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??OBJECTIVE To rapidly identify the related substances in doxycycline hyclate tablets and investigate the possible degradation pathways of doxycycline hyclate solution by 2D-LC-IT-TOF/MS. METHODS Firstly,the chromatography was carried out using the 1stD InertSustain^TM C₁₈(4.6 mm??150 mm,5 ??m) column and a mobile phase containing a mixture of buffer solution(0.25 mol??L^-1 ammonium acetate solution-0.1 mol??L^-1 ethyldiaminetetraacetic acid disodium: triethyiamine=100??10??1)-acetonitrile(85??15). Solution pH was adjusted to 8.8 with glacial acetic acid. Then a InertSustain^TM C₁₈(2.1 mm??50 mm,2 ??m) column was used with 0.1% formic acid-water as mobile A and 0.1% formic acid-acetonitrile as mobile B in a gradient elution mode in 2ndD. Electrospray ionization (ESI) source was tested in both positive and negative ion modes. Nebulized gas flow was 1.5 L?? min^-1. Dry gas flow was 10 L?? min^-1. The desolvation tube temperature was kept at 200 ??. Related substances were characterized according to multi-level MS behaviors. The 2D-LC-IT-TOF/MS method was employed to identify the structures of impurities in forced degradation solutions and illuminate the degradation pathways of doxycycline hyclate solution. RESULTS A total of eight related substances were detected in doxycycline hyclate tablets. Four of them had a content of more than 0.1%, and their structures were identified to be 4-epidoxycycline, metacycline, ??-epidoxycycline and 2-acetyl-2-decarbamoyldoxycycline. The solution of doxycycline hyclate easily degraded under alkaline condition and generated an open loop compound taken off the keto group. The solution was sensitive to heat, and generated 4-epidoxycycline with a little amount of 4-epi-6-epidoxycycline; but the solution was stable under illumination and acidic condition. CONCLUSION The established method is suitable for rapid identification of impurities in doxycycline hyclate, which can be applied as a useful analytical tool for quality control and drug process optimization of doxycycline hyclate.     

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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.     

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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 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 method for determination of the contents of eplerenone and its related substances. METHODS A RP-HPLC method was developed by using a C₁₈ column (Agilent Poroshell,4.6 mm??100 mm, 2.7 ??m), the mobile phase consisted of H₂O-CH₃CN-MeOH (64??18??18), the flow rate was 1.0 mL??min^-1, and the detection wavelength was set at 240 nm. The diluent was H₂O-CH₃CN-MeOH (50??25??25). RESULTS Eplerenone was well separated from the impurities. For the determination of related substances, the LODs of eplerenone, its intermediate (?? and ??) and related substances (A-E) were 0.03, 0.13, 0.25, 0.05, 0.05, 0.26, 0.07 and 0.12 ng, and the LOQs were 0.12, 0.42, 0.84, 0.18, 0.18, 0.86, 0.22 and 0.41 ng, respectively. The calibration curves of eplerenone and related substances (A-E) had good linear relationship within 0.13-1.53, 0.25-3.03, 0.25-2.99, 0.13-1.61, 0.14-1.63, and 0.13-1.54 ??g??mL^-1(r=0.999 9, n=7), and the correction factors of the related substances (A-E) were all in the range of 0.90-1.10. The average recoveries of the related substances (A-E) were all within 95.0%-105.0% (n=9). For the content determination of eplerenone, the validation test showed good linearity over the range of 12.60-75.63 ??g??mL^-1 (r=0.999 6, n=6) with good repeatability (RSD=0.19%, n=6). CONCLUSION The developed method proves to be simple, accurate, specific and reliable. It can be applied to the determination of eplerenone and its related substances.     

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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 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 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 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.     

