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??This review provided the research progresses of the strategies of the modification and application of poly (lactic-co-glycolic acid, PLGA) nanoparticles . The preparation and modification METHODS of PLGA nanoparticles and their application according to the reported foreign literature were analysed and summaried. PLGA is a biodegradable and biocompatible polymer which is one of the most widely used materials in preparation of sustained and controlled microparticles as drug vectors. This review focuses on the METHODS of modified nanoparticles and the advantages. Emulsification method, nanoprecipitation, emulsification solvent diffusion, solvent injection method and supercritical anti-solvent technique are the main five preparation METHODS of PLGA nanoparticles.The strategies of modifition include covalent cross-link, electrostatic interaction and hydrophobic interaction. The applications of modified nanoparticles to the optimal formulation, sustained release profile, improved cellular uptake, and targeting to specific organs or cells are introduced.     

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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 develop a comprehensive strategy integrateded with chemometrics METHODS for quality evaluation of Glechoma longituba(GL) from different geographical origins. METHODS The chemical differentiation of 22 batches of GL were performed by UPLC/QTOF-MS^E coupled with UNIFI^TM software. To evaluate the quality of GL from different geographical origins, the principal component analysis (PCA) was developed to analyze the MS^E data of 22 batches of GL. To find the markers, the orthogonal partial least squares discriminant analysis (OPLS-DA) was adopted to analyze the MS^E data of 22 batches of GL. RESULTS A total of 31 compounds including 8 phenolic acids, 14 flavones, 7 terpenes, 1 organic acid and 1 coumarin were unambiguously or tentatively identified in the GL from Guangxi province. And 14 compounds were reported for the first time. Twenty-two batches of GL were well gathered and segregated into two different groups scattering in the score plot of PCA by MarkerLynx XS. The result of PCA showed that the chemical compositions of GL from Anhui province had obvious difference with those from other provinces. Based on the S-Plot from the score plot of OPLS-DA, the differential component of GL from Anhui province was tentatively identified as terpene. CONCLUSION The integrated strategy facilitates authentication of herbal medicines of different origins in a more efficient and more intelligent manner.     

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??OBJECTIVE To establish an HPLC-MS/MS method for the determination of melatonin in human plasma. METHODS The plasma samples were extracted with ethyl acetate, using melatonin-D7 as the internal standard (IS). Then the ethyl acetate layer was evaporated in vacuum concentrator. Dried samples were redissolved in mobile phase (0.1% formic acid-methanol= 56:44, V/V), vortexed and centrifuged. The redissolved solution was transferred to an auto sampler vial and the supernatant was injected to the HPLC-MS/MS system. The MS/MS analysis was carried out in positive ionization mode by multiple reactions monitoring (MRM) at m/z 233.1??174.1 for melatonin and m/z 240.2??178.0 for IS, respectively. RESULTS The calibration curve of melatonin in human plasma was linear over the concentration range of 0.020 00-30.00 ng??mL^{- 1}. The lower limit of quantitation was 0.020 00 ng??mL^{- 1}. The RSDs of within-day and between-day were less than 15%. The extraction recoveries were between 59.0%-65.0%. The matrix effects were between 95.5%-98.9%. CONCLUSION The method is proved to be convenient, sensitive and accurate. It can be applied to study the pharmacokinetics of melatonin prolonged-release tablets in healthy Chinese volunteers.     

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??OBJECTIVE To establish an HPLC-MS/MS method for determination of ASC-J9 in rat blood. METHODS After liquid-liquid extraction, ASC-J9 was separated on a Symmetry C₁₈colume, with mobile phase of acetonitrile-water containing 0.1% formic acid and 10 mmol??L^-1 ammonium formate.The flow rate was 1.0 mL??min^-1, mass shunt was 0.4 mL??min^-1, and column temperature was main tained at 35 ??. Quantification was performed in positive ion multiple-reaction-monitoring(MRM) mode. RESULTS The calibration curve of ASC-J9 had good linearity in the concentration range of 3.54-1 180 ng??mL^-1. The extraction recovery rate was within 83.19% to 87.27%,and the intra-day and inter-day RSDs were both less than 8.89%. CONCLUSION This method is specific, sensitive and suitable for determination of ASC-J9 in rat blood.
     

