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Background: Cytidine Disodium Triphosphate (CTP-2Na) for injection is mainly used for treating nervous system diseases. Currently, there are few studies focused on the separation and identification of polar impurities in CTP-2Na for injection, which is important for ensuring drug safety and efficacy. Objective: The study aimed to establish an HPLC-Q/TOF method for the separation and identification of polar impurities in CTP-2Na for injection. Methods: Chromatographic separation was achieved on a Waters Atlantis T3 column using 5 mM aqueous ammonium acetate solution as the mobile phase in an isocratic elution mode. A postcolumn compensation technology was used to improve the ionization efficiency of impurities in the spray chamber. Results: Three polar impurities (disodium cytidine tetraphosphate, disodium cytidine diphosphate, disodium cytidine monophosphate) were detected in CTP-2Na for injection. The former one is probably the overreaction product during the production of CTP-2Na, the latter two were reported as degradation products. The fragmentation patterns of cytidine phosphate compounds in negative ion mode are summarized. Conclusion: This study provides a good reference for the separation and identification of polar impurities in nucleotide drugs.

The polymerized impurities in oxacillin sodium can induce allergic reaction, which seriously threaten the health of patients. Gel filtration chromatography (GFC) was widely used for the analysis of polymerized impurities at present, however it has drawbacks. To effectively control the polymerized impurities in oxacillin sodium, a high-performance size exclusion chromatography (HPSEC) method and a RP-HPLC method were established to replace the classical GFC method.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. The column switching two-dimension liquid chromatography was applied to eluted polymerized impurities from the HPSEC method for oxacillin sodium. The 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.The structures of twenty-five unknown impurities in oxacillin sodium were elucidated based on the high-resolution MSThe forming mechanisms of degradation impurities in oxacillin sodium were also 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.

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.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.Basso, Beattie and Bresnahan scores, hematoxylin and eosin (HE) 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).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.

Related substances, such as process-related substances and degradation products, may affect the efficacy of drugs and cause adverse reactions. Therefore, identifying and controlling them is of the importance. A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method has been developed for the separation and characterization of related substances in Candesartan cilexetil tablets. Chromatographic separation was achieved by using ZORBAX Eclipse XDB-C18 column (2.1 mm× 100 mm, 1.8 µm), with two mobile phases consisting of acetonitrile/water (57:43, v/v) (including 0.1% formic acid) as solvent A and acetonitrile/water (90:10, v/v) (including 0.1% formic acid) as solvent B in a gradient mode of elution. This method is applied to the separation of related substances in commercial tablets obtained from two companies. Moreover, accelerated stability studies (60℃, 90% RH and 4500lx for ten days) and forced degradation studies (acidic, alkaline, oxidative and photolytic degradation conditions) were conducted as described in ICH Q1. A total of eleven related substances (including one process-related impurity and ten degradation products) were detected. The related substances were characterized through Q-TOF fragmentation studies, and their fragmentation pathways were proposed. Among them, four related substances have not been reported in the literature yet, and one of them (RS7) was further confirmed by reference substance. In addition, plausible mechanisms for the formation of these related substances are discussed.

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.

Regulatory requirements for the analysis of 24 elemental impurities (Li, V, Cr, Co, Ni, Cu, As, Se, Mo, Ru, Rh, Pd, Ag, Cd, Sn, Sb, Ba, Os, Ir, Pt, Au, Hg, Tl and Pb) within pharmaceutical products are now included in the guidelines of the USP and ICH. Accurately screening all 24 elements in a single analysis is a challenging task due to different physico-chemical properties of the analytes and diverse compositions of available pharmaceuticals. In the present work, a new sample preparation method was developed for accurate analysis of all 24 USP target elements in five oral drug products (terbutaline sulfate tablets, sertraline hydrochloride tablets, cilostazol tablets, rosuvastatin calcium tablets and sitaglipin phosphate/metformin hydrochloride tablets). Method development particularly involved the stabilization and reliable determination of Os, Se and Au. Sample preparation was performed by microwave-assisted acid digestion using aqua regia followed by a two-step dilution procedure. The digest was first diluted with a simple thiourea solution in order to stabilize Os, and then an aliquot was diluted with a diluted acid solution to eliminate the matrix effect of Se and Au before quantitation by ICP-MS. The influences of different digestion solutions and different post-digestion diluents were investigated. The ICP-MS methodology covering the determination of all 24 elements was successfully validated in terms of linearity, accuracy, precision, the LOD and the LOQ. It is expected that the proposed method can be implemented for routine analysis of a broad spectrum of oral drug products, and help official medicine control laboratories in the quality assessment of drug products in the market.

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.

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.

Objectives: Spinal cord injury (SCI) is a disastrous central nervous system (CNS) disorder. The aim of this study was to explore the effects of repetitive trans-spinal magnetic stimulation (rTSMS) act on different segments of the spinal cord on movement function and expression of GAP43 and 5-HT in rats after acute cord injury and to preliminarily discuss the best treatment site of rTSMS, so as to provide theoretical foundation and experimental evidence for the clinical application of rTSMS in spinal cord injury. Methods: In the present study, we used a rat model of T10 laminectomy with transient violent oppression by aneurysm clip. The rats were classified into A group (sham surgery), B group (acute SCI without stimulation), C group (T6 segment stimulation), D group (T10 segment stimulation) and E group (L2 segment stimulation). Results: In vivo the magnetic stimulation was found to protect motor function and alleviate myelin sheath damage, decrease the expression levels of NgR and Nogo-A, increase the expression levels of growth-associated protein-43 (GAP43) and 5-hydroxytryptamine (5-HT), and inhibite TUNEL-positive cells as well as the expressions of apoptosis-related protein of rats following 8 weeks post-operation.Conclusions: This study suggests that rTSMS can promote the expression of GAP-43 and 5-HT and axonal regeneration in the spinal cord which is beneficial to the recovery of motor function after acute spinal cord injury.

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.

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.

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.

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.

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.

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.