[Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide].

Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom, and the main active ingredients are snake venom thrombin-like enzymes (svTLEs). The svTLEs derived from different snake species differ in their structures, hemostatic mechanisms, and pharmacological effects. Therefore, accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology, the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study, and an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox, enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (Nano LC-Q-Exactive-MS). The mass spectrum data were analyzed by Proteome Discoverer 2.2 software. The maker peptide "EAYNGLPAK", which characterized the svTLE from Bothrops atrox, was finally screened and validated by comparison of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. For the marker peptide, the enzymolysis temperature, enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 4 h; and amount of enzyme, 10 µL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly, 20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then, 25 mmol/L ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted; subsequently, addition of 10 µL trypsin solution was added, followed by shaking, and the mixture was placed in an incubator for 4 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, centrifuged at 12000 r/min for 10 min, and analyzed by LC-MS. Separation was performed on the UPLC system with a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm, 3.0 µm) under the gradient elution of acetonitrile containing 0.1% (v/v) acetic acid and water containing 0.1%(v/v) acetic acid, at a flow rate of 0.3 mL/min, column temperature of 30 ℃, and injection volume of 2 µL. The maker peptides were determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes using the external standard curve method. The detection ions were m/z 481.9> 315.2 and 481.9> 485.2. There was a good linear relationship between the mass concentration of the marker peptide and the chromatographic peak area in the range of 2.5-30 ng/mL, and the correlation coefficient (r) was 0.9996, The limit of detection (S/N=3) and limit of quantification (S/N=10) were 2.5 mg/kg and 6.25 mg/kg, respectively. At spiked levels of 40, 80, and 120 mg/kg, the recoveries of the marker peptides were 95.5%-101.9%, while the relative standard deviations (RSDs) of the results for parallel analyses at various spiked levels were 1.1%-3.2%. The developed method is simple, rapid, sensitive, and specific, and it can be used for the identification of Bothrops atrox venom species and determination of the svTLE content. The findings of this study would help ensure the quality of hemocoagulase products from the relevant source and provide a reference for the quality control of other snake venom products.

[Determination of dimethyl sulfate genotoxic impurities in tertiary amine drugs by ultra-high performance liquid chromatography-tandem mass spectrometry].

Dimethyl sulfate is an important chemical raw material that is widely used in the synthesis of drugs, dyes, spices, and pesticides. The highly toxic and corrosive dimethyl sulfate residue in medicines is harmful to the human body, and hence, the residue level should be strictly controlled. Traditional detection methods use high-purity acetonitrile and anhydrous as the solvents, which limits the choice of detection solvents and degrades the versatility and accuracy of detection. Therefore, a simple and accurate method for the determination of dimethyl sulfate residues is urgently needed. Dimethyl sulfate is usually detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with pyridine as the methylation substrate. In this study, a new method for the detection of dimethyl sulfate was established using tertiary amines such as aminophenazone, which has many advantages over pyridine, as the methylation substrate. For example, the hybrid orbital and electron cloud of the N atom are different, resulting in stronger nucleophilicity of aminophenazone. High temperatures that are detrimental to the stability of dimethyl sulfate are not required when using aminophenazone, and the aliphatic quaternary ammonium salt product is more stable, with good stability, low interference, good ionization properties, and high response. The separation was performed on a Waters Atlantis HILIC C18 column (100 mm×2.1 mm, 3.0 µm) using a mobile phase consisting of 10 mmol/L ammonium acetate solution-0.1% formic acid methanol solution (50∶50, v/v) at a flow rate of 0.3 mL/min. The column temperature was set at 40 ℃, and the sample size was 1 µL. Dimethyl sulfate was determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes. Dimethyl sulfate showed good linear relationships within the range of 0.9935 to 7.9480 ng/mL (r=0.9997). The limit of detection and limit of quantification for dimethyl sulfate were 0.50 ng/mL and 1.15 ng/mL, respectively. The recoveries (n=3)of dimethyl sulfate were 94.9% to 106.4%. The relative standard deviations (RSDs) were 1.44% to 5.51%. The RSD of the methylated aminophenazone peak area was 4.32%, indicating good stability of the reaction product. Dimethyl sulfate genotoxic impurities were not detected in 9 batches of aminophenazone, caffeine, and tegafur samples, which indicated that the drug manufacturers paid attention to the control of these impurities. The proposed method is advantageous over the existing techniques in terms of the better ion peak shape and higher molecular weight, without interference from other fragments. The method is specific, sensitive, simple, rapid, and accurate, and it can be used for the determination of dimethyl sulfate genotoxic impurities in aminophenazone and other medicines.

