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Oleic acid is a pharmaceutical excipient and has been widely used in many dosage forms. It remains unclear in terms of the fatty acids (FAs) profile. In this study, a sensitive and direct method based on high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was developed to study the compositions of oleic acid. The chromatographic conditions were optimized to achieve good separation and high sensitivity. The components of oleic acid were identified by ion trap/time of flight mass spectrometry (MS-IT-TOF). Twenty-seven FAs were identified based on the exact mass-to-charge ratio and fragments, among which 13 FAs were confirmed with the reference standards. Nine FAs in the oleic acid samples including oleic acid, linolenic acid, myristic acid, palmitoleic acid, linoleic acid, palmitic acid, stearic acid, arachidic acid and behenic acid were simultaneously determined by the developed HPLC-CAD, which showed good linearity with r2>0.999. The limit of detection (LOD) and limit of quantification (LOQ) of 9 FAs were 0.006-0.1 μg mL-1 and 0.032-0.22 μg mL-1, respectively. The components with concentration level not less than 0.03 % (referring to the sample concentration of 1.0 mg mL-1) can be quantified. The mean recovery values of 9 FAs ranged from 96.5%-103.6% at three concentration levels of 80 %, 100 % and 120 %. The repeatability and intermediate precision were less than 5.0 % for oleic acid and components with concentration levels more than 0.05 %. In contrast to the conventional pre-column derivatization gas chromatography (GC), HPLC-CAD could unbiasedly and directly detect more components, especially the FAs with long carbon chains. Overall, the developed novel HPLC-CAD method can ameliorate the deficiency of the indirect GC method recorded in current pharmacopeias, thus having great potential for the comprehensive understanding and quality control of oleic acid.

Zhi-Zi-Da-Huang decoction (ZZDHD) has been used for treatment of alcoholic liver disease in China for thousands of years. In order to reveal the dynamic biotransformation of the decoction in vivo, a high-throughout, sensitive and special method based on high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (HPLC–DAD–TOF/MS) and high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC–QqQ/MS) was developed and validated. 25 parent compounds and 28 metabolites were characterized, among which, two metabolites were found for the first time and tentatively identified by neutral-loss scan and product-ion scan. All the compounds were assigned to iridoids, flavones, anthraquinones, coumarin or p-coumaric acid, and their biotransformation pathways were found to involve glucuronidation, sulfation, reduction and ring cleavage. Glucuronidation occurred as a major metabolic pathway of genipin and flavanone and the conjugates could be detected almost during the whole sampling duration. To compounds such as anthraquinones, coumarin and p-coumaric acid, sulfation is the only transformation pathway and the metabolites were found at 0–12, 4–18, 4–48 h respectively after administration. Reduction and/or ring cleavage of genipin glucuronide and naringin were also observed obviously. The phenomena that parts of parent compounds and metabolites were able to be detected even 48 h after administration implied that the accumulating effect of these constituents in vivo would happen and the potential toxicity of the decoction might appear if multiple dosing is adopted. The strategy used in this paper was proved helpful to offer important information for the clinical safe use of ZZDHD.

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitative analysis of 1,2:5,6-dianhydrogalactitol (DAG) in mouse plasma and tissues. Sodium diethyldithiocarbamate (DDTC) was used as the derivatization reagent to improve its LC-MS/MS behavior. Analytes were separated on a Welch Ultimate XB-CN column with a mobile phase consisting of acetonitrile and 0.1% formic acid solution (65:35). The MS analysis was conducted by positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity (r2 > 0.9958) was observed over the concentration range of 1–1000 ng/mL in plasma and tissue homogenates (brain, liver, heart, spleen, lung and kidney). The intra- and inter-batch precision and accuracy of DAG in plasma and brain samples were all within the acceptable limits. The extraction recovery was stable and no significant matrix effects were observed. The method was successfully applied to study the pharmacokinetic and tissue distribution of DAG in mice after intravenous administration. DAG could cross the blood-brain barrier and had limited liver distribution. Rat primary hepatocytes in vitro experiments demonstrated that DAG had a safe profile in liver.

