Study on the impurity profiles of cloxacillin and flucloxacillin based on liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

RATIONALE: Cloxacillin and flucloxacillin are prone to degradation and polymerization in humid and hot environments, and their polymers have long been recognized to trigger allergic manifestations. A series of the degradation and polymerized impurities in cloxacillin and flucloxacillin were separated and characterized to ensure safe use of these drugs by the public. METHODS: By studying the chromatographic behavior of the degradation impurities and polymerized impurities in reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution, the impurities in cloxacillin and flucloxacillin were effectively separated and eluted. RP-HPLC tandem ion trap/time-of-flight mass spectrometry (MS) was applied to characterize the structures of unknown impurities eluted from the RP-HPLC methods for cloxacillin and flucloxacillin. The mechanisms of formation of the impurities in cloxacillin and flucloxacillin were also investigated. RESULTS: The structures of 10 unknown impurities in cloxacillin and 8 unknown impurities in flucloxacillin were elucidated based on the high-resolution MSn data at positive and negative modes, respectively. Six polymerized impurities were found and characterized, of which three were from the polymerization of cloxacillin and three were from the polymerization of flucloxacillin. CONCLUSIONS: The study on the impurity profiles of cloxacillin and flucloxacillin provided a scientific basis for improving their production processes and quality control.

Universal response method for the quantitative analysis of photodegradation impurities in lomefloxacin hydrochloride ear drops by liquid chromatography coupled with charged aerosol detector.

In terms of risk assessment especially for the impurities with different ultraviolet responses, quantitative analysis without the availability of corresponding reference substances currently poses a challenge. In this study, a universal response method was established for the quantitative analysis of photodegradable impurities in lomefloxacin hydrochloride ear drops by high performance liquid chromatography-charged aerosol detector (HPLC-CAD) for the first time. The chromatographic conditions and CAD parameters were optimized for a good separation and sensitivity. The uniform response of developed method was validated by impurity reference substances with different ultraviolet responses. In the gradient compensation HPLC-CAD method validation, good linearities were obtained with coefficient of determination (R2) all greater than 0.999 for lomefloxacin and impurity reference substances. The average recoveries of the impurities were 98.63%- 102.18% by UV and 97.92%- 102.57% by CAD, respectively. RSDs all were less than 2.5% for intra-day and inter-day precision by UV and CAD, with good precision and accuracy. The correction factor experimental results showed that the developed method provided a uniform response to the impurities with differences chromophores in lomefloxacin. The effects of packaging materials and excipients on the photodegradation were also investigated using the developed method. The results of correlation analysis showed that the packaging materials with low light transmittance and the organic excipients (glycerol and ethanol) could significantly improve the stability of lomefloxacin hydrochloride ear drops. The developed HPLC-CAD quantification method was a reliable and universal response method for quantitative analysis of impurities in the lomefloxacin. This study also revealed the key factors affecting the photodegradation of lomefloxacin hydrochloride ear drops, which guided enterprises to improve drug prescription and packaging materials and ensure the public medication safety.

Study on the impurity profile and influencing factors of photodegradation in non-aqueous ofloxacin ear drops using liquid chromatography combined with ion trap/time-of-flight mass spectrometry.

Ofloxacin ear drops contain a large proportion of organic solvents, which have a great effect on the photodegradation of ofloxacin. The photodegradation impurities of ofloxacin in aqueous solution has been studied, however, the photodegradation of ofloxacin in non-aqueous solution with a high proportion of organic solvents has not been reported. In this article, the impurity profile in non-aqueous ofloxacin ear drops was studied for further improvement of official monograph in pharmacopoeia and quality control of drug. The liquid chromatography combined with ion trap/time-of-flight mass spectrometry was applied to separate and characterize the structures of the impurities in non-aqueous ofloxacin ear drops. Mass fragmentation pattern of ofloxacin and its impurities were studied. The structures of seventeen impurities in ofloxacin ear drops were elucidated based on the high-resolution MSn data in positive ion modes, and ten of them were unknown impurities. The results showed that the impurity profile of non-aqueous ofloxacin solution was significantly different from that of aqueous ofloxacin solution. The effects of packaging materials and excipients on the photodegradation of ofloxacin ear drops were also investigated. The results of correlation analysis showed that the packaging materials with low light transmittance could reduce the light degradation, and ethanol of excipients could significantly decrease the light stability of ofloxacin ear drops. This study revealed the impurity profile and key factors affecting the photodegradation of non-aqueous ofloxacin ear drops, and guided enterprises to improve drug prescription and packaging materials to ensure the safety of drug use by the public.