高效液相色谱-电喷雾-离子阱质谱法快速推定硫酸奈替米星中有关物质的结构

 目的 应用高效液相色谱-电喷雾-离子阱质谱（HPLC-ESI-IT-MSn）法及TFAfix技术,快速鉴定硫酸奈替米星样品中有关物质的结构。方法 Agilent SB-C₁₈(4.6 mm×150 mm , 3.5 μm)色谱柱,水-三氟乙酸-甲醇(84∶1∶15)为流动相,流速0.5 mL·min^-1;离子肼质谱仪正离子检测,柱后分流（3∶2）进样,电喷雾离子源,离子源温度350 ℃,雾化室压力275.8 kPa,干燥气流速9 L·min^-1,采用TFAfix技术,即在柱后添加丙酸-异丙醇（20∶80）溶液,改善流动相中三氟乙酸对电喷雾离子化的抑制作用;对有标准品的有关物质,其结构通过与对照品的色谱质谱行为来确定;对无对照品的有关物质,主要以奈替米星和西索米星质谱行为为模板,根据它们多级质谱信息来推定。结果 硫酸奈替米星原料中检出9个有关物质,推定出其中7个物质的结构,分别为西索米星、去甲基西索米星、1-N-乙基-加洛糖胺、5-O-乙基-奈替米星、2′-N-乙基-奈替米星、3″-N-乙基-奈替米星、3-N-丙基-依替米星,解析了另两物质的部分结构。结论 建立的方法可以用于硫酸奈替米星原料中有关物质结构的快速推定,为奈替米星质量控制和工艺优化研究提供了可靠快速的分析手段。     

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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.     

HPLC-ELSD和HPLC-MSn分析硫酸奈替米星及其注射液

 目的建立高效液相色谱-蒸发光散射检测法(HPLC-ELSD)测定硫酸奈替米星及注射液的含量和有关物质,并采用高效液相色谱-电喷雾离子阱质谱联用(HPLC-MSn),鉴定硫酸奈替米星及其有关物质。方法采用Agilent SB-C₁₈(4.6 mm×250mm,5μm)色谱柱,流动相为0.05 mol·L^-1三氟乙酸溶液-丙酮(90∶10),流速为1.0 mL·min^-1,漂移管温度为50℃,雾化气体压力为3.5×10⁵Pa,在正离子检测方式下,用电喷雾离子阱质谱法对奈替米星和有关物质进行多级质谱分析。结果奈替米星和有关物质分离良好。奈替米星在28～870 mg·L^-1内呈较好的线性关系(r=0.999 9),重复性试验RSD=1.1%,回收率为99.9%,检测限为5.6 mg·L^-1,结合多级质谱裂解初步推断奈替米星中主要一个未知有关物质是奈替米星分子中发生B环与C环之间的糖苷键断裂,生成的脱去C环(氨基葡萄糖)的降解产物。结论本方法快速、简便,结果准确可靠,重现性好。     

HPLC-ELSD和HPLC-MSn分析硫酸西索米星及其注射液

目的建立高效液相色谱-蒸发光散射检测器(HPLC-ELSD)法测定硫酸西索米星及注射液的含量和有关物质,并采用高效液相色谱-电喷雾离子阱质谱(HPLC-MSn)联用,鉴定硫酸西索米星及其有关物质。方法采用Agilent SB-C18(4.6 mm×250 mm,5μm)色谱柱,流动相为0.05 mol.L-1三氟乙酸溶液-甲醇(90∶10),流速为1.0 mL.m in-1,漂移管温度为50℃,雾化气体压力为3.5×105Pa,在正离子检测方式下,用电喷雾离子阱质谱法对西索米星和有关物质进行多级质谱分析。结果西索米星和有关物质分离良好。西索米星在14~1 100 mg.L-1内呈较好的线性关系(r=0.999 5),重复性实验RSD=1.0%,回收率为99.8%,检测限为5.5 mg.L-1,结合多级质谱裂解初步推断西索米星中主要一个未知有关物质是西索米星分子中发生B环与C环之间的糖苷键断裂,生成的脱去C环(氨基葡萄糖)的降解产物。结论本方法快速、简便,结果准确可靠,重现性好。     

HPLC-ELSD和HPLC-MS^分析硫酸西索米星及其注射液

目的 建立高效液相色谱-蒸发光散射检测器（HPLC-ELSD）法测定硫酸西索米星及注射液的含量和有关物质，并采用高效液相色谱-电喷雾离子阱质谱（HPLC-MS^n）联用，鉴定硫酸西索米星及其有关物质。方法 采用Agilent SB—C18（4．6mm×250mm，5μm）色谱柱，流动相为0．05mol·L^-1三氟乙酸溶液-甲醇（90：10），流速为1．0mL·min^-1，漂移管温度为50℃，雾化气体压力为3．5×10^5Pa，在正离子检测方式下，用电喷雾离子阱质谱法对西索米星和有关物质进行多级质谱分析。结果 西索米星和有关物质分离良好。西索米星在14～1100mg·L^-1内呈较好的线性关系（r=0．9995），重复性实验RSD=1．0％，回收率为99．8％，检测限为5．5mg·L^-1，结合多级质谱裂解初步推断西索米星中主要一个未知有关物质是西索米星分子中发生B环与C环之间的糖苷键断裂，生成的脱去C环（氨基葡萄糖）的降解产物。结论 本方法快速、简便，结果准确可靠，重现性好。     

