Determination of nitrobenzene potential genotoxic impurities in nifedipine with GC-MS.

There is an increasing scientific interest in the detection of genotoxic impurities (GTIs), with nitrobenzene compounds being considered potential genotoxic impurities due to their structural alerts, which demonstrates a threat to drug safety for patient. While current reports on the detection of nifedipine impurity primarily focus on general impurities in nifedipine. In this study, an effective and simple gas chromatography-mass spectrometry (GC-MS) method was established and verified for the separation and quantification of 2-nitrotoluene, 2-nitrobenzyl alcohol, 2-nitrobenzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, and 2-nitrobenzyl bromide in nifedipine, which have not been previously reported. The validation of this GC-MS method was conducted following the International Conference of Harmonization (ICH) guidelines, exhibiting good linearity within the range of 2-40 µg/g and accuracy between 84.6 % and 107.8 %, the RSD% of intra-day and inter-day precision was in the range of 1.77-4.55 %, stability and robustness also met acceptance criteria. This method filled the gap in detection method for nitrobenzene compounds in nifedipine, offering a novel method and technical support for nifedipine quality control.

Study of the impurity profile of photodegradation in lomefloxacin hydrochloride ear drops using liquid chromatography combined with ion trap/time-of-flight mass spectrometry.

RATIONALE: Lomefloxacin hydrochloride ear drops are highly unstable to light and prone to produce photodegradation impurities. These impurities might be related to the phototoxicity of lomefloxacin, which could seriously threaten the health of patients. In this article, the photodegradation impurity profile in lomefloxacin hydrochloride ear drops was studied for further improvement of quality control of the drug. METHODS: By studying the chromatographic behavior of photodegradation impurities, the photodegradation impurities in lomefloxacin hydrochloride ear drops were separated and detected effectively. Liquid chromatography combined with ion trap/time-of-flight mass spectrometry was applied to characterize the structures of the photodegradation impurities in lomefloxacin hydrochloride ear drops. RESULTS: The structures of 17 impurities in lomefloxacin hydrochloride ear drops were elucidated based on high-resolution MSn data in positive ion mode, 12 of them being unknown impurities. CONCLUSIONS: The structural characteristics and fragmentation patterns of the photodegradation impurities were also studied. The study of the photodegradation impurity profile in lomefloxacin hydrochloride ear drops provides a scientific basis for quality control of these ear drops and ensures the safety of drug use by the public.

Separation and characterization of the polymerized impurities in oxacillin sodium by 2D HPSEC and HPLC IT-TOF MS.

RATIONALE: The polymerized impurities in oxacillin sodium can induce allergic reaction, which can seriously threaten the health of patients. Gel filtration chromatography (GFC) is currently widely used for the analysis of polymerized impurities, but it has drawbacks. To effectively control the polymerized impurities in oxacillin sodium, a high-performance size exclusion chromatography (HPSEC) method and a reversed-phase high performance liquid chromatography (RP-HPLC) method were established to replace the classical GFC method. METHODS: By studying the chromatographic behavior of polymerized impurities and small molecular weight impurities in both methods with different chromatographic separation mechanisms, the polymerized impurities in oxacillin sodium were separated and detected effectively. Column-switching two-dimensional liquid chromatography was applied to eluted polymerized impurities from the HPSEC method for oxacillin sodium. Ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and unknown impurities eluted from the HPSEC/RP-HPLC method for oxacillin sodium. RESULTS: The structures of 25 unknown impurities in oxacillin sodium were elucidated based on the high-resolution massn data. Thirteen polymerized impurities were found and characterized. The corresponding relationship of impurities between the two methods was established and the specificity of the two methods was compared. The RP-HPLC method for analysis of the polymerized impurities not only has higher column efficiency and more specificity than the HPSEC method, but also higher sensitivity. CONCLUSIONS: The mechanisms of the formation of degradation impurities in oxacillin sodium were studied. The newly established RP-HPLC methods could effectively separate and detect polymerized impurities and unknown impurities in oxacillin sodium. The study of the impurity profile in oxacillin sodium provided a scientific basis for the improvement of official monographs in pharmacopoeias.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection.

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Relationship between gut microbiota and lymphocyte subsets in Chinese Han patients with spinal cord injury.

