RP-HPLC method for the quantitation of β-Sitosterol in Elaeagnus Gon yanthes Benth

Objective To establish an RP-HPLC method for the determination of β-Sitosterol in Elaeagnus Gonyanthes Benth. Methods The separation was performed on a luna C8 (2) (150 mm×4.6 mm, 5μm) column with the mobile phase of methanol-water (88∶12, v/v) at a flow rate of 1.0 mL/min, the detection wavelength was set at 210 nm, and the temperature of the column was maintained at 35 ℃. Results The calibration curve of β-Sitosterol was linear over the concentration range of 0.075-0.375 mg/mL (r=0.9999) and the average recovery of β-Sitosterol was 96.30% with RSD of 3.60%(n=3). Conclusion The method is simple, rapid, and accurate, and can be used for the quality control of Elaeagnus Gonyanthes Benth.     

LC-ESI-MS/MS, a modified method for simultaneous quantification of isoflavonoids, flavonoids, alkaloids and saponins in a Chinese herbal preparation Gegen-Qinlian decoction

A sensitive and reliable liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method was established to simultaneously quantitate four categories of compounds (isoflavonoids, flavonoids, alkaloids and saponins) in Gegen-Qinlian decoction (GQD). These compounds were separated by a Shiseido CAPCELL PAK C18 column with a linear gradient consisting of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B), and delivered at a flow rate of 0.3 mL/min. All the analytes were determined by electrospray positive ionization tandem mass spectrometry in a multiple reaction monitoring (MRM) mode. Linearity, accuracy, precision, recovery and stability of the method were evaluated with the validation over the range of 4.0-538 5 ng/mL. The proposed method was applied to the analysis of a Chinese herbal preparation GQD successfully.     

Simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture by high performance liquid chromatography

A reversed phase high performance liquid chromatography （HPLC） method was established for the simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture. A Grace Apollo Cl8 column （250 mm × 4.6 mm, 5 μm） was used as the stationary phase and the mobile phase was composed of acetonitrile and aqueous phosphoric acid （0.2%, v/v）. Gradient elution was carried out at the flow rate of 1.0 mL/min and the column temperature was 30 ℃. An ultraviolet （UV） detector was used with a selected wavelength of 240 nm. Calibration curves were linear within the concentration range of 4.6-45.75 μg/mL for 12, 13-dihydroxyeuparin （r〉0.9999） and 106.9-1068.9μg/mL for glycyrrhizic acid （r〉0.9999）, respectively. Recoveries were 102.18% for 12, 13-dihydroxyeuparin and 101.17% for glycyrrhizic acid. The method developed could be applied to the simultaneous determination of 12, 13- dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture.     

A rapid method for the determination of dopamine in porcine muscle by pre-column derivatization and HPLC with fluorescence detection

A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle. Methanol and deionized water （0.1% acetic acid, v/v） with a ratio of 60：40 was used as mobile phase. The flow rate was 0.8 mL/min and dopamine was eluted within 15 min. The linearity range was 0.003-8 μg/mL with r=0.9992. The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg. Recovery studies were carried out at 0.1, 0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%. The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.     

丹皮药材指纹图谱定性和有效成分定量分析方法研究

目的　建立丹皮药材的反相高效液相色谱定性和定量分析方法。方法　RP HPLCPlanetsilC18分析柱 (15 0mm× 4 .6mm ,5 μm) ,流动相为甲醇 水 冰醋酸 (44∶5 6∶0 .1) ,流速为 1.0mL·min-1,检测波长为 2 4 0nm ,柱温为室温 ,对丹皮药材进行指纹图谱定性分析 ,并对丹皮中的有效成分丹皮酚进行定量测定。结果　在此色谱条件下 ,13份不同丹皮药材的RP HPLC指纹图谱中可检出 11个相对位置稳定的色谱峰 ,通过色谱峰间峰面积的相关性分析 ,可以确定 10个峰可作为定性鉴别的指标峰 ;丹皮药材中丹皮酚含量为 1.32 %～ 2 .78%。结论　丹皮RP HPLC指纹图谱定性和有效成分定量分析方法具有较好的针对性和准确性 ,可用于丹皮药材的质量控制。     

治带片中苦参碱及氧化苦参碱的HPLC法测定

目的建立治带片中苦参碱及氧化苦参碱的高效液相色谱(HPLC)测定方法。方法色谱柱:Lichrospher-NH2(4.6 mm×250 mm,5μm),C18保护柱;流动相:乙腈-无水乙醇-0.5 mol/L磷酸水溶液(80∶10∶10);检测波长212 nm;流速1.0 mL/min;柱温:室温;进样量20μL。结果苦参碱和氧化苦参碱线性范围均为1.0-10.0μg/mL,回归方程苦参碱为:C=1.201×10-4A+0.161,r=0.9992;氧化苦参碱为:C=1.366×10-4A+0.221,r=0.9996,平均回收率分别为99.9%和99.4%,RSD分别为1.48%和4.33%。结论本法简便快捷,结果准确,可用于该制剂的质量控制。     

