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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 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 a high performance size exclusion chromatography(HPSEC) method for the separation and analysis of polymers in cefotaxime sodium and cefotaxime sodium for injection, and determine the structures of the impurities by LC-MS. METHODS HPSEC was performed by using Sepax SRT SEC-150(7.8 mm??300 mm, 5 ??m)column. The mobile phase was 0.1 mol??L^-1 disodium hydrogen phosphate and 0.1 mol??L^-1 phosphate buffer solution. The flow rate was 0.8 mL??min^-1, the detection wavelength was set at 235 nm, the injection volume was 10 ??L, and the column temperature was maintained at 35 ??. The concentration of polymers was quantified by external standard method. The LC-MS/MSⁿ system conditions were as following: the mobile phase was 20 mmol??L^-1 amonium acetate, the flow rate was 0.8 mL??min^-1, ESI source with positive and negative ion scan was utilized, the scanning range was m/z 200-1 600, and the post-column diversion ratio was 1??4. RESULTS Eight impurity peaks were obtained in total; the resolutions were all greater than 1.5. The linear range of cefotaxime was 1-100 ??g??mL^-1(r=1.000 0). The RSD repeatability was 1.2%(n=6). The limit of detection was 0.2 ??g and the limit of quantitation was 0.4 ??g. Three polymers were identified by LC-MS. CONCLUSION The HPSEC method can be used for the quantitative and qualitative analyses of individual polymer impurities. It is also sensitive for the control of polymers in cefotaxime.     

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??OBJECTIVE To compare and optimize the analytical methods for detection of related substances of etimicin sulfate injection. METHODS For the HPLC-CAD (charge aerosol detector)method, the mobile phase was 0.2 mol·L^-1 trifluoroacetic acid aqueous solution-methanol (95:5), the flow rate was 1.0 mL·min^-1 and the column temperature was maintained at 30 ??. The nebulization temperature for the CAD was maintained at 30 ?? and the gas pressure was 0.24 MPa.The HPLC-ELSD and HPLC-PAD methods adopted by the Ch.P 2015 were also used to detect the related substances of etimicin sulfate injection for the purpose of comparison. RESULTS Compared with the HPLC-ELSD method, the HPLC-CAD method showed higher selectivity and sensitivity; compared with the HPLC-PAD method, the results of the determination of the impurities were more accurate for the HPLC-CAD method. CONCLUSION The separation capability of the new HPLC-CAD method for detection of the related substances of etimicin sulfate injection is superior to HPLC-PAD and HPLC-ELSD methods and can detect more impurities, which is suitable for the quality control of etimicin sulfate injection.     

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??OBJECTIVE To develop an HPLC-ELSD method for determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection. METHODS The column was Waters Symmetry 300 C₁₈(4.6 mm??150 mm, 5 ??m;pore size:300). The mobile phase was methonal-tetrahydrofuran-0.17 mol??L^-1 ammonium acetate(93??6??1). The flow rate was 1.0 mL??min^-1. The column temperature was 25 ?? and the injection volume was 10 ??L. The ELSD conditions were as follows: Alltech 2000ES ELSD detector; drift temperature: 110 ??; rate: 2.6 L??min^-1. RESULTS This method had good specificity. The linear ranges of the calibration curves for MPEG-DSPE and HSPC were 0.03-0.48 mg??mL^-1 (r=0.999 8) and 0.1-1.0 mg??mL^-1 (r = 0.999 8), respectively. The average recovery rates of MPEG-DSPE and HSPC were 100.0% (n=3??3) and 101.0% (n=3??3), respectively. The LODs of MPEG-DSPE and HSPC were 13 and 52 ng, respectively. The repeatability and intermediate precision of MPEG-DSPE were 0.9% (n=5) and ??1.9%(n=3), respectively. The repeatability and intermediate precision of HSPC were 1.1%(n=5) and ??1.3%(n=3) , respectively. CONCLUSION The established method is accurate, reliable, repeatable and suitable for the determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection.     

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??OBJECTIVE To establish an RP-HPLC analytical method for simultaneous determination of crysoeriol and centaureidin in the aeries parts of Echinops integrifolius. METHODS The separation was achieved on a Kromasil C₁₈ column ( 4. 6 mm??250 mm,5 ??m)at 30 ?? using acetonitrile-water-acetic acid solution (35??65??2) as mobile phase at a flow rate of 1.0 mL??min^-1 and 350 nm as the detection wavelength. RESULTS The linear ranges of crysoeriol and centaureidin were 0.4-2.4 mg??L^-1(r=0.999 8), 0.6-2. 6 mg??L^-1 (r=0.999 9), respectively. The average recoveries (n=6) were 91.6% and 92.7%, and RSDs were 1.37% and 1.08% respectively. CONCLUSION The methodology validation shows that this method is accurate,simple and reliable,which is applicable for the simultaneous determination of two flavonols crysoeriol and centaureidin in the aeries parts of Echinops integrifolius.     