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N-oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC-MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C18 column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500-5000 ng/mL for CBZ and 0.525-2100 ng/mL for CBN. The extraction recoveries were constant and the intra- and inter-batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague-Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose-proportional increases in exposure.

Patrinia scabra Bunge has long been used in clinic as a traditional Chinese medicine for treating leukemia and cancer and regulating host immune response. Despite their wide use in China, no report on system analysis on their chemical constituents is available so far. The current study was designed to profile the fingerprint of ethyl acetate extract of it, and in addition, to characterize the major fingerprint peaks and determine their quantity. Therefore, a detailed gradient high-performance liquid chromatography was described to separate more than 30 compounds with satisfactory resolution in P. scabra Bunge. Based on the chromatograms of 10 batches samples, a typical high-performance liquid chromatographic (HPLC) fingerprint was established with 23 chromatographic peaks being assigned as common fingerprint peaks. Furthermore, a quadrupole time of flight mass spectrometry (Q-TOF/MS) was coupled for the characterization of major compound. As (+)-nortrachelogenin was the most predominant compound in P. sca...

The chemical stability of cabozantinib (CBZ) was investigated using a novel stability-indicating LC method. Forced degradation of CBZ was carried out under acidic, basic, thermal, oxidative and photolytic stress conditions. Hydrolysis and oxidation were the primary pathways for this compound and three major unknown degradation products were characterized by LC/TOF-MS and LC-MS/MS. The major oxidative degradation product was isolated by preparative LC and identified by UV, HRMS and NMR techniques to be N-{4-[(N-oxide-6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N'-(4-fluorophenyl)-cyclopropane-1,1-dicarboxamide. The developed method was validated as per ICH guidelines and then successfully applied to investigate the degradation kinetics of CBZ. Degradation of CBZ followed first-order kinetics under all experimental conditions. A V-shaped pH-rate profile over the pH range 2-10 was observed with maximum stability at pH 6. The effect of temperature on the rate of CBZ degradation was characterized using the Arrhenius equation. The activation energy for hydrolysis was 57.31kJmol(-1) in alkaline solution.

YiGan San (YGS) has long been used in traditional Japanese and Chinese folk medicine and serves as a potent and novel therapeutic agent to treat Alzheimer's disease. In the present study, a rapid and sensitive method based on HPLC coupled with diode-array detection and quadrupole TOF MS (Q-TOF-MS) was designed to reveal the chemical constituents of YGS. Thirty-six compounds were identified and assigned in YGS, including 14 alkaloids, nine γ-lactones, six flavonoids, three triterpenoid saponinares, two small molecular organic acids, and two other types of compounds. In addition, the accurate fragment weight and MS/MS fragmentation reactions of a subtype indole alkaloid in Uncariae ramulus cum uncis were summarized for the first time to realize rapid identification without reference substances. For the first time, 11 major constituents were comprehensively quantified with a HPLC coupled with triple-quadrupole MS method. A three-section switch was used to realize such multicomponent identification. The contents of saikosaponin B2 and isoliquiritin, which produce anti-inflammatory and antidepressant-like effects, were extremely different, up to 700 times, in two sources of YGS. The developed qualitative and quantitative method was proved to be precise, accurate, and reproducible.