色谱相关光谱法识别莫西沙星HPLC分析中的有关物质色谱峰

 目的建立识别HPLC色谱图中莫西沙星有关物质色谱峰的色谱相关光谱方法。方法采用高效液相色谱-二极管阵列检测器(HPLC-DAD),提取各有关物质峰的标准光谱,建立有关物质标准光谱库;通过计算莫西沙星有关物质色谱峰的光谱与标准光谱库中光谱的相似系数,对有关物质进行识别。结果光谱识别的最低检出限为20 ng,相似系数阈值为0.995。结论色谱相关光谱法可有效地对HPLC分析中的色谱峰进行识别,具有专属性强,准确度高,耐用性好的特点,适用于对由于色谱柱、流动相等色谱条件的改变,无法利用相对保留时间定性的色谱峰的归属。     

HPLC/MS/MS法分离鉴定罗通定在大鼠胆汁中的代谢产物

目的:研究罗通定在大鼠胆汁中的主要代谢产物。方法:灌胃给药后,收集大鼠胆汁,液液萃取,采用HPLC/MS/MS法分析鉴定罗通定的代谢产物。色谱柱Lichrospher-C18(5μm,4.6mm×250mm),流动相:甲醇-水-三乙胺(37630.063,V/V/V),冰醋酸调pH至6.3,检测波长281nm。结果:在大鼠胆汁中发现2个罗通定代谢产物,初步推测其结构为罗通定单羟基化后再与葡萄糖醛酸结合的产物。结论:罗通定在大鼠胆汁中主要以羟基化后的葡萄糖醛酸结合物形式排泄。     

HPLC-HRMS鉴定抗癌先导物T-OA在鼠尿中的代谢产物

目的：研究抗癌先导化合物T-OA（齐墩果酰基-3,5,6-三甲基吡嗪-2-甲酯）在大鼠尿液中的代谢产物,初步推断其在大鼠体内的代谢方式。方法：收集空白组、原料组（川芎嗪TMP与齐墩果酸OA摩尔等量）及T-OA组大鼠尿液;尿液冷冻干燥,固体经乙酸乙酯超声提取后,提取物用色谱乙腈复溶;HPLC-HRMS联用技术在ESI⁺和ESI^-模式下寻找可能的质谱峰,通过对比3组谱图的异同得出代谢产物的相关信息。结果：原料组鉴定出1个OA的代谢产物和2个TMP的代谢产物;T-OA 组未检测到原料的代谢产物,而得到1个Ⅱ相代谢产物。结论：在大鼠尿液中首次鉴定出1个T-OA的Ⅱ相代谢产物,1个OA的Ⅱ相代谢产物,初步推断T-OA在体内不以原料的形式发挥药效;所建立的HPLC-HRMS方法可用于相关衍生结构的代谢产物鉴定;该文也可为以齐墩果酸为母体的前药设计提供一定的借鉴。     

LC/MS/MS法鉴别神速宁胶囊中添加的盐酸氯丙嗪成分

目的:建立神速宁胶囊中非法添加盐酸氯丙嗪成分的鉴别方法。方法:采用色谱柱C18(250mm×2.0mm);乙腈-10mmol.L-1醋酸铵-三乙胺(65∶35∶0.5)(用醋酸调pH6.2)为流动相;流速:0.2mL.min-1;电喷雾离子化(ESI)扫描方式;二级质谱母离子m/z319,碰撞能量15V。结果:在高效液相色谱、紫外光谱、质谱中,样品出现与盐酸氯丙嗪成分一致的色谱峰、光谱图、质谱峰。结论:本方法简单可行,结果准确可靠,可用于神速宁胶囊中添加盐酸氯丙嗪成分的定性鉴别。