This study is to investigate the changes of lymphocyte subsets and the gut microbiota in Chinese Han patients with spinal cord injury (SCI). We enrolled 23 patients with SCI and 21 healthy controls. Blood and fecal samples were collected. The proportion of lymphocyte subsets was detected by flow cytometry. 16S rDNA sequencing of the V4 region was used to analyze the gut microbiota. The changes of the gut microbiota were analyzed by bioinformatics. Correlation analysis between gut microbiota and lymphocyte subsets was performed. CD4 + cells, CD4 + /CD8 + ratio and CD4 + CD8 + cells in peripheral blood of SCI patients were significantly lower than those of the control group (P < 0.05). There was no significant difference in B cells and CIK cells between the SCI group and the control group. The gut microbiota community diversity index of SCI patients was significantly higher than that of healthy controls. In SCI patients, the relative abundance of Lachnospiraceae (related to lymphocyte subset regulation), Ruminococcaceae (closely related to central nervous system diseases), and Escherichia-Shigella (closely related to intestinal infections) increased significantly, while the butyrate producing bacteria (Fusobacterium) that were beneficial to the gut were dramatically decreased. Correlation analysis showed that the five bacterial genera of SCI patients, including Lachnospiraceae UCG-008, Lachnoclostridium 12, Tyzzerella 3, Eubacterium eligens group, and Rumencocciucg-002, were correlated with T lymphocyte subsets and NK cells. In the SCI group, the flora Prevotella 9, Lachnospiraceae NC2004 group, Veillonella, and Sutterella were positively correlated with B cells. However, Fusobacterium and Akkermansia were negatively correlated with B cells. Moreover, Roseburia and Ruminococcaceae UCG-003 were positively correlated with CIK cells. Our results suggest that the gut microbiota of patients with SCI is associated with lymphocyte subsets. Therefore, it is possible to improve immune dysregulation in SCI patients by modulating gut microbiota, which may serve as a new therapeutic method for SCI.

The effect of Iridoids effective fraction of Valeriana jatamansi Jones on movement function in rats after acute cord injury and the related mechanism.

OBJECTIVES: Spinal cord injury (SCI) is a disastrous central nervous system (CNS) disorder, which was intimately associated with oxidative stress. Studies have confirmed that Iridoids Effective Fraction of Valeriana jatamansi Jones (IEFV) can scavenge reactive oxygen species. This study aimed to confirm the efficacy of IEFV in ameliorating SCI. METHODS: For establish the SCI model, the Sprague-Dawley rats underwent a T10 laminectomy with transient violent oppression by aneurysm clip. Then, the rats received IEFV intragastrically for 8 consecutive weeks to evaluate the protective effect of IEFV on motor function, oxidative stress, inflammation and neurotrophic factors in SCI rats. RESULTS: Basso, Beattie and Bresnahan scores, hematoxylin and eosin (H&E) staining and transmission electron microscopy experiments found IEFV protected motor function and alleviated neuron damage. Meanwhile, IEFV treatment decreased the release of malondialdehyde, interleukin-6 (IL-6), cyclooxygenase-2 and tumor necrosis factor-α. Moreover, IEFV treatment elevated the expression levels of brain-derived neurotrophic factor and nerve growth factor of SCI rats. Finally, administration of IEFV significantly inhibited the expression of p-p65 and toll-like receptor 4 (TLR4). CONCLUSIONS: This study suggests that IEFV could attenuate the oxidative stress and inflammatory response of the spinal cord after SCI, which was associated with inhibition of the TLR4/nuclear factor-kappaB signaling pathway.

Safety of Every-Other-Day Fasting in the Treatment of Spinal Cord Injury: A Randomized Controlled Trial.

ABSTRACT: Every-other-day fasting is effective for a variety of major human diseases, but the safety of these interventions is uncertain for patients with spinal cord injury. A randomized controlled study was conducted to investigate the safety of every-other-day fasting in patients with spinal cord injury. Participants who met the diagnostic inclusion and exclusion criteria were randomly divided into the control and every-other-day fasting groups. In the every-other-day fasting group, fasting lasted from 09:00 p.m. on day 1 to 06:00 p.m. on the following day (day 2). Dinner on day 2 was restricted to approximately 30% of the average daily calorie intake. The changes in plasma glucose were recorded daily for 2 days and every other day from the third day after every-other-day fasting intervention. The changes in albumin, prealbumin, plasma potassium, serum sodium, blood calcium, body weight, and body mass index were monitored at the baseline and at the end of the every-other-day fasting intervention. The results showed that compared with the control group, the mean blood glucose levels were significantly decreased from the second week after every-other-day fasting intervention. The body weight of patients in the every-other-day fasting group was notably reduced compared with that at baseline, whereas in body mass index, no obvious differences were observed before and after treatment with every-other-day fasting. In general, every-other-day fasting could be considered as a safe approach for individuals with spinal cord injury.