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??OBJECTIVE To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS The interference sequence targeting at rat SERCA gene was designed and synthesized. pGag/Pol, pRev, and pVSV-G were co-transfected into 293T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the RESULTS were compared with those in the control group. RESULTS The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3??10⁸ U??mL^-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that shSERCA could significantly reduce the mRNA and protein levels of SERCA (P<0.01). CONCLUSION The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.     

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??OBJECTIVE To establish an HPLC-ELSD method for determination of mesaconine.METHODS Gemini C₁₈ 110A column (4.6 mm??150 mm,5 ??m) was adopted with the mobile phase consisting of methanol-0.1%TFA (25:75) at the flow rate of 0.6 mL??min^-1. The detector was Alltech ELSD 2000ES, with the drift tube temperature of 90 ?? and the airflow velocity of 2.3 L??min^-1. RESULTS Under this conditions, mesaconine and the related substances were well separated. The linear range of mesaconine was 0.40-0.60 mg??mL^-1 (r=0.999 6). RSD of injection precision and repeatability test were 0.04% (n=5) and 0.7% (n=6), the intra day and inter day precision RSD were 0.4% and 0.6%, respectively. The average recovery was 101.0% (RSD=1.1%, n=9). CONCLUSION The established method was specific, simple, accurate, and suitable for determination of mesaconine.     

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??OBJECTIVE To identify rapidly the chemical constituents in Tanreqing injection and Tanreqing capsules by ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS^E).METHODS The separation was performed on a Waters Acquity UPLC BEH C₁₈ column (1.0 mm??100 mm, 1.7 ??m) with acetonitrile-0.1% formic acid as mobile phase in gradient elution. ESI ion source was employed in negative ion mode. The differences of chemical compositions between the two preparations and the sources of these compounds were illustrated based on their retention time, accurate mass measurements and the mass fragments by comparison with those in the literature or database and the reference standards.RESULTS A total of 111 compounds including 13 unknown components were identified or tentatively characterized. Among these compounds, 14 were derived from Scutellariae Radix (SR) intermediate, 36 were from Bear Bile Powder(BBP) intermediate, 7 were from Caprae Hircus Cornu(CHC) intermediate, 34 were from Lonicerae japonicae Flos(LJF) intermediate and 22 were from Forsythiae Fructus(FF) intermediate. Moreover, quinic acid and rutin were simultaneously detected in LJF and FF intermediates, 28 constituents were unambiguously confirmed by their reference standards. However, 71 compounds were observed both in injection and capsules, while 24 compounds were only found in Tanreqing injection and 16 compounds only in the capsules.CONCLUSION The differences of chemical constituents between Tanreqing injection and capsules are effectively characterized by UPLC/Q-TOF-MS^E method, which will facilitate the quality control of the two preparations.     

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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 develop a method for the determination of iridoid,phenylpropanoid glycosides,and organic acids in Scrophulariae Radix from different habitats and commercial herbs by UPLC-QTRAP-MS/MS. METHODS The analysis was carried out on a BDS HYPERSIL C₁₈ column (4.6 mm×250 mm,5 μm) with elution by mobile phase of acetonitrile-water at a flow rate of 1.0 mL·min^-1. The column temperature was maintained at 35 ??. The target compounds were analyzed by the negative ion multiple reaction monitoring (MRM) mode. RESULTS Twelve multiple constituents showed good linearity (r>0.999 4) in the range of the tested concentration. The average recoveries of the 12 components were 99.59%-101.24% with relative standard deviations of 0.93%-1.60%. CONCLUSION The established method is accurate and precise,which provides a reliable and effective technique for the quality evaluation of Scrophulariae Radix.     