Determination of elemental impurities in lomefloxacin hydrochloride ear drops by inductively coupled plasma-mass spectrometry using oxygen reaction mode and study of their catalytic effect on photodegradation reaction.

RATIONALE: This study developed a method for the simultaneous determination of 12 element impurities in lomefloxacin hydrochloride ear drops using inductively coupled plasma-mass spectrometry (ICP-MS) in accordance with the requirements of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q3D. However, this product contains high concentrations of organic compounds such as glycerol and ethanol, and soluble carbon interference exists when it is determined by direct injection method. Therefore, we applied ICP-MS using oxygen reaction mode to reduce the effect of high organic matter on plasma body. The catalytic effect of trace metal ions on the photodegradation of lomefloxacin hydrochloride was also investigated. METHODS: The sample was dissolved, diluted, and directly determined by direct injection using oxygen reaction mode of ICP-MS with 45 Sc, 73 Ge, and 115 In as internal standards. Direct injection using oxygen reaction mode was equipped with argon-oxygen mixer. To investigate the catalytic effect of metal ions on photodegradation, a series of combinations of lomefloxacin hydrochloride mixed with different metal ions were designed. After being irradiated under ultraviolet light for 10 days, the photodegraded impurities in combinations were determined using high-performance liquid chromatography. RESULTS: The linear relationships in the corresponding concentration range for 12 element impurities were good (r > 0.9993). Limits of detection were between 0.002 and 1.294 ng ml-1 . The average recoveries were 93.7%-108.2%, and the precision relative standard deviation was 0.04%-0.55% (n = 6). The experimental results showed that metal ions had a certain catalytic effect on photodegradation of lomefloxacin when EDTA, Mg2+ , and Cu2+ coexisted. CONCLUSIONS: ICP-MS using oxygen reaction mode is suitable for the direct determination of the sample rich in organic matter without digestion and can effectively eliminate the interference of high organic matter in this product. The established method was rapid, sensitive, and accurate and can be used for the quality control of elemental impurities in lomefloxacin hydrochloride ear drops.

Comparative Pharmacokinetic Study of Forchlorfenuron in Adult and Juvenile Rats.

Forchlorfenuron (CPPU) is a plant growth regulator extensively used in agriculture. However, studies on CPPU pharmacokinetics are lacking. We established and validated a rapid, sensitive, and accurate liquid chromatography-mass spectrometry method for CPPU detection in rat plasma. CPPU pharmacokinetics was evaluated in adult and juvenile rats orally treated with 10, 30, and 90 mg/kg of the compound. The area under the plasma drug concentration-time curve from 0 to 24 h (AUC), at the final time point sampled (AUC0-t), and the maximum drug concentration of CPPU increased in a dose-dependent manner. The pharmacokinetic parameters AUC0-t and absolute bioavailability were higher in the juvenile rats than in adult rats. The mean residence time and AUC0-t of juvenile rats in the gavage groups, except for the 10 mg/kg dose, were significantly higher in comparison to those observed for adult rats (p < 0.001). The plasma clearance of CPPU in juvenile rats was slightly lower than that in the adult rats. Taken together, juvenile rats were more sensitive to CPPU than adult rats, which indicates potential safety risks of CPPU in minors.

Structural optimization towards promising ß-methyl-4-acrylamido quinoline derivatives as PI3K/mTOR dual inhibitors for anti-cancer therapy: The in vitro and in vivo biological evaluation.

Built upon the 4-acrylamido quinoline derivative 4, a previously discovered PI3K/mTOR dual inhibitor, structural modification was undertaken in this study with the attempt to improve its oral exposure via introducing steric hindrance to the 4-acrylamido functionality. Consequently, 14d, as the representative among the synthesized compounds, exhibited IC50 values of 0.80, 0.67, 1.30, 1.30 and 5.0 nM against PI3Kα, PI3Kß, PI3Kγ, PI3Kδ and mTOR, respectively. Besides, 14d displayed comparable anti-proliferative activity against both PC3 and U87MG cell lines to that of the positive reference GSK2126458 with respective GI50 value of 0.36 and 0.14 µM. Kinase selectivity assay showed that 14d was selective to PI3K family. In U87MG cells, 14d can strongly down-regulate PI3K/Akt/mTOR pathway via blocking both PI3K and mTOR signaling at the concentration as low as 25 nM. Importantly, following a PO dose of 5 mg/kg in male SD rats, 14d displayed favorable oral exposure (AUC0-t = 1336.16 h × ng/mL, AUC0-∞ = 1447.63 h × ng/mL) and high maximum plasma concentration (Cmax = 903.00 ng/mL). In a U87MG glioblastoma xenograft model, tumor growth inhibition of 93.5% and tumor regression were observed at PO dose of 30 and 60 mg/kg, respectively. Meanwhile, no overt loss of body weight was observed in the 14d-treated groups. Taken together, 14d, by virtue of its attractive performance, merits further development as a potential anti-tumor candidate.

