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.

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.

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.

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.

Impurity profile and spectrum characteristics of the isomers in cefamandole nafate using high- performance liquid chromatography/high-performance size exclusion chromatography tandem ion trap/time-of-flight mass spectrometry.

RATIONALE: Reversed-phase, high-performance liquid chromatography (RP-HPLC) and high-performance size exclusion chromatography (HPSEC) methods were developed to effectively separate unknown impurities and polymerized impurities in cefamandole nafate. The liquid chromatography-tandem ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) was applied to characterize the structures of the impurities. Ultraviolet (UV) spectrum characteristics and mass spectrum characteristics of △3 -isomer and 7-epimer in cefamandole nafate were studied to distinguish the isomers. METHODS: RPLC-IT-TOF-MS was used to characterize the structures of unknown impurities and polymerized impurities eluted from the C18 column. On this basis, the two-dimensional (2D) HPSEC-IT-TOF-MS was used to confirm the structures of polymerized impurities eluted from the TSK-gel G2000SWxl column. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of the impurities. RESULTS: The structures of 19 unknown impurities in cefamandole nafate were elucidated based on the high-resolution MSn data with both positive and negative modes, assisted by the UV spectra and stress testing, of which 2 impurities were polymerized impurities. Cefamandole nafate produced a series of degradation impurities, and another principal component cefamandole acid also produced a series of similar degradation impurities. The disciplines between mass fragmentation pattern/UV spectrum and structure for △3 -isomer and 7-epimer were presented to distinguish their structures. CONCLUSIONS: The results of this study provided a scientific basis for the improvement of official monographs in pharmacopoeias to effectively control the impurities and ensure drug safety for the public. This study also revealed the formation mechanisms of degradation impurities in cefamandole nafate, which may guide industry to improve the manufacturing process and storage conditions to reduce the content of impurities in products.

Quantitative analysis of the impurities in Etimicin using hydrophilic interaction liquid chromatography coupled with charged aerosol detector.

Etimicin is a typical aminoglycoside antibiotic (AG). High performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD) method is a commonly used method for determining impurities in Etimicin. However, due to the poor reproducibility, low sensitivity and narrow linear range of the ELSD, high-throughput quantitative analysis of impurities in Etimicin currently poses a challenge. In this study, a sensitive method using hydrophilic interaction liquid chromatography coupled with charged aerosol detector (HILIC-CAD) was developed for the analysis of the impurities in Etimicin. The liquid phase conditions for determination impurities in Etimicin were optimized using Box Behnken design (BBD) and response surface methodology (RSM), resulting in satisfactory separation and optimal CAD output signal. We also studied the influence of CAD parameters on the signal-to-noise ratio and linearity of Etimicin and its impurities. This method has also been proven to be effective in separating impurities from two other typical AGs, Isepamicin and Amikacin. In the method validation, the coefficient of determination (R2) of Etimicin, Isepamicin and Amikacin and their impurities were all greater than 0.999, within the range of 0.5-50 µg/mL. The average recoveries of the impurities of three typical AGs were 99.03 %-101.22 %, RSDs all were less than 2.5 % for intra-day and inter-day precision, with good precision and accuracy. The developed HILIC-CAD quantification method was sensitive, accurate and highly selective for quantitative analysis of impurities in the AGs without need ion-pairing reagents, which is ensure the public medication safety. The method is first reported application of HILIC-CAD method for quantitative analysis of the impurities in AGs.

Sex-specific impacts of thimerosal on the behaviors and brain monoaminergic systems in zebrafish (Danio rerio).

Thimerosal (THI) is the most widely used form of organic mercury in pharmaceutical and personal care products, and has become a major source of ethylmercury pollution in aquatic ecosystems. However, knowledge about its potential risk to aquatic species is limited. In this study, zebrafish were exposed to THI for 7 days, and variations in their behavioral traits, brain monoaminergic neurotransmitter contents, and related gene expression were investigated. After the 7-day exposure, THI reduced locomotor activity and thigmotaxis in males but not females. Exposure to THI increased the social interaction between females but decreased that between males. The THI exposure also significantly reduced the serotonin (5-HT), 5-hydroxyindoleacetic acid, dopamine (DA), and 3,4-dihydroxyphenylacetic acid contents in the brain of males, but only significantly decreased the DA content in females. Correlation analysis revealed that the neurochemical alterations in the brain of zebrafish play critical roles in the behavioral abnormalities induced by THI exposure. Moreover, THI also significantly altered the expression of some genes associated with the synthesis, metabolism, and receptor binding of 5-HT and DA in the brain of zebrafish. The differences in these gene expressions between female and male zebrafish exposed to THI seem to be an important mechanism underlying their sex-specific responses to this chemical. This is the first report on the sex-specific effects of THI on behaviors and brain monoaminergic neurotransmitter contents in zebrafish, which can further improve our understanding of its toxic effects on teleost.