HPLC法测定健宝胶囊中淫羊藿苷含量

目的　建立健宝胶囊中淫羊藿苷的含量测定方法。方法　采用高效液相色谱法 ,色谱柱为C18(15 0mm×4.6mm) ,流动相为乙腈 0 .0 75mol·L-1磷酸溶液 (2 5∶75 ) ,流速 1.0mL·min-1,检测波长为 2 70nm。结果　淫羊藿苷进样量在 0 .0 6～ 0 .30 μm范围内 ,线性关系良好 (r=0 .9991) ,方法回收率为 98.5 0 %(n =5 ,RSD =1.2 0 %)。结论　本方法准确、简便、快速 ,适合于该药的质量分析检验。     

Rapid analysis of components in Rhizoma Anemarrhenae by HPLC-DAD-MS and HPLC-DAD-TOFMS

A global quality control method based on high performance liquid chromatography (HPLC) coupled with diode array detection (DAD), single quadrupole mass spectrometry (MS) and time-of-flight mass spectrometry (TOFMS) was developed for simultaneous determination of seven major components (mangiferin, neomangiferin, timosaponin E1, timosaponin E, timosaponin BⅡ, timosaponin BⅢ, and timosaponin AⅢ) and identification of most components in extracts of Rhizoma Anemarrhenae (RA). HPLC analysis was performed on an Agilent SB-C18 column (4.6 mm×150 mm, 5 μm) by gradient elution using acetonitrile and water-acetic acid(100∶0.05, v/v) as the mobile phase. Seven major components in RA were successfully separated. This quantitative method was fully validated in respect of the following performance criteria: linearity, precision, repeatability, stability, accuracy, limits of detection (LOD) and quantification (LOQ). A formula database of known compounds in RA was established, against which, most of the reported components in this herbal extract were identified effectively based on the extract masses acquired by TOFMS. This qualitative and quantitative method was successfully used to analyze the components in 10 batches of RA samples collected from different regions in China. This global quality control method, which consisted of HPLC-DAD-MS assay of seven major components and unambiguous identification of nineteen components, is suitable for routine quantification and comprehensive quality control of RA.     

Quantitative analysis of a novel antimicrobial peptide in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry

We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide （phylloseptin, PSN-1）. Chromatographic separation was accomplished on a Waters bridged ethyl hybrid （BEH） C18 （50mm× 2.1 mm, 1.7 μm） column with acetonitrile-water （25：75, v/v） as isocratic mobile phase. Mass spectrometry detection was performed in the positive electrospray ionization mode and by monitoring of the transitions at m/z 679.6/120, 509.6/120 （PSN-1） and m/z 340.7/165 （Thymopentin, IS）. Protein precipitation was investigated and the recovery was satisfactory （above 82%）. The method was shown to be reproducible and reliable with intra-day precision below 5.3%, inter-day precision below 14.2%, and linear range from 0.02 to 2 lag/mL with r〉0.994. The method was successfully applied to a pharmacokinetic study of PSN-1 in rats after intravenous administration.     

RP-HPLC法测定更昔洛韦血清浓度

为建立 HPL C法测定更昔洛韦血药浓度的方法。采用反相高效液相色谱法 ,以阿昔洛韦为内标 ,流动相为甲醇水溶液 (5∶ 95) ,检测波长为 2 52 nm。结果在 0 .2～ 15μg· ml-1范围内呈良好的线性关系。方法平均回收率为 10 0 .52 % ,RSD日内为 2 .7% ,日间为 2 .5%。说明本方法灵敏度高 ,适合于更昔洛韦的血药浓度测定及药代动力学研究。     

RP-HPLC法测定人血浆中水飞蓟宾的浓度

建立了 RP- HPLC法测定水飞蓟宾在人血浆中浓度的方法。用乙腈沉淀血浆蛋白 ,离心后 ,取上清液 10 μl进样分析。色谱条件 :C18柱 (10 μm) 2 2 0 mm× 4 .6 mm;流动相为 :V(甲醇 )∶ V(水 )∶ V(0 .5mol· L-1磷酸二氢钾 ) =10∶ 10∶ 1(用 0 .2 mol· L-1磷酸调 p H=4 .0 ) ;流速为 1.0 ml· min-1;检测波长为 2 90 nm。结果水飞蓟宾 A、B平均回收率分别为 98.96 %、97.93% ;日内 RSD分别为3.0 8%、2 .15% ;日间 RSD分别为 4 .4 8%、2 .6 4 %。     

葛根中有效部位及有效成分的高效液相色谱分析

目的建立全面控制葛根及其制剂的定性和定量分析方法。方法用反相高效液相色谱法(RPHPLC),以甲醇水(42∶58)为流动相在250nm波长下检测,对葛根有效部位(醋酸乙酯提取部位)进行指纹图谱定性分析,对葛根有效部位中的有效成分葛根素和大豆苷元进行定量测定,并对11种不同来源的葛根药材进行分析测定。结果在定性分析条件下,葛根有效部位的HPLC指纹图谱中有5个共有峰,可以作为其定性鉴别的指标峰;在以葛根素和大豆苷元为定量指标分析中,其方法的回收率分别为98.0%和95.4%,相对标准偏差(RSD)分别为1.30%和1.61%,葛根药材中葛根素和大豆苷元的含量分别在0.213%～3.626%和0.012%～0.049%范围内。结论以葛根有效部位指纹图谱定性分析和有效成分定量分析方法,可以准确有效地控制其质量。     