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??OBJECTIVE To establish an HPLC-UV-ESI-MSⁿ method for the study of impurity profile of amoxicillin and clavulanate potassium tablets. METHODS Agilent 1100 LC/MSD Trap liquid chromatography-mass spectrometry was used, and the column was Shim pack CLC-ODS RP18(4.6 mm??250 mm, 5 ??m). The mobile phase A was 20 mmol??L^-1 ammonium acetate (pH adjusted to 6.0), and the mobile phase B was 20 mmol??L^-1 ammonium acetate-acetonitrile (20??80) (pH adjusted to 6.0). Gradient elution was performed at a flow rate of 1.0 mL??min^-1. ESI source was used. Positive and negative ion scan was conducted with a scanning range of m/z 100-1 500. The nebulizing pressure was 275.8 kPa, dry gas flow was 9 L??min^-1, and post-column diversion ratio was 1??5. Some related substances were identified by comparing the retention time in the chromatography, [M+H]⁺ spectrum and MS² spectrum with those of the reference substances, while the others which do not have reference substances were deduced or speculated by analyzing the MS² or MSⁿ fragmentation with the help of a rule summarized from the MS² fragmentation of amoxicillin, clavulanic acid and system suitability impurity reference substances. RESULTS A total of 15 related substances were separated and characterized including nine known impurities like amoxicilloic acid, amoxicillin dimer, etc. and six unknown impurities. CONCLUSION The method can be applied in the identification and qualitative analysis of the related substances in amoxicillin and clavulanate potassium tablets and is helpful for the quality control and optimization of the synthetic process.     

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??OBJECTIVE To establish an HPLC-MS/MS method to identify the unknown impurities in polymyxin B sulfate. METHODS The analysis was performed on Agilent 1260-6550 Q/TOF-MS with a Diamonsil Plus C₁₈ column(4.6 mm??250 mm, 5 ??m). Mobile phase A was 0.01 mol??L^-1 trifluoroacetic acid-acetonitrile(95??5), and mobile phase B was acetonitrile containing 0.1% formic acid. Mobile phase A and B were set at the volume ratio of 79??21 at a flow rate of 1 mL??min^-1 under isocratic elution. The detection wavelength was set at 254 nm. ESI source was used. Positive ion scanning was conducted in the range of m/z 50-1 700 for MS and MS/MS. The unknown components were identified by comparing the MS and MS/MS with the known reference standards like polymyxin B₁ and B₂. The photochemical Paterno-B??chi reaction was performed using a low-pressure mercury lamp as the light source at emission wavelength of 254 nm with acetone/water(50/50, V/V) as the reaction solvent. RESULTS The structures of seven unknown related substances in polymyxin B sulfate were identified. The most abundant impurity was identified to be vinyl polymyxin B₁, for which the double bond was at the end of the fatty acyl residue. CONCLUSION Vinyl polymyxin B₁ is reported for the first time. The method provides a good idea for the identification of related substances in drugs.     

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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 establish a method for the determination of various tetracycline antibiotics residues in calf pulmonary surfactant extract. METHODS UPLC-MS/MS method was adopted with, Waters HSS T3 column(2.1 mm??100 mm,1.8 ??m) as solid phase. A acetonitrile solution(containing 0.1% formic acid) and aqueous solution(containing 0.1% formic acid) were used as mobile phase A and mobile phase B, respectively. Gradient elution was conducted at, the flow rate of 0.3 mL??min^-1, the column temperature was maintained at 30 ??, and the injection volume was 1 ??L. RESULTS This method can simultaneously determine 11 antibiotics, including tetracycline, chlortetracycline, oxytetracycline, doxycycline, metacycline, minocycline, ??-doxycycline, epi-tetracycline, epi-chlortetracycline, anhydrotetracycline, and epi-anhydrotetracycline. The linear range, sensitivity, precision and recoveries of each compound meet the analytical requirements. Tetracycline and oxytetracycline were detected in the calf pulmonary surfactant extract samples. The amount of oxytetracycline residues was about 0.03 mg??kg^-1, and the tetracycline did not reach the limit of quantitation(0.006 mg??kg^-1). CONCLUSION The method can be used for detecting tetracycline antibiotic residues in calf pulmonary surfactant extract.     