Universal response method for accurate quantitative analysis of the impurities in quinolone antibiotics using liquid chromatography coupled with diode array detector and charged aerosol detector.

HPLC method is the standard method for the separation and quantification of impurities from quinolone antibiotics. However, due to the large differences in the UV absorption of the impurities in quinolone antibiotics, quantitative analysis without the availability of corresponding reference substances currently poses a challenge. A sensitive and direct method using high performance liquid chromatography coupled with diode array detector and charged aerosol detector (HPLC-DAD-CAD) was developed for the analysis of impurities in quinolone antibiotics. The chromatographic conditions were optimized for good separation and output signal of CAD detector by response surface method (RSM). The systematic variation of CAD parameter settings, such as nebulization temperature, filter constant and power function value (PFV), were used to study the effect of on the detector response of signal-to-noise ratios (S/N) and linearity for ofloxacin, ciprofloxacin and their impurities. In the method validation, good linearity of each component was obtained with coefficient of determination (r) greater than 0.999 in the range of 0.5-300 µg mL-1. The average recoveries of each component were 99.02-102.39 % by DAD, were 98.22-101.91 % by CAD, RSDs were less than 2.5 % for intra-day and inter-day precision by DAD-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 and could unbiasedly and directly detect the impurities in quinolone antibiotics. The method is first reported application of HPLC-DAD-CAD method for the analysis of impurities in quinolone antibiotics and it can be used for quality control of quinolone antibiotics.

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.

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.

Functional screening and rational design of compounds targeting GPR132 to treat diabetes.

Chronic inflammation due to islet-residing macrophages plays key roles in the development of type 2 diabetes mellitus. By systematically profiling intra-islet lipid-transmembrane receptor signalling in islet-resident macrophages, we identified endogenous 9(S)-hydroxy-10,12-octadecadienoic acid-G-protein-coupled receptor 132 (GPR132)-Gi signalling as a significant contributor to islet macrophage reprogramming and found that GPR132 deficiency in macrophages reversed metabolic disorders in mice fed a high-fat diet. The cryo-electron microscopy structures of GPR132 bound with two endogenous agonists, N-palmitoylglycine and 9(S)-hydroxy-10,12-octadecadienoic acid, enabled us to rationally design both GPR132 agonists and antagonists with high potency and selectivity through stepwise translational approaches. We ultimately identified a selective GPR132 antagonist, NOX-6-18, that modulates macrophage reprogramming within pancreatic islets, decreases weight gain and enhances glucose metabolism in mice fed a high-fat diet. Our study not only illustrates that intra-islet lipid signalling contributes to islet macrophage reprogramming but also provides a broadly applicable strategy for the identification of important G-protein-coupled receptor targets in pathophysiological processes, followed by the rational design of therapeutic leads for refractory diseases such as diabetes.

Study of the polymerized impurities in cefotaxime sodium and cefepime by applying various chromatographic modes coupled with ion trap/time-of-flight mass spectrometry.

Polymerized impurities in ß-lactam antibiotics can induce allergic reaction, which seriously threaten the health of patients. In order to study the polymerized impurities in cefotaxime sodium and cefepime, a HPSEC method with TSK-gel G2000SWxl column and a RP-HPLC method with C18 column were established to replace the classical gel filtration chromatography with Sephadex G-10 gel as stationary phase. Two-dimensional (2D) liquid chromatography was employed to further investigate the HPSEC and RP-HPLC method and ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities eluted from the two methods. The structures of the polymerized impurities in cefotaxime sodium and cefepime were deduced based on the MSn data, six of which were previously unknown. The corresponding relationship of impurities between the two methods was established, and the specificity of the two methods was compared. The results showed that the polymerized impurities in cefotaxime sodium and cefepime were co-eluted with other small molecular weight impurities with high polarity in HPSEC, leading to a poor specificity. The newly established RP-HPLC methods could effectively separate and detect polymerized impurities and were suitable for the quality control in daily analysis.