Metabolic networks of plasma and joint fluid base on differential correlation.

Whether osteoarthritis (OA) is a systemic metabolic disorder remains controversial. The aim of this study was to investigate the metabolic characteristics between plasma and knee joint fluid (JF) of patients with advanced OA using a differential correlation metabolic (DCM) networks approach. Plasma and JF were collected during the joint replacement surgery of patients with knee OA. The biological samples were pretreated with standard procedures for metabolite analysis. The metabolic profiling was conducted by means of liquid mass spectrometry coupled with a AbsoluteIDQ kit. A DCM network approach was adopted for analyzing the metabolomics data between the plasma and JF. The variation in the correlation of the pairwise metabolites was quantified across the plasma and JF samples, and networks analysis was used to characterize the difference in the correlations of the metabolites from the two sample types. Core metabolites that played an important role in the DCM networks were identified via topological analysis. One hundred advanced OA patients (50 men and 50 women) were included in this study, with an average age of 65.0 ± 7.6 years (65.6 ± 7.1 years for females and 64.4 ± 8.1 years for males) and a mean BMI of 32.6 ± 5.8 kg/m2 (33.4 ± 6.3 kg/m2 for females and 31.7 ± 5.3 kg/m2 for males). Age and BMI matched between the male and female groups. One hundred and forty-five nodes, 567 edges, and 131 nodes, 407 edges were found in the DCM networks (p < 0.05) of the female and male groups, respectively. Six metabolites in the female group and 5 metabolites in the male group were identified as key nodes in the network. There was a significant difference in the differential correlation metabolism networks of plasma and JF that may be related to local joint metabolism. Focusing on these key metabolites may help uncover the pathogenesis of knee OA. In addition, the differential metabolic correlation between plasma and JF mostly overlapped, indicating that these common correlations of pairwise metabolites may be a reflection of systemic characteristics of JF and that most significant correlation variations were just a result of "housekeeping" biological reactions.

Methyl Gallate Improves Hyperuricemia Nephropathy Mice Through Inhibiting NLRP3 Pathway.

Hyperuricemia nephropathy (HN) is a form of chronic tubulointerstitial inflammation, caused by the deposition of monosodium urate crystals (MSU) in the distal collecting duct and medullary interstitium, associated with a secondary inflammatory reaction. Numerous published reports indicated that NLRP3 inflammasome pathway play crucial roles in HN symptoms. The present study aims to investigate the protective effects of methyl gallate on HN mice and the underlying mechanisms. An HN model was established by intraperitoneal injection of potassium oxide (PO) to assess the effect of methyl gallate on renal histopathological changes, renal function, cytokine levels and expressions of NLRP3-related protein in HN mice. Moreover, in vitro models of lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) and human peripheral blood mononuclear cells (PBMCs) were established to explore the mechanism of methyl gallate on NLRP3 inflammasome activation. The results showed that methyl gallate significantly ameliorated HN by inhibiting uric acid production and promoting uric acid excretion as well as ameliorating renal injury induced by NLRP3 activation. Mechanistically, methyl gallate is a direct NLRP3 inhibitor that inhibits NLRP3 inflammasome activation but has no effect on the activation of AIM2 or NLRC4 inflammasomes in macrophages. Furthermore, methyl gallate inhibited the assembly of NLRP3 inflammasomes by blocking the ROS over-generation and oligomerization of NLRP3. Methyl gallate was also active ex vivo against ATP-treated PBMCs and synovial fluid mononuclear cells from patients with gout. In conclusion, methyl gallate has a nephroprotective effect against PO-induced HN through blocking the oligomerization of NLRP3 and then exerting anti-inflammatory activity in the NLRP3-driven diseases.