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??OBJECTIVE To establish an HPLC-PAD method to determine the related substances of sisomicin sulfate injection and compare with the statutory method. METHODS IonPac AMG C₁₈(4.0 mm??150 mm, 3 ??m)chromatographic column was used with acetonitrile-0.1 mol??L^-1 trifluoroacetic acid (containing 0.025% of pentafluoropropionic acid, 5 mL of 50% NaOH solution without carbonate, pH of the aqueous solution adjusted to 2.3 with 50% NaOH solution.)as mobile phase at a flow rate of 0.7 mL??min^-1. NaOH solution of 0.76 mol??L^-1 was added post column at a flow rate of 0.35 mL??min^-1. The column temperature was maintaine at 30 ??. PAD detector was operated with the cell temperature set at 35 ??. The working electrode was a gold electrode (diameter of 3 mm)and a quadruple-potential waveform was selected as detection waveform. The reference electrode was Ag/AgCl, the detection potential was four potential. The determination result of the related substances of sisomicin sulfate injection was compared with that of the statutory method. RESULTS The peaks of sisomicin sulfate, gentamicin C_1a and netilmicin could be completely separated, and other impurities could also be effectively separated. The blank sample had no interferences. The LOD and LOQ of etimicin were found to be 2 and 6 ng respectively, and the RSD of precision test (n=6) was 0.9%. Paired-samples t-test showed significance levels of P=0.034, P=0.364 and P=0.605 for total amount of impurities (%), the biggest single impurity (%)and content (%)respectively between the statutory method and the method of HPLC-PAD. CONCLUSION Compared with the statutory method, this HPLC-PAD method shows higher sensitivity, and is accurate and reliable. It can be applied to the determination of related substances in sisomicin sulfate injection.     

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??OBJECTIVE To compare the HPLC-PAD method and HPLC-ELSD method for determination of related substances in spectinomycin hydrochloride for injection, and determine 31 batches of samples by the two methods. METHODS The following conditions were used for both methods:the analytical column was Apollo C₁₈ column (4.6 mm??250 mm,5 ??m), mobile phase was 0.1 mol??L^-1 trifluoroacetic acid solution, the column temperature was maintained at 30 ??, and the sample volume was 20 ??L. For the PAD detection, gold electrode (3 mm)was used as the detection electrode, Ag-AgCl as reference electrode, titanium alloy as counter electrode, and integrated amperometric detector was employed with four waveform detection potential. For the ELSD detection, the drift tube temperature was set at 110 ??, and the carrier gas flow rate was 2.6 L??min^-1, with gain of 1. RESULTS The two methods were consistent in the species and number of detected impurities. The detection limits of the PAD method and ELSD method were 2.4 and 72.8 ng, respectively (S/N=3). For the PAD method there existed a direct linear relationship in the concentration range of 0.15-150 ??g??mL^-1, while a double logarithmic linear relationship was shown in the concentration range of 17-170 ??g??mL^-1 for the ELSD method. When 31 batches of samples were determined for related substances by the two kinds of methods, the contents of impurity D and E were basically the same, but there were significant differences in the contents of impurity A and (4R)-dihydrospectinomycin. After correction, the contents of impurity A and total impurities determined by the PAD method were consistent with those by the ELSD method. CONCLUSION The two methods can both effectively detect the related substances of spectinomycin to achieve the purpose of controlling related substances. When using HPLC-PAD method, the impurity A and (4R)-dihydrospectinomycin should be adjusted by using response factor or the reference substances. The ELSD method needs to use double log linear regression.     

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??OBJECTIVE To establish an improved LC method combined with pulsed electrochemical detection for the analysis of etimicin sulfate. METHODS The mobile phase was composed of 40 mL of acetonitrile and 960 mL of an aqueous solution containing 15 mL??L^-1 of trifluoroacetic acid,500 ??L??L^-1 of pentafluoropropionic acid, 8 mL??L^-1of sodium hydroxide (50%) and 1.5 g??L^-1of sodium sulfate. The pH of the aqueous solution was adjusted to 3.5 with 50% NaOH solution. A pulsed electrochemical detector, which was kept at 35 ?? in a hot air oven was adopted. The electrochemical cell consisted of a working electrode, a pH-Ag/AgCl reference electrode and a titanium counter electrode. The working electrode was a gold electrode (diameter 3 mm)and a quadruple-potential waveform (QPW) was selected as detection waveform. The 0.8 mol??L^-1 NaOH solution was added post column at a flow rate of 0.4 mL??min^-1. RESULTS In total, 22 impurities could be separated. The LOD and LOQ of etimicin were found to be 2 ng and 6 ng respectively. The linearity of the calibration curve for etimicin ranged from 0.24 to 45 ??g??mL^-1 with a coefficient of correlation equal to 0.999 7. The repeatability RSDs (n=6) of the content and total impurities in one sample were 0.7% and 1.72% respectively. The inter-day repeatability RSDs (n=18) of the content and total impurities in one sample were 0.98% and 1.71% respectively. The sample solution was stable within 12 h. CONCLUSION Compared with previously published methods, this improved method shows higher sensitivity, better separation ability and robustness and has been incorporated by the Chinese Pharmacopoeia (Ch.P.) 2015 for analysis of etimicin sulfate.     