Analysis of the polymerized impurities in cefmetazole sodium based on novel separation principle by liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

To effectively control the polymerized impurities in cefmetazole sodium, novel high performance gel filtration chromatography (HPSEC) with TSK-gel G2000SWxl column and RP-HPLC method with C18 column were used in replace of classical gel filtration chromatography with Sephadex G-10 gel. By studying the chromatographic behavior of polymerized impurities in both chromatographic systems with different chromatographic separation principles, the polymerized impurities in cefmetazole sodium were separated and detected effectively. The two-dimensional liquid chromatography tandem ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) was applied to characterize the structures of polymerized impurities eluted from HPSEC method, and liquid chromatography tandem ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and other unknown impurities eluted from RP-HPLC method. The structures of fourteen unknown impurities in cefmetazole sodium were deduced based on the MS n data, nine of which were polymerized impurities. The corresponding relationship between impurities in the HPSEC method and RP-HPLC method was established, and the specificity of the two methods was evaluated. The RP-HPLC method for analysis of the polymerized impurities has higher column efficiency and specificity than the HPSEC method. The RP-HPLC method is suitable for quality control of the polymerized impurities in cefmetazole sodium. The forming mechanisms of degradation impurities in cefmetazole sodium were also studied.

In vitro and in vivo safety studies indicate that R15, a synthetic polyarginine peptide, could safely reverse the effects of unfractionated heparin.

Unfractionated heparin (UFH) is an anionic glycosaminoglycan that is widely used to prevent blood clotting. However, in certain cases, unwanted side effects can require it to be neutralized. Protamine sulfate (PS), a basic peptide rich in arginine, is the only approved antagonist for UFH neutralization. Many adverse reactions occur with the clinical application of PS, including systemic hypotension, pulmonary hypertension, and anaphylaxis. We previously described R15, a linear peptide composed of 15 arginine molecules, as a potential UFH antagonist. In this study, the in-depth safety of R15 was explored to reveal its merits and associated risks in comparison with PS. In vitro safety studies investigated the interactions of R15 with erythrocytes, fibrin, complement, and rat plasma. In vivo safety studies explored potential toxicity and immunogenicity of R15 and the UFH-R15 complex. Results showed that both PS and R15 can induce erythrocyte aggregation, thicken fibrin fibers, activate complement, and cause anticoagulation in a concentration-dependent manner. However, those influences weakened in whole blood or in live animals and were avoided when R15 was in a complex with UFH. We found dramatically enhanced complement activation when there was excess UFH in analyses involving UFH-PS complexes, and a slight increase in those involving UFH-R15 complexes. Within 2 h, R15 was degraded in rat plasma in vitro, whereas PS was not. Enhanced creatinine was found after a single intravenous injection of PS or R15 (900 U·kg-1 , body weight), suggesting possible abnormal renal function. The UFH-PS complex, but not the UFH-R15 complex, exhibited obvious immunogenicity. In conclusion, R15 is nonimmunogenic and potentially safe at a therapeutic dose to reverse the effects of UFH.

Preclinical pharmacokinetics of M10 after intragastrical administration of M10-H and M10-Na in Wistar rats.

As a myricetin derivative, M10 is a potent agent of anti-chronic colonic inflammation. It has better activity than myricetin in preventing azoxymethane/dextran sulfate sodium - induced ulcerative colitis. Here, we introduce a sensitive quantification method based on ultra performance liquid chromatography-tandem mass spectrometry for the determination of M10-H and M10-Na in Wistar rat plasma. Samples were treated with L - ascorbic acid and phosphate buffer solution to maintain stability and with acetonitrile to remove the proteins in the plasma. The supernatant was separated with BEH C18 column and eluted with ultrapure water and acetonitrile both containing 0.1% formic acid. The detection was performed by a triple quadrupole mass spectrometer with positive electrospray ionization mode in multiple reactive monitoring. This method was validated for the carryover effect, selectivity, accuracy, precision, matrix effect, stability, and recovery. A linear correlation was established between concentration and response by the calibration curves over 10-2000 ng·mL-1 (r > 0.99). This method was applied to a pharmacokinetic study of intragastrical administration of M10-H and M10-Na in Wistar rats. In addition, the relative bioavailability of M10-H to M10-Na in Wistar rats was 60 ±â€¯19%, calculated by the ratio of area under concentration (AUC) of M10-H to M10-Na after intragastrical administration of a single dose (100 mg·kg-1 for M10-H and M10-Na, respectively) in Wistar rats.