A Species-Specific Strategy for the Identification of Hemocoagulase Agkistrodon halys pallas Based on LC-MS/MS-MRM.

Hemocoagulase Agkistrodon halys pallas is a complex mixture composed of snake venom thrombin-like enzymes (svTLEs) and small amounts of thrombokinase-like enzymes. It has been widely used as a hemostatic with rapidly growing marketing due to its advantage of localized clotting fibrinogen other than systemic coagulation. However, svTLEs from different species have various structures, functions, and hemostatic mechanisms. To ensure the efficacy and safety of Hemocoagulase Agkistrodon halys pallas, an exclusive and sensitive method has been developed to identify specific marker peptides based on liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM) mode. By combining transcriptomics and proteomics, a series of species-specific peptides of Agkistrodon halys pallas were predicted and examined by LC-MS/MS. After reduction, alkylation, and tryptic digestion were performed on Hemocoagulase Agkistrodon halys pallas, a target peptide TLCAGVMEGGIDTCNR was analyzed by LC-MS/MS-MRM. It offers a new and effective approach for the quality control of Hemocoagulase Agkistrodon halys pallas products. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy, and throughput. The strategy can also be applied in studying other important protein-based medicines.

[Determination of donkey skin ingredients in Asini Corii Colla by ultra-high performance liquid chromatography-tandem mass spectrometry].

In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sample was weighed and placed in a 50 mL volumetric flask; then, 1% ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted into a 5 mL volumetric flask, followed by the addition of 1.0 mL trypsin solution and 100 µL mixed internal standard working solution. This mixture was diluted to the scale using 1% ammonium bicarbonate solution, shaken, and placed in an incubator for 16 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, filtered through a 0.22 µm membrane, and analyzed by LC-MS. Separation was performed on an UPLC system with a BEH C18 column (100 mm×2.1 mm, 2.5 µm) under gradient elution using acetonitrile containing 0.1% (v/v) formic acid (B) and water containing 0.1% (v/v) formic acid (A) as the mobile phases at a flow rate of 0.3 mL/min. The column temperature was 30 ℃, and the sample size was 2 µL. The gradient elution conditions are: 0-1 min, 10%B; 1-5 min, 10%B-30%B; 5-5.1 min, 30%B-70%B; 5.1-7 min, 70%B; 7-7.1 min, 70%B-10%B; 7.1-10 min, 10%B. The marker peptides were determined in positive electrospray ionization (ESI +) and multiple reaction monitoring (MRM) modes using the isotopic internal standard method. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 16 h; and amount of enzyme, 1 mL. The two marker peptides showed good linearities in the range of 50 to 1250 mg/L; the correlation coefficients (r) were greater than 0.996, and the limits of quantitation (S/N=10) were 20 mg/kg. At spiked levels of 300 mg/kg, 600 mg/kg, and 900 mg/kg, the average recovery ratios of the two marker peptides were 103.2% to 108.3%, while the relative standard deviations (RSDs) of 1.0%-3.0%. This method was favorable for testing actual samples. Asini Corii Colla from 29 production companies was detected by this method, and the sum contents of the two marker peptides was different because the production process and raw materials were different. The sum contents of the samples were 0.096% to 0.180% with an average of 0.151%. The developed method is simple, reliable, and reproducible, and it is suitable for detecting the donkey hide components Asini Corii Colla.

[Determination of ambroxol hydrochloride in human plasma by ultra high performance liquid chromatography-tandem mass spectrometry and bioequivalence evaluation of its preparation].