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??OBJECTIVE To optimize vancomycin regimen in children with MRSA infection. METHODS Vancomycin AUC_0-24/MIC predictions were performed across a range of dosages (20-70 mg??kg^-1??d^-1) using a Monte Carlo simulation (n=10 000). AUC_0-24 was calculated as daily dose divided by vancomycin clearance, and daily dose was fixed for a given simulation. The MIC distribution for MRSA was obtained from the RESULTS of clinical laboratory, the First Affiliated Hospital of Guangxi Medical University, from 2012 to 2014 (n=430;30%??0.5 mg??L^-1; 58.6%= 12 mg??L^-1; and 11.2%=2 mg??L^-1; 0.2%=4 mg??L^-1). RESULTS With increasing vancomycin daily dose, the percentage of patients predicted to achieve AUC_0-24/MIC >400 similarly increased. At 35 mg??kg^-1??d^-1, the percentage predicted to achieve AUC_0-24/MIC >400 was 99.41% when MIC was 0.5 mg??L^-1. However, the dosage rose to 65 mg??kg^-1??d^-1 when MIC was 1 mg??L^-1. At this regimen, the percentage predicted to achieve AUC_0-24/MIC >400 was 97.55%. At a MIC of 2 mg??L^-1 and more, none of the dosages predicted to achieve AUC_0-24/MIC>400. CONCLUSION Recommended empiric vancomycin dosing in children should be above 35 mg??kg^-1??d^-1 when MIC is 0.5 mg??L^-1. At the MIC is 1 mg??L^-1, the recommended regimen should be over 65 mg??kg^-1??d^-1.     

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??OBJECTIVE To establish a method of on-line matrix elimination together with ion chromatographic for determination of residual tetraethylammonium bromibde(TEAB) which acts as phase transfer catalyst in clopidogrel sulfate. METHODS An ion excluding column Dionex IonPac NG1 was used for the separation of TEAB from clopidogrel sulfate, using 20 mmol??L^-1 MSA as eluent. A pre-concentration column Dionex CG17 was used to enrich the trace TEAB. After being switched to analytical system, the separation was performed on Dionex ICS3000 using Dionex IonPac CS17 as analytical column, and suppressor was not required. 5 mmol??L^-1 MSA containing 35% acetonitrile was used as mobile phase. RESULTS The RSDs of retention time and peak area were good. The recoveries of TEAB were between 97.8%-103.3%. The calibration curves of analytes were linear in the range between 0.4 and 10.0 ??g??mL^-1. The limit of detection reached 0.2 ??g??mL^-1. CONCLUSION This method can be applied to detect trace kation in pharmaceutical chemicals.     

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??OBJECTIVE To develop and verify a magnetic beads-based extraction combined with quantitative PCR(q-PCR)method for determination of residual host cell DNA in recombinant human albumin products expressed in Pichia pastoris. METHODS The residual Pichia pastoris host cell DNA in samples were extracted by magnetic beads-based extraction method and then determined by Taqman probe-based q-PCR. The residual DNA content was calculated according to the standard curve. The developed method was verified for accuracy and precision with different derivation albumin matrixes and concentrations, and the residual DNA of 3 batches of recombinant human albumin products expressed in Pichia pastoris were detected. RESULTS The minimum detection limit of Pichia pastoris residual DNA by the developed method was 3 fg??L^-1, the linear range was 3 fg??L^-1-300 pg??L^-1, and the correlation coefficient(r²) was 0.998 3. The recovery rates of spiked samples in rHA matrix were 93.58%(RSD 19.6%, n=4)at 100 fg??L^-1 and 215.56%(RSD 42.9%, n=4) at 10 fg??L^-1, respectively. The recovery rates of spiked samples in HSA matrix were 67.09%(RSD 6.9%,n=3)at 100 fg??L^-1 and 113.40%(RSD 11.1%, n=3) at 10 fg??L^-1, respectively. The residual Pichia pastoris DNA contents in 3 batches of recombinant human albumin products expressed in Pichia pastoris determined by the developed method were 5.98, 4.16, 4.49 fg??L^-1(n=7) respectively and not more than 1 ng per 10 g protein. CONCLUSION Magnetic beads extraction method combined with fluorescence quantitative PCR method solves the technical problem of quantitative determination of trace DNA in recombinant human albumin products with ultra-high concentration protein. The method is accurate and reproducible, and can be used for quantitative determination of DNA residue in recombinant human albumin expressed by Pichia pastoris.     

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??OBJECTIVE To develop an immunoaffinity column clean-up and high performance liquid chromatography coupled with triple quadrupole mass spectrometry(HPLC-MS/MS) method to determine aflatoxins in gelatin drugs. METHODS The analysis was performed by an HPLC-MS/MS system with X-Brigde-C₁₈(3.0 mm??50 mm,3.5 ??m)column. Multiple-reaction monitoring (MRM) was performed to identify and quantify aflatoxin B₁,B₂,G₁ and G₂, which were extracted from Asini Coril Colla, Cervil Cornus Colla, and Testudinis Carapacis Colla with 60% methanol solution. RESULTS Linear calibration curves were obtained with r??0.997 9. The precision of the method was showed by RSDs (n=6) ranging from 1.2% to 4.1%. The recoveries were determined at three concentration levels and ranged from 77.3% to 94.6%. The ranges of LOQs were from 0.5 to 0.8 ??g??L^-1 and the RSDs (n=9) of intra-day precision and inter-day precision were from 1.1% to 2.9% and from 1.5% to 2.8%, respectively. CONCLUSION The method is specific, simple and rapid to detect aflatoxins in gelatin drugs.