Good separation of the four arsenic species was achieved within 15 min on an anion-exchange column with isocratic elution using 15 mmol/L KH2PO4 (pH 5.9) as eluent at a flow rate of 1.0 mL/min. The assay was linear over the range of 1.25–200, 1.56–200, 1.34–172, and 2.50–200 ng/mL with the detection limits of 0.80, 1.00, 0.86 and 2.00 ng/mL for As(III), DMA, MMA and As(V), respectively. The method was validated for selectivity, precision, accuracy and recovery and then applied to a comparative pharmacokinetic study of the arsenic species in beagle dogs after a single oral administration of Realgar (24.32 mg/kg, equivalent to 11.31 mg As/kg) alone or Niu Huang Jie Du Pian (a patent traditional Chinese medicine (TCM), 380 mg/kg, equivalent to 28.45 mg As/kg), respectively. DMA was found to be the predominant species in the dog plasma after dosing, with As(V) appeared as the quickly eliminating one. No traces of MMA and As(III) were detected at any sampling time points. The main pharmacokinetic parameters found for DMA p.o. administration of Realgar and Niu Huang Jie Du Pian were as follows: Cmax (14.7 ± 4.2) and (57.0 ± 32.0) ng/mL, Tmax (2.4 ± 0.5) and (2.5 ± 0.5) h, AUC0–36 (151.1 ± 12.9) and (635.9 ± 418.2) ng h/mL, AUC0–∞ (206.0 ± 44.5) and (687.2 ± 425.1) ng h/mL, t1/2 (16.2 ± 7.9) and (9.4 ± 2.2) h, respectively. The influence of compounding in Niu Huang Jie Du Pian on the pharmacokinetics of arsenics was shown with increased transformation of DMA and its faster elimination rate.Highlights► An HPLC–HG–AFS method for determination of 4 arsenic species in dog plasma was developed. ► The comparative pharmacokinetic study of arsenic species was carried out after oral administration of Realgar and NHJDP. ► DMA and As(V) were found to be the main arsenic species in dog plasma after oral administration of both Realgar and NHJDP. ► The concentration–time profiles and pharmacokinetic parameters of DMA in beagle dogs were presented.

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.

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.