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??OBJECTIVE To establish an HPLC-MS method for the analysis of the impurity profile of cefotaxime sodium. METHODS Shimadzu-LCMS-IT-TOF was used with Waters XBridge Shield (RP18,4.6 mm??250 mm, 5 ??m) column. Mobile phase A was 20 mmol??L^-1 ammonium acetate (pH adjusted to 6.25)-methanol (92:8), and mobile phase B was set at 20 mmol??L^-1 ammonium acetate-methanol (60:40) (pH adjusted to 6.25).Gradient elution was performed at a flow rate of 1.0 mL??min^-1. ESI source was used.Positive and negative ion scanning was conducted in the range of m/z 150-900.The heating temperature was 200 ??, CDL temperature was maintained at 200 ??, atomization gas flow rate was 1.5 L??min^-1, dry gas pressure was 94.0 kPa, and the post-column diversion ratio was 1:4.Some related substances in cefotaxime sodium were identified by comparing the retention time in chromatography,[M+H]⁺ spectrum and MS² spectrum with those of reference substances, the others which haven't reference substances were deduced or speculated by analyzing the MS² or MSⁿ fragmentation with the help of a rule summarized from the MS² fragmentation of cefotaxime sodium and the reference substances of system suitability impurities. RESULTS Twenty-six related substances were separated and detected in the sample, all of which were identified or deduced. They were cefotaxime sodium isomeric compounds and homologs generated during the production process or degradation products. CONCLUSION The method can be applied in the identification and qualitative analysis of the related substances of cefotaxime sodium and the quality control and optimization of the synthesis of cefotaxime sodium.     

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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 determine two toxic impurities, namely bromoacetic acid and 4-aminobenzonitrile, in the intermediate of dabigatran etexilate by UHPLC-MS. METHODS The separation was performed on a Shimadzu Shim-Pack GIS C₁₈ column (2.1 mm??50 mm,2 ??m) with mobile phase consisting of 0.1% formic acid aqueous solution (A) and 0.1% formic acid methanol (B) by gradient elution at a flow rate of 0.4 mL??min^-1. The detection was achieved by triple quadrupole mass spectrometry with rapid polarity switching using MRM mode. RESULTS The calibration curves were linear in the ranges of 0.2-40 and 0.4-40 ng??mL^-1 for bromoacetic acid and 4-aminobenzonitrile, respectively. The values of LOQ of bromoacetic acid and 4-aminobenzonitrile were 0.1 and 0.4 ng??mL^-1, respectively. The recoveries of bromoacetic acid and 4-aminobenzonitrile were 100.9% and 99.6%, respectively. CONCLUSION The method is accurate, rapid, sensitive, and reliable to determine the two toxic impurities bromoacetic acid and 4-aminobenzonitrile in the intermediate of dabigatran etexilate for quality control.     

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??OBJECTIVE To establish the self contrast and correction factor method for the content determination of the related substances in compound ezetimibe and rosuvastatin calcium tablets simultaneously. METHODS RP-HPLC method was adopted. The determination was performed on Kromasil 100-5 C₁₈ Dimensions column (4.6 mm??250 mm, 5 ??m) with mobile phase A consisting of methanol-acetonitrile-0.05 mol??L^-1 potassium dihydrogen phosphate (adjusted to pH 4.0 with phosphoric acid) (10??30??60) and mobile phase B consisting of tetrahydrofuran-acetonitrile-0.05 mol??L^-1 potassium dihydrogen phosphate (adjusted to pH 4.0 with phosphoric acid) (10??50??40) at a flow rate of 1.0 mL??min^-1. The detection wavelength was set at 242 nm. The injection volume was 20 ??l. The slope of linear equation was used to determine the correction factors between ROS impurities 1, 2, 3, EZT impurities 1, 2, 3, 4, 5, 6, 7 and ezetimibe or rosuvastatin calcium. The relative retention time was used to determine the positions of impurities. RESULTS The relative retention time of ROS impurities 1, 2, 3, 4 and EZT impurities 1, 2, 3, 4, 5, 6, 7 to rosuvastatin calcium was 1.5, 1.9, 2.1, 1.1, 1.7, 2.5, 2.6, 2.8, 2.9, 3.0, and 4.0, respectively.The correction factors of ROS impurities 1, 2, 3, 4 and EZT impurities 1, 2, 3, 4, 5, 6, 7 were 1.1, 1.1, 1.0, 1.0, 1.3, 1.1, 1.0, 1.3, 1.4, 0.5, and 1.0,respectively.The content of ROS impurity 4 was 0.15% in three batches of samples, the other impurities were less than 0.1%, and the contents of total impurities were 0.27%, 0.27%, and 0.26%, respectively. CONCLUSION The method is simple,efficient, and accurate for analyzing the related substances in compound ezetimibe and rosuvastatin calcium tablets.     

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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.