Uncaria rhynchophylla (UR) is a species of Uncaria that is distributed mainly in China and Japan. In this study, the chemical constituents, including alkaloids, flavonoids, and quinic acids, in UR have been systematically identified and quantified by a developed method of high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF-MS). Tetracyclic monoterpenoid oxindole alkaloids (TMOAs) are characteristic compounds in this herb, and their fragmentations in Q-TOF-MS have been investigated. Diagnostic fragmentation ions (DFIs) were first delineated for isorhynchophylline-type (7S, C20-ethyl) and corynoxeine-type (7R, C20-vinyl) TMOAs, and these were used for identification of these alkaloids in UR. In this study, a total of 29 compounds, comprising 18 alkaloids, six flavonoids, and five quinic acids, were identified. Among them, there are four novel TMOAs, named as 22-O-β-glucopyranosyl isorhynchophyllic acid (10), 22-O-β-glucopyranosyl rhynchophyllic acid (11), 9-hydroxy isocorynoxeine (16), and 9-hydroxy corynoxeine (20), which have not been reported previously. Furthermore, eight marker compounds, namely chlorogenic acid (3), catechin (8), epicatechin (9), isocorynoxeine (24), rhynchophylline (25), isorhynchophylline (27), vincoside lactam (28), and corynoxeine (29), have been simultaneously quantified. The developed method has been validated and successfully applied to analyze three samples of UR from Jiangxi Province. The contents of the marker compounds have been detected and compared.

YK-1101, with its structure as S-((E)-4-((7S,10S,Z)-4-ethylidene-7-isopropyl-2,5,8,12-tetraoxo -9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-en-1-yl) ethanethioate, is synthesized as a potential histone deacetylase inhibitor. Its quality and stability under various stress conditions are not fully understood. In this study, a high performance liquid chromatographic (HPLC) method was established and validated for the analysis of YK-1101 bulk drug samples. The chromatographic separation was performed on a C18 column with acetonitrile and water as mobile phase in a gradient elution. Based on the established method, the stability studies of YK-1101 under various stress conditions were carried out. YK-1101 was shown to undergo degradation under basic and acidic stress conditions, while it was stable under oxidative, photolytic and thermal conditions. In addition, a time of flight mass spectrometer (TOF/MS) was coupled to HPLC for the characterization of major degradation products produced under basic and acidic stress conditions. Their degradation pathways were also discussed. Keywords: YK-1101, Degradation products, Stress study, Liquid chromatography, Time of flight mass spectrometer

Although the chemical synthesis of wogonin (5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one) has been established, the impurity profile of this pharmaceutical agent is still not fully understood. In this study, a novel process related impurity present in the synthetic wogonin was isolated by preparative HPLC. Its structure elucidation was unambiguously carried out by NMR, HRMS and IR spectra, and it was characterized as 6-chloro-5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one ( imp-1 ), a new compound which had never been reported before. Moreover, forced degradation studies were also carried out, and wogonin was shown to undergo isomerzation under basic stress condition to form a major degradant, 5,7-dihydroxy-6-methoxy-2-phenyl-4H-chromen-4-one ( imp-2 ) using HPLC–Q-TOF–MS/MS technique. Finally, a selective and simple routine HPLC method was developed for simultaneous quantification of wogonin and the two related compounds. The proposed method is useful for purity control during the process development of wogonin and laboratory-prepared preparations. Their formation pathways were also discussed.

Oroxylin A shows reversal activity on multidrug resistance and has an attractive safety profile. A highly sensitive and specific method based on liquid chromatography coupled with tandem mass spectroscopy was developed and validated for simultaneous determination of oroxylin A and its major metabolite, oroxylin A-7-O-glucuronide in rat plasma. The assay procedure involved precipitation of plasma samples with acetonitrile after adding luteolin as an internal standard. Chromatographic separation was achieved on a C18 column using methanol and 0.5% formic acid as the mobile phase, eluting in a gradient system. Detection was achieved with selected reaction monitoring using the electrospray ionization technique. The method was validated by evaluation of selectivity, linearity, precision, accuracy and limit of quantification. Its applicability was demonstrated through the quantification of oroxylin A and oroxylin A-7-O-glucuronide in plasma after intravenous administration of oroxylin A at a dose of 20 mg kg−1 to rats. The developed quantification method can now be used for pharmacokinetic studies after intravenous infusion of oroxylin A injection in rats.

The tissue distribution of wogonin was investigated for study of its anticancer activity. A robust, reliable, and sensitive LC method was developed for simultaneous analysis of wogonin and its major metabolite, wogonoside, in tissues of tumor-bearing nude mice. To reach the required level of detection in different tissues, sample pretreatment was by solid-phase extraction. Good LC separation was achieved on a C18 column with methanol–0.5% formic acid 68:32 (v/v) as mobile phase. UV detection was performed at 275 nm and calibration plots for both analytes were linear over the range 0.16–80.0 μg g−1 for all the biological samples studied with correlation coefficients >0.9980. Inter-day and intra-day precision were no more than 15% at the limit of quantification and 10% at other concentrations; recovery from all tissues was 92.3–101.4% for wogonin and 66.7–74.3% for wogonoside. The method was ideal for this tissue distribution study and results obtained reflect the potential of wogonin for further pharmaceutical development as an anticancer agent.

A sensitive, specific and rapid high performance liquid chromatography - tandem mass spectrometric (LC/MS/MS) method for the determination of cyclovirobuxine D in human plasma was developed and validated. The triple-quadrupole tandem mass spectrometric detector with an electrospray interface (ESI) was operated under the selected reaction monitoring (SRM) mode. After the addition of citalopram as an internal standard (IS), plasma samples were extracted with ethyl acetate. Chromatographic separation of the analytes was performed on a Kromasil CN column with a mobile phase of methanol/water (88/12, v/v) containing 0.4% formic acid. Linearity was established for the range of concentration 0.2-40ng/ml. Under optimized conditions, the mean recovery was 86.6%. The intra-day precision ranged from 4.56% to 7.81%, while the intra-day accuracy ranged from 2.75% to 11.0%. The inter-day precision was in the range 3.87-10.7%, and the inter-day accuracy was in the range -4.00% to 2.50%. The cyclovirobuxine D was stable in human plasma after three freeze-thaw cycles, under storage at room temperature for 12h, in a freezer at -20 degrees C for 15 days and during processing (in autosampler) at 10 degrees C for 24h. The validated method is suitable for quantitative determination of cyclovirobuxine D in human plasma in pharmacokinetics study and has been successfully applied to the analysis of clinical samples.

To identify the major metabolites of resveratrol in rat, rat urine samples were pretreated by using solid-phase extraction technique (SPE) with polyamide cartridges. And a LC-MS/MS method with electrospray ionisation (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of resveratrol. According to the results of our experiment, we found that the main metabolites of resveratrol were resveratrol monoglucuronide (M1), dihydroresveratrol monosulfate (M2), resveratrol monosulfate (M3) and dihydroresveratrol (M4).

Three Callicarpa species, namely Callicarpa nudiflora Hook. et Arn., Callicarpa macrophylla Vahl. and Callicarpa kwangtungensis Chun. are astringency and hemostasis herbs in the traditional Chinese medical systems. Despite their wide use in Chinese medicine, no report on system comparison on their chemical constituents is available so far. High-performance liquid chromatography coupled with diode array detector and electrospray ionization trap mass spectrometry (HPLC–DAD–ESI-Trap MS) technique was used for qualitative and quantitative analyses of the three Callicarpa herbs. Phenylpropanoid glycosides, flavonoids and organic acids were identified by comparing with reference standards or according to their MS/MS fragmentation behaviors. A total of 33 compounds were identified identified or tentatively identified, and 23 of them were reported from these herbs for the first time. Phenylpropanoid glycosides were featured in the three species with their types and contents presenting significant differences. Furthermore, quantitative analysis was conducted by determining four marker phenylpropanoid glycosides (forsythoside B (14), acteoside (15), poliumoside (19), isoacteoside (21)) and two flavonoids (luteolin (30), apigenin (32)). Three flavonoid glucuronides (luteolin-diglucuronide-glucuronide (5), luteolin-diglucuronide (12), apigenin-7-O-β-glucuronide (24)) were semi-quantified according to their corresponding aglycones. The total contents of the nine major compounds in the three species varied significantly from 8.92 to 40.89 mg/g.