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??OBJECTIVE To establish a gradient elution method for determination of related substances of azithromycin for injection. METHODS Gradient elution was used for the analysis. A C₁₈ (CAPCELL PAK MG??, 4.6 mm??250 mm,5 ??m)column was used. The mobile phase A consisted of 0.05 mol??L^-1 K₂HPO₄ solution (pH 8.2, adjusted with 20% phosphoric acid)-acetonitrile (45??55), and the mobile phase B was methanol. The flow rate was 1.2 mL??min^-1, and the detection wavelength was set at 210 nm. The column temperature was maintained at 30 ??. The gradient elution program was as follows: 0-35 min, A:75%??95%; 35-64 min, A:75%; 64-65 min, A:95%??75%; 65-71 min, A:75%. RESULTS Azithromycin, near peaks and known impurities were well separated from each other. All acid radicals of azithromycin for injection did not influence the determination of related substances of azithromycin. CONCLUSION This method is more specific than the exising method for determination of related substances of azithromycin, which can more effectively control the quality of the drug.     

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??OBJECTIVE To establish an HPLC-HRMS/MS method for analyzing the structures and sources of the related substances in etimicin sulfate. METHODS The chromatographic separation was achieved on a broad pH range column with a basic elution system composed of[H₂O-ammonia-glacial acid(96??3.6??0.4)]-methanol(70??30). Twenty percent of the eluent was detected under positive electrospray ionization(ESI) by Q-Exactive mass spectrometry. The fragmentation pathways were elucidated according to the HRMS and HRMS/MS fragmentation of etimicin and some known compounds. Then the unknown related substances were identified by analyzing their HRMS and HRMS/MS fragmentation with the help of the rule. RESULTS Sixty-five related substances were detected by the HPLC-HRMS/MS method in the two samples from different companies,among which 38 were detected in the product of company A, 59 in that of company B, and 32 were detected for both companies. Fifty-four related substances were identified or deduced, and the other 11 were not able to be identified due to limited information. Based on the significant difference of impurity spectra between the two enterprises, the key parameters of the synthesis process were analyzed. CONCLUSION An HPLC-HRMS/MS method, which showes excellent accuracy, is developed to identify the related substances in etimicin sulfate. The resources and structures of the related substances are analyzed, which will be helpful to the process optimization.     

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??OBJECTIVE To establish an ion chromatography method for measuring the content of sodium sulfite as an antioxidant in etimicin sulfate injection. METHODS DionexIonPac AS11-HC (4 mm??250 mm) and DionexIonPac AG1-HC (4 mm??50 mm) were employed as anion analytical column and anion guard column to separate sodium sulfite and determine its content. Gradient elution was carried out with potassium hydroxide solution at the flow rate of 1.0 mL??min^-1. Conductivity detector was used with the suppressor current set at 99 mA, the conductivity pool was maintained at 35 ?? and the column temperature was maintained at 30 ??.RESULTS A good linear relationship was found between the peak response and the concentration range of 8-80 ??g??mL^-1. The detection limit was determined to be 0.02 ??g??mL^-1 (S/N=3), and the quantitation limit was 0.09 ??g??mL^-1 (S/N=10). CONCLUSION In this paper, we set up an analytical technique with such characteristics as rapidity, accuracy and reproducibility, which will serve to control the dosage of sodium sulfite. So far, it seems appropriate to use sodium sulfite as an antioxidant, but its amount in pharmaceutical preparations should be further optimized and decreased in the future.     

五氟苯基柱HPLC-PAD法测定硫酸巴龙霉素含量及有关物质

目的 建立一种用于直接测定硫酸巴龙霉素含量和有关物质的高效液相色谱-脉冲安培电化学检测器(HPLC-PAD)法。方法 采用Agilent Pursuit^®PFP(4.6 mm×250 mm,5 μm)色谱柱,以体积分数2.0%三氟乙酸溶液[含0.15%(体积分数)五氟丙酸,用50%氢氧化钠溶液(质量分数)调pH值至3.5]的水溶液为流动相,积分脉冲安培电化学检测器的四电位波形测定硫酸巴龙霉素有关物质。结果 五氟苯基柱比传统的十八烷基硅烷键合硅胶填充色谱柱分离效果更优,代表性样品中分离出21个峰,有巴龙霉素Ⅱ与其同分异构体巴龙霉素Ⅰ和另外的19个有关物质;在0.49~118.30 μg·mL^-1内巴龙霉素质量浓度与峰面积呈良好的线性关系(r>0.999 3),检测限(LOD)为0.49 μg·mL^-1,定量限(LOQ)为0.99 μg·mL^-1,重复性和精密度RSD均小于2.0%。结论 建立的HPLC-PAD方法专属性好、灵敏度高、稳定性强、耐用性好,为巴龙霉素质量控制和工艺优化研究提供了可靠的分析手段。     