Characterization of eight unknown impurities and analysis of their source in xinfujunsu and injection by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry.

Eight unknown impurities in xinfujunsu and its injection were characterized by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS). In order to determine the m/z values of the molecular ions and predict the formulas of all detected impurities, full scan LC-MS in positive ion mode was firstly executed to obtain the m/z value of the molecules. Then LC-MS2, LC-MS3 and LC-MS4 were carried out on target compounds to obtain as much structural information as possible. Based on MSn spectral data and exact mass measurements, the chemical structures of eight unknown impurities were characterized, among which three impurities were degradation impurities and five impurities were process impurities. In addition, the source of impurities and the correlation between process and impurities were also studied. The production of degraded impurities was caused in the high pressure sterilization process of xinfujunsu injection. Based on characterization of impurities, this study revealed the cause of impurity production and provided guidance for enterprises to improve the process to reduce impurity content. Furthermore, this study also provided scientific basis for the further improvement of official monographs in pharmacopoeias.

Effects of repetitive magnetic stimulation on motor function and GAP43 and 5-HT expression in rats with spinal cord injury.

OBJECTIVES: Spinal cord injury (SCI) is a disabling central nervous system disorder. This study aimed to explore the effects of repetitive trans-spinal magnetic stimulation (rTSMS) of different spinal cord segments on movement function and growth-associated protein-43 (GAP43) and 5-hydroxytryptamine (5-HT) expression in rats after acute SCI and to preliminarily discuss the optimal rTSMS treatment site to provide a theoretical foundation and experimental evidence for clinical application of rTSMS in SCI. METHODS: A rat T10 laminectomy SCI model produced by transient application of an aneurysm clip was used in the study. The rats were divided into group A (sham surgery), group B (acute SCI without stimulation), group C (T6 segment stimulation), group D (T10 segment stimulation), and group E (L2 segment stimulation). RESULTS: In vivo magnetic stimulation protected motor function, alleviated myelin sheath damage, decreased NgR and Nogo-A expression levels, increased GAP43 and 5-HT expression levels, and inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and apoptosis-related protein expression in rats at 8 weeks after the surgery. CONCLUSIONS: This study suggests that rTSMS can promote GAP43 and 5-HT expression and axonal regeneration in the spinal cord, which is beneficial to motor function recovery after acute SCI.

Determination and pharmacokinetic study of skimmin by UHPLC-MS/MS in rat plasma.

Skimmin, a major active ingredient derived from Hydrangea paniculata, has been considered to possess various pharmacological activities, including renoprotective activity, anti-inflammatory, anti-cancer, and antiamoebic properties. However, no investigation has reported the quantification and pharmacokinetics of skimmin in biomatrices. In the present study, we established and validated an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the estimation of skimmin in rat plasma, which was successfully applied to explore the oral and intravenous pharmacokinetics of skimmin. All plasma samples were obtained following blood collection from the rat' tail vein and prepared using the protein precipitation method with acetonitrile. Separation of the analyte and internal standard (IS) magnoflorine was achieved by a reversed phase T3 column. The mobile phase consisted of water containing 0.1 % formic acid and acetonitrile with a gradient elution program. The analytical run time was 4 min with a flow rate of 0.3 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI).Multiple reaction monitoring transitions were performed at m/z of 325.34 → 163.00 and 342.24 → 57.98 for skimmin and IS, respectively. The method demonstrated good linearity in the range of 2-2000 ng/mL and was validated by US FDA bioanalytical guidelines. A pharmacokinetic study of skimmin was then successfully conducted using the validated method. Hence, the absolute bioavailability of skimmin was approximately 25.08 % with rapid absorption and elimination. This study will be beneficial in better understanding the pharmacological properties and the further development of skimmin.

Separation and characterization of forced degradation products in homoharringtonine injection by UHPLC-Q-TOF-MS.

A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed for separation and characterization of the degradation products in HHT injection. Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 HD column (2.1 × 100 mm, 1.8µm) using methanol- ammonium formate (pH 3.0; 30 mM) (30:70, v/v) as mobile phase in an isocratic mode of elution. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, photolytic and thermal stress conditions as described in ICH. A total of eleven forced degradation products were detected and the drug was found to be susceptible to all the tested stress conditions. The degradation products were characterized through Q-TOF fragmentation studies and their fragmentation pathways were proposed. Seven of them have not been reported or described as degradation product before, and one of them was further confirmed by reference substance. In addition, plausible mechanisms for the formation of the degradation products were proposed.