A rapid, simple and sensitive ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the determination of ambroxol hydrochloride in human plasma, and bioequivalence of its preparation was evaluated. The 50 µL-plasma sample was treated with methanol for protein precipitation, while ambroxol-d5 was used as an internal standard (IS). The separation was carried out on a Waters XBridge BEH C18 column (50 mm×2.1 mm, 2.5 µm) by gradient elution at a flow rate of 0.4 mL/min, with 0.1% (v/v) formic acid aqueous solution and methanol containing 0.1% (v/v) formic acid as the mobile phases. The analyte was detected using an electrospray ionization source in positive ion multiple reaction monitoring (MRM) mode. The calibration curves were linear in the range of 2-400 ng/mL (r=0.998). The intra- and inter-run accuracies were 97.1%-108.7%, the intra- and inter-run precisions were 1.0%-5.6%. The method was applied to the determination of the plasma concentration of the six healthy subjects after the oral administration of 30 mg of test and reference preparations. The bioavailability was (102.3±14.8)%. The 90% confidence intervals of the test preparation's pharmacokinetic parameters were 80.0%-125.0% of the reference preparation's corresponding parameters. Thus, it is proved that the test preparation and reference preparation are bioequivalent.

Simultaneous analysis of 23 illegal adulterated aphrodisiac chemical ingredients in health foods and Chinese traditional patent medicines by ultrahigh performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

This paper presents an application of ultra high-performance liquid-chromatography-quadrupole-TOF high resolution mass spectrometry (UHPLC-Q-TOF HRMS) for simultaneous analysis of 23 illegal adulterated aphrodisiac type chemical ingredients in health foods and Chinese Traditional Patent Medicines (CTPMs). The mass spectrometer was operated in Information Dependent Acquisition (IDA) mode, which provides crucial information for the elemental composition analysis, structure elucidation and quantitative analysis simultaneously. Quantitative analysis was performed using the peak areas of the precursor ions in the XICs. The method validation included assessment of selectivity, sensitivity, calibration curve, accuracy, precision, recovery, matrix effect and stability. The results show good linear relationship with the concentrations of the analytes over wide concentration ranges (e.g., 0.05-10 µg/g for sildenafil) as all the fitting coefficients of determination r2 are >0.9984. The detection limits (LODs) were in the range of 0.002-0.1 µg/g. The recoveries were able to reach 82.5-103.6%, while the matrix effects ranged from 87.7 to 109.3%. The intra- and inter-day accuracies were in the range of 82.3-113.8%, while the intra- and inter-day precision ranged from 0.4 to 13.6%. Among 40 batches of health foods and 32 batches of CTPMs (including 28 capsules, 32 tablets, 10 liquid and 2 pills) samples, 28 batches of heath foods were positive. The detected chemical ingredients involved sildenafil, tadalafil, aildenafil and sulfoaildenafil. This method can be used for the screening, identification and quantification of illegal adulterated aphrodisiac chemical ingredients in health foods and CTPMs. Moreover, the LC-Q-TOF MS is very useful to structural elucidation of unknown compound.

Synthesis and antibacterial activity of novel 4''-O-arylalkylcarbamoyl and 4''-O-((arylalkylamino)-4-oxo-butyl)carbamoyl clarithromycin derivatives.

Novel series of novel 4''-O-arylalkylcarbamoyl and 4''-O-((arylalkylamino)-4-oxo-butyl)carbamoyl clarithromycin derivatives were designed, synthesized and evaluated for their in vitro antibacterial activities. These derivatives retained excellent activity against the erythromycin-susceptible strains and showed significantly improved activity against all of the tested erythromycin-resistant strains. Among them, compound 4c was the most effective (0.06 microg/mL) against Streptococcus pneumonia encoded by the erm gene and compound 4a was had the most potent activity (0.25 microg/mL) against S. pneumonia encoded by the erm and mef genes.

Synthesis and antibacterial activity of 4'',11-di-O-arylalkylcarbamoyl azithromycin derivatives.

A series of new 4'',11-di-O-arylalkylcarbamoyl azithromycin derivatives were designed, synthesized and evaluated for their in vitro antibacterial activities. Some derivatives exhibited greatly improved activity against erythromycin-resistant bacteria. Among them, compounds 5f and 5k were found to have potent activity against erythromycin-resistant Streptococcus pneumoniae whose resistance was encoded by the erm or mef gene.