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??OBJECTIVE To establish an LC/MSⁿ method for identification of the related substances in etimicn sulfate detected under the chromatographic condition described in Chinese Pharmacopoeia 2010.METHODS The HPLC separation was carried out on a Welch Ultimate LP-C₁₈ column(4.6 mm??300 mm,5 ??m)with mobile phase consisting of 0.2 mol??L^-1trifluoroacetic acid(containing 0.1% propionic acid )-methanol(84??16) at a flow rate of 1.0 mL??min^-1. Thirty percent of the eluent was detected by ion trap mass spectrometry, and the parent ions and the corresponding product spectra of all the related substances in etimicin sulfate were determined and elucidated.RESULTS Addition of 0.1% propionic acid into the mobile phase significantly enhanced the sensitivity of MS detector without altering the chromatographic behavior such as retention time and elution order of the related substances. Twenty-eight related substances were separated and detected by the LC/MSⁿ method in a typical sample. Nine of them were identified with the help of corresponding impurity reference substances and 14 of them were elucidated by MS fragment information, while the other five were not identified due to limitated information. CONCLUSION The established method can be applied to the identification of the related substances in etimicn sulfate detected under the chromatographic condition described in Chinese Pharmacopoeia 2010, which is helpful to the quality improvement and process optimization of etmicin sulfate.     

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??OBJECTIVE To establish an improved reversed-phase high performance liquid chromatography method coupled with pulsed electrochemical detection for determining the related substances of netilmicin sulfate injection. METHODS Agilent Proshell 120 SB-C₁₈ column (4.6 mm×150 mm,2.7 μm)and gradient elution were used. Mobile phase A was 0.2 mol·L^-1 trifluoroacetic acid in 0.1 mol·L^-1 sodium hydroxide solution-acetonitrile (97:3), mobile phase B was 0.1% pentafluoropropionic acid-acetonitrile (97:3), and the flow rate was 0.8 mL·min^-1. A pulsed electrochemical detector was adopted, and the temperatures of detector and column were kept at 35 ??. The working electrode was a gold electrode with diameter of 3 mm and a quadruple-potential waveform (QPW)was selected as detection waveform. The injection volume was 25 μL. NaOH solution of 0.8 mol·L^-1 was added post-column at a flow rate of 0.3 mL·min^-1. RESULTS A total of 28 impurities could be detected and effective separation was achieved in the typical sample and most of which could not be separated in the method of Ch.P 2015. The linearity of the calibration curve for netilmicin ranged from 0.25 to 15 μg·mL^-1 with a coefficient of determination equal to 0.999 1. The LOD and LOQ of netilmicin were found to be 0.25 ng and 1.25 ng, respectively. The repeatability RSD(n=6) of the single largest impurity and total impurities were 0.9% and 0.8%, respectively. The sample solution was stable within 24 h. CONCLUSION Compared with previously published investigations, the improved method shows higher sensitivity, better separation ability and good reproducibility, especially for differentiating the origin of bulk drug for netilmicin sulfate injection, thus is more suitable for the determination of related substances of netilmicin sulfate injection.     

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??OBJECTIVE To establish an HPLC method to determine the related substances of vortioxetine hydrobromide and identify the degradation products of vortioxetine hydrobromide by HPLC-MS. METHODS An HPLC method was developed by using a CN column(Agilent Zorbax SB-CN,4.6 mm??250 mm, 5 ??m),the mobile phase consisted of CH₃CN-HCOONH₄(50??50, pH adjusted to 5.0 with phosphoric acid),the flow rate was 1.0 mL??min^-1, and the detection wavelength was set at 228 nm. The column temperature was maintained at 25 ??, and the injection volume was 20 ??L. RESULTS Vortioxetine hydrobromide was well separated from the related substances. The detection sensitivity of vortioxetine hydrobromide and its related substances met the determination requirements. The calibration curves of vortioxetine hydrobromide and related substances had good linearity. The repeatability of the method was good(RSD=6.89%, n=6). The average recoveries of the sample were all within 95%-105%. The degradation product produced by oxidation was identified by mass spectrometry as related substance D. CONCLUSION The developed method proves to be simple,accurate,specific and reliable. It can be applied to the determination of vortioxetine hydrobromide and its related substances.