Identification of the Novel Capecitabine Metabolites in Capecitabine-Treated Patients with Hand-Foot Syndrome.

Hand-foot syndrome (HFS), the most common side effect of capecitabine, is a dose-limiting cutaneous toxicity with only rare therapeutic options. The causative mechanisms of HFS are still unclear. Many studies suggested that capecitabine or its metabolites caused the toxicity. This study is attempting to determine if there are any new metabolites that may be present and be linked to toxicity. For this purpose, 25 patients who ingested capecitabine orally were enrolled and divided into HFS positive and negative groups. Urine and plasma samples were collected before administration and five cycles after administration. Eleven phase I and phase II metabolites of capecitabine were detected and identified by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry with a metabolomic approach and MetaboLynxXS. Nine novel metabolites of capecitabine were identified herein, which were not observed in the HFS negative group. Their structures were confirmed by chemical synthesis and nuclear magnetic resonance spectroscopy. The cytotoxities of capecitabine and its metabolites on HaCaT cells were measured. Among them, M9/10 exhibited significant inhibitory activity, and they were produced via acetylation mainly by N-acetyltransferase 2. Our study comprehensively described the metabolism of capecitabine in patients with HFS and detected the novel pathways of capecitabine, which was a positive significance for the mechanism of HFS.

Possible Pathways of Capecitabine-Induced Hand-Foot Syndrome.

Capecitabine, an oral prodrug of 5-fluorouracil, inhibits DNA synthesis and has received FDA approval for treatment of metastatic colorectal and breast cancers. Hand-foot syndrome (HFS) is a serious dose-limiting toxicity and the most frequently reported side effect of capecitabine. Because of the lack of knowledge about the causative mechanism of HFS, scarce information is available for effective treatment or prevention. Data are based on published literatures and reports available from the HFS development program database. The purpose of this Review is to provide information regarding definition, clinical manifestation, and the possible mechanisms of HFS induced by capecitabine. Ethnic variations in the clinical presentation of HFS warrant further attention. Several physiological and pharmacological mechanisms have been investigated, such as cyclooxygenase (COX) inflammatory-type reaction, accumulation of capecitabine metabolites, and enzymes and transporters involved in the metabolism and absorption. Although current studies describe the possible mechanisms of HFS induced by capecitabine, much remains to be determined. It appears from this scientific evidence that additional study is needed to determine the effect of skin-mediated metabolism in the possible mechanism of HFS induced by capecitabine.

Application of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to identify curcumin metabolites produced by human intestinal bacteria.

Curcumin, a yellow pigment derived from the rhizomes of Curcuma longa Linn, is a natural antioxidant that exhibits a variety of pharmacological activities and therapeutic properties. However, as curcumin is generally conjugated when absorbed through the intestine, free curcumin is present at extremely low levels in the body. Thus, curcumin metabolites are presumed to be responsible for curcumin bioactivity. In this study, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx(XS)) for rapid analysis of the metabolic profile of curcumin in human intestinal flora. The results show that curcumin undergoes extensive phase I and phase II metabolism. A total of 23 curcumin metabolites were detected and identified in vitro. Furthermore, we identified a number of novel metabolic pathways of curcumin in the human intestinal microflora system.

Metabolism of chamaechromone in vitro with human liver microsomes and recombinant human drug-metabolizing enzymes.

Chamaechromone is a major component in the dried roots of Stellera chamaejasme with antihepatitis B virus and insecticidal activity. In this study, metabolic profiles of chamaechromone were investigated in human liver microsomes. One monohydroxide and two monoglucuronides of chamaechromone were identified. The enzyme kinetics for both hydroxylation and glucuronidation were fitted to the Michaelis-Menten equation. The hydroxylation of chamaechromone was inhibited by α-naphthoflavone, and predominantly catalyzed by recombinant human cytochrome P450 1A2, whereas the glucuronidation was inhibited by quercetin, 1-naphthol, and fluconazole, and mainly catalyzed by recombinant human UDP-glucuronosyltransferase 1A3, 1A7, 1A9, and 2B7.

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Stellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. MATERIALS AND METHODS: Twenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg(-1)), one intravenous injection group (5 mgkg(-1)), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and ß-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. RESULTS: A total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. CONCLUSIONS: The present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites.