Trace Level Quantification of 4-Methyl-1-nitrosopiperazin in Rifampicin Capsules by LC-MS/MS.

Rifampicin is a first-line anti-tuberculosis drug. However, in August 2020, the presence of 1-methyl-4-nitrosopiperazine (MNP), a nitrosamine impurity, was detected by the United Stated Food and Drug Administration (US FDA) in rifampicin capsules. Consequently, the development of efficient methods for the detection of MNP is an important objective. In this study, the MNP present in rifampicin capsules was detected using LC-MS/MS. A total of 27 batches from nine manufacturers in the Chinese market were tested, with MNP (0.33-2.36 ppm) being detected in all samples at levels exceeding the maximum acceptable intake limit of 0.16 ppm initially set by the FDA. However, after considering the associated benefits and risks, the FDA-approved limit was revised to 5 ppm; hence, all the samples examined herein exhibited MNP levels well below the required limit. Furthermore, the results of forced degradation experiments suggest that MNP is formed by the thermal degradation of rifampicin.

Identification, cloning and expression patterns of the genes related to phosphate solubilization in Burkholderia multivorans WS-FJ9 under different soluble phosphate levels.

As important plant growth-promoting rhizobacteria, phosphate-solubilizing bacteria (PSB) fix nitrogen, dissolve potassium, promote growth, improve the soil micro-environment, and enhance soil fertility. A high-efficiency PSB strain from the pine tree rhizosphere, Burkholderia multivorans WS-FJ9, was screened in our laboratory. In this study, using a Bio Screener fully automatic microbial growth curve meter to determine the growth of the WS-FJ9 strain in phosphate-removing medium, the growth and mineral phosphate solubilization of WS-FJ9 were measured by Mo-Sb colorimetry and organophosphate-solubilization plate assays. Second-generation sequencing technology was used to obtain genomic information and to analyze possible phosphate decomposition genes. The related expression levels of these genes under different soluble phosphate levels were determined by quantitative real-time PCR. The results showed that WS-FJ9 had strong adaptability and capacity for mineral phosphate solubilization at low soluble phosphate levels, which is characterized by its low soluble phosphate induction and high soluble phosphate inhibition. The amount of solubilized mineral phosphate could exceed 140 mg/L. The total length of the WS-FJ9 genome was 7,497,552 bp after splicing, and the GC content was 67.37%. Eight phosphate-related genes were selected to determine their expression patterns at different soluble phosphate levels. Among them, AP-2, GspE and GspF were only related to organic phosphate, HlyB was only related to inorganic phosphate, and PhoR, PhoA, AP-1 and AP-3 were related to both. The WS-FJ9 strain utilizes multiple pathways for mineral phosphate solubilization, and the solubilization processes of different phosphate sources are interrelated and independent, indicating that the WS-FJ9 strain can adapt to different phosphate source environments and has good potential for future applications.

Indole-3-Acetic Acid in Burkholderia pyrrocinia JK-SH007: Enzymatic Identification of the Indole-3-Acetamide Synthesis Pathway.

Burkholderia pyrrocinia JK-SH007 is a plant growth-promoting bacteria (PGPB), that can promote the growth of poplar and other trees, and, production of the plant hormone indole-3-acetic acid (IAA) is one of the reasons for this effect. Therefore, the aims of this study were to evaluate the effect of the external environment on the synthesis of IAA by B. pyrrocinia JK-SH007 and to perform a functional analysis of its IAA synthesis pathway. In this study, IAA and its synthetic intermediates indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), tryptamine (TAM), and indole-3-acetonitrile (IAN) were detected in B. pyrrocinia JK-SH007 fermentation broth by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS), and these indolic compounds were also found in the cell-free extraction of B. pyrrocinia JK-SH007, but the genomic analysis of B. pyrrocinia JK-SH007 indicated that IAA biosynthesis was mainly through the IAM and TAM pathways. The effects of L-tryptophan (L-Trp), temperature and pH on the synthesis of IAA were investigated, and the results showed that L-Trp exerted a significant effect on IAA synthesis and that 37°C and pH 7 were the optimal conditions IAA production by B. pyrrocinia JK-SH007. In addition, the protein expression of tryptophan 2-monooxygenase and indoleacetamide hydrolase, which are the key enzymes of the indole acetamide-mediated IAA synthesis pathway, was analyzed, and their activity was verified by substrate feeding experiments. The results revealed the existence of an IAA synthesis pathway mediated by IAM and indicated that this pathway plays a role in B. pyrrocinia JK-SH007. This study lays the foundation for further exploration of the specific pathway and mechanism of IAA synthesis in B. pyrrocinia JK-SH007.

Development, validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC-MS/MS to simultaneously quantify dopamine, serotonin and γ-aminobutyric acid in four rat brain regions.

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate-matrix and surrogate-analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC-MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5-HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5-HT were paralleled in striatum and hippocampus almost 2-fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate-matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.

The direct analysis of drug distribution of rotigotine-loaded microspheres from tissue sections by LESA coupled with tandem mass spectrometry.

The direct analysis of drug distribution of rotigotine-loaded microspheres (RoMS) from tissue sections by liquid extraction surface analysis (LESA) coupled with tandem mass spectrometry (MS/MS) was demonstrated. The RoMS distribution in rat tissues assessed by the ambient LESA-MS/MS approach without extensive or tedious sample pretreatment was compared with that obtained by a conventional liquid chromatography tandem mass spectrometry (LC-MS/MS) method in which organ excision and subsequent solvent extraction were commonly employed before analysis. Results obtained from the two were well correlated for a majority of the organs, such as muscle, liver, stomach, and hippocampus. The distribution of RoMS in the brain, however, was found to be mainly focused in the hippocampus and striatum regions as shown by the LESA-imaged profiles. The LESA approach we developed is sensitive enough, with an estimated LLOQ at 0.05 ng/mL of rotigotine in brain tissue, and information-rich with minimal sample preparation, suitable, and promising in assisting the development of new drug delivery systems for controlled drug release and protection. Graphical abstract Workflow for the LESA-MS/MS imaging of brain tissue section after intramuscular RoMS administration.

Charge Variants of an Avastin Biosimilar Isolation, Characterization, In Vitro Properties and Pharmacokinetics in Rat.

The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin.

Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS.

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The optimized enzyme reaction condition contained 1.5 µg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra- and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005-1.000 µg/mL for MVAL and 0.010-0.500 µg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 µg/mL for MVAL and 0.010 µg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95% to 2.39% and 2.26% to 3.38% for MVAL, 1.46% to 3.89% and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

[Classification of Cimicifuga species based on 1H-NMR fingerprint combined with pattern recognition technique].

The metabolomic analysis of three Cimicifuga species was performed using H-NMR spectroscopy and pattern recognition (PR) techniques. A broad range of metabolites could be detected by 'H-NMR spectroscopy without any chromatographic separation. The analysis using principal component analysis (PCA) and discriminant partial least square (DPLS) of the 1H-NMR spectrum showed a clear discrimination between C. foetida and the other two species. The major metabolites responsible for the discrimination were triterpenoid saponins and saccharides. These results indicated that the combination of 1H-NMR and PR provides a useful tool for chemotaxonomic analysis and authentification of Cimicifuga species, and could used for the quality control of plant materials.

Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry.

A highly sensitive ultra-high-performance liquid chromatographic-tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantification of the synthetic peptide drug of exenatide in monkey plasma. Sample preparation was carried out by solid-phase extraction (SPE), and bivalirudin was used as the internal standard (IS). An excellent chromatographic separation was obtained on a reversed-phase C18 column with a gradient elution. Detection utilized a Qtrap 5500 system operated in the positive ion mode with multiple reaction monitoring (MRM). The proposed method was validated by assessing the specificity, linearity, intra- and inter-day precision and accuracy, recovery, and stability. The method resulted in a linear calibration range of 0.10-30 ng/mL, extracting with only 50 µL monkey plasma aliquots. The intra- and inter-day precisions (as relative standard deviation) were less than 7.5% and 9.6%, respectively. The method could be successfully utilized for the pharmacokinetic study of exenatide in monkeys following a single subcutaneous injection of Byetta.

Pharmacokinetics and bioavailability of cimicifugosides after oral administration of Cimicifuga foetida L. extract to rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifuga foetida L., a traditional Chinese medicine, has been used as an anti-inflammatory, antipyretic and analgesic remedy. The primary active constituents are believed to be present in the triterpene glycoside fraction. MATERIALS AND METHODS: To develop an LC-MS/MS assay for four major cimicifugosides [cimicifugoside H-1 (Cim A), 23-epi-26-deoxyactein (Cim B), cimigenolxyloside (Cim C) and 25-O-acetylcimigenoside (Cim D)] obtained from C. foetida L. and apply it to investigate their pharmacokinetic (PK) properties and bioavailabilities through oral administration of C. foetida L. extract (12.5, 25 and 50mg/kg) and single intravenous (i.v.) doses (5mg/kg) of the individual cimicifugosides in rat. PK parameters were estimated by non-compartmental analysis. RESULTS: All calibration curves showed excellent linear regressions (all r>0.995) within the range of tested concentrations. The intra- and inter-day variations were <15% in terms of RSD. The molar ratio of Cims A, B, C, and D in the extract was 20.7:1.4:2.9:1. PK parameters for Cims A, B, C, and D following oral administration of the extract were respectively: C(max) 4.05-17.69, 90.93-395.7, 407.1-1180 and 21.56-45.09pmol/mL; T(max) 0.46-1.28, 2.00-4.67, 14.67-19.67 and 8.08-14.27h; absolute oral bioavailability (F) 1.86-6.97%, 26.8-48.5%, 238-319% and 32.9-48%. PK parameters after i.v. administration of individual cimicifugosides were respectively: elimination half-life 1.1, 2.5, 5.7 and 4.2h; clearance 15.7, 0.48, 0.24 and 1.13mL/hkg. CONCLUSIONS: Systemic exposure to Cims B, C and D following oral administration of the extract was significantly greater than to Cim A despite the predominance of Cim A in the extract. Significantly different clearance and interconversion from Cim A to Cim C probably accounts for the different exposure to the four cimicifugosides.

Pharmacokinetic characteristics of vincristine sulfate liposomes in patients with advanced solid tumors.

AIM: To evaluate the single- and multiple-dose pharmacokinetics of vincristine sulfate liposomes (VSLI) in patients with advanced solid tumors. METHODS: In single-dose pharmacokinetic study, 16 patients were administered VSLI (1.5, 2.0, or 2.3 mg·m(-2)) through intravenous infusion. Another 6 patients receiving vincristine sulfate (VCR, 2.0 mg) were taken as the control. In multiple-dose pharmacokinetic study, 12 patients were administered VSLI (1.5 or 1.8 mg·m(-2)) through intravenous infusion weekly for 4 consecutive weeks. The plasma concentration of VSLI was determined using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: After intravenous infusion of the single dose of VSLI, the plasma concentrations were characterized by bi-exponential decline curves. No statistically significant differences were observed between the main pharmacokinetic parameters in the 3 dose groups. Compared with the patients receiving VCR, the patients treated with VSLI displayed an increase in the area under the plasma concentration vs time curve (AUC), and a decrease in plasma clearance rates. On the 4th cycle in the multiple-dose study, the plasma concentration of VCR in all subjects prior to the weekly administration was below the lower limit of quantification (LLOQ). The calculated pharmacokinetic parameters from the subjects in the multiple- and single-dose (1.5 mg·m(-2)) groups had no significant differences. Although the administration of liposomal VCR may significantly elevate the plasma concentration of VCR, VSLI-associated adverse events were similar to those associated with conventional VCR. CONCLUSION: VSLI exhibits a lower clearance and a higher AUC compared with conventional VCR. No accumulation was observed in patients exposed to VSLI for 4 consecutive weeks. VSLI was generally tolerated in the subjects. The phase II dose of VSLI may be recommended as 4 doses of 1.5 mg·m(-2) for treatment of patients with advanced solid tumors.

[Studies on the constituents from the herba of Erigeron acer].

OBJECTIVE: To investigate the chemical constituents of the aerial part of Erigeron acer. METHODS: The chemical constituents were isolated by various columns and thin layer chromatographic methods. The structures were identified by spectral data. RESULTS: Five compounds were isolated and identified as alpha-amyrin (1), beta-amyrin (2), caffeic acid (3), quercetin (4), 4'-hydroxywogonin-7-O-beta-D-glucuronic acid glycoside (5). CONCLUSION: The five compounds are obtained from this plant for the first time. The signals of 13C-NMR of the compound(5) are reassigned by means of DEPT, HMQC, HMBC, the signals of 1H-NMR of 2" - 5"-H of the glucuronic acid moiety are assigned by means of HMBC, HMQC, 1H-1H COSY for the first time.

Pharmacokinetics and in vitro and in vivo correlation of huperzine A loaded poly(lactic-co-glycolic acid) microspheres in dogs.

The purpose of this study was to investigate the pharmacokinetics and in vitro/in vivo correlation (IVIVC) of huperzine A loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in dogs. Several huperzine A loaded PLGA microspheres were prepared by an O/W method and three of them (single dose) were injected intramuscularly (i.m.) or subcutaneously (s.c.) to five beagle dogs, respectively. With the increase of the molecular weight of PLGA and the particle size of microspheres, the in vitro and in vivo release periods of huperzine A were prolonged. After s.c. injection, the release of huperzine A from microspheres was faster than that after i.m. injection. The IVIVC models of huperzine A loaded PLGA microspheres were established successfully and after i.m. administration the linear relationship between the in vitro and the in vivo releases was better than that after s.c. administration. It was also found when the particle size of the microspheres was smaller; the values of correlation coefficient were higher.