Analysis and determination of diterpenoids in unprocessed and processed Euphorbia lathyris seeds by HPLC-ESI-MS

Euphorbia lathyris （Caper spurge） is a toxic and potent Chinese materia medica （T/PCMM）. This study sought a method for identifying five diterpenoids （Euphorbia factors LI-L3, L7a, and Ls） with the spectra of UV and mass, quantifying three diterpenoids L1, L2, and L8 in crude extracts of unprocessed and processed E. lathyris seeds by liquid chromatography/ electrospray ionization mass spectrometry （LC-ESI-MS）. The analysis was achieved on an Agilent Eclipse XDB-C18 column （4.6 mm× 150mm i.d., 5 μm） with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 ℃ and UV detection was set at 272 nm. An ESI source was used with a positive ionization mode. The calibration curve was linear in the ranges of 9.9-79 μg/mL for Euphorbia factor Lb 3.8-30.5μg/mL for Euphorbia factor L2, and 1.0-20.6 μg/mL for Euphorbia factor LB. The average recoveries （n=6） of three diterpenoids were 98.39%, 91.10% and 96.94%, respectively, with RSD of 2.5%, 2.4% and 2.1%, respectively. The contents of the three diterpenoids in processed E. lathyris seeds were 3.435, 1.367 and 0.286 mg/g, respectively, which decreased more sharply than those in unprocessed E. lathyris seeds which were 4.915, 1.944 and 0.425 mg/g, respectively. The method is simple, accurate, reliable and reproducible, and it can be applied to control the quality of unprocessed and processed E. lathyris seeds.     

引阳素胶囊中淫羊藿苷的含量测定

目的建立引阳素胶囊中淫羊藿苷的高效液相色谱(HPLC)含量测定方法。方法采用HPLC法。色谱柱Li-chrospher C18(4.6 mm×250 mm,5μm),C18保护柱;流动相为乙腈-20 g/L冰醋酸水溶液(30∶70);检测波长270 nm;流速1.0 mL/min;柱温为室温;进样量10μL。样品加50%(体积分数)乙醇超声提取,浸提液过0.45μm滤膜后测定。结果淫羊藿苷线性范围为10.0-60.0 mg/L,回归方程为C=2.042×10-5A+0.554,r=0.9993,平均回收率为99.8%,RSD为0.65%。结论本法用于引阳素胶囊中淫羊藿苷的含量测定时简便快捷、准确。     

健脾补血片中阿魏酸的质量浓度测定

目的建立健脾补血片中阿魏酸的高效液相色谱(HPLC)测定方法。方法采用HPLC法。色谱柱Lichrospher C18(4.6 mm×250 mm,5μm),C18保护柱;流动相:乙腈-0.378 mol/L冰醋酸水溶液(30∶70);检测波长321 nm;流速0.8 mL/min;柱温:室温;进样量10μL。结果阿魏酸质量浓度在1.0-10.0 mg/L范围内,与峰面积呈良好的线性关系(r=0.9996),平均回收率为99.8%,RSD为0.26%。结论HPLC可用于健脾补血片中阿魏酸的质量浓度测定。     

川芎药材活性部位的高效液相色谱指纹图谱定性分析方法

目的　建立川芎药材活性部位的指纹图谱 ,为其定性鉴别提供依据。方法　川芎药材经乙醇超声提取后 ,用醋酸乙酯萃取 ,采用ODS柱为分析柱 ,以甲醇 水 冰醋酸 (30∶70∶0 .5 )为流动相 ,在 2 76nm条件下进行分析 ,建立其指纹图谱。并对 7种不同来源的川芎药材进行高效液相色谱指纹图谱定性分析比较。结果　本研究建立的分析方法的精密度、重现性较好 ,以阿魏酸峰计 ,RSD分别为 1.6 8%和 1.0 4 % ;不同川芎药材指纹图谱中主要峰群的整体图貌基本一致 ,但各成分含量的相对比值有所不同 ,不同来源的川芎药材共有峰面积的比值及共有峰面积和均有一定的差异。结论　HPLC指纹图谱分析法可简便、快速地鉴别区分不同来源的川芎药材     

钙拮抗剂尼群地平的反相高效液相色谱分析及其在家兔体内的分布

对兔血浆及组织器官中钙拮抗剂尼群地平的分析方法进行了研究，建立了反相高效液相色谱（RP-HPLC）的内标测定法。样品在弱碱性条件下经环已烷-异丙醇（96．5：3．5）提取，用YWGC18反相柱，以地尔硫作内标，甲醇：50mmol／L磷酸盐缓冲液（70：30）pH6．8为流动相，UV236nm检测。方法最低检测限约为10ng，血药浓度在21．6～216μg／L之间，尼群地平的量与其峰面积比值之间，线性关系良好，回归方程为Y＝0．00672X－0．013，r＝0．9958。方法回收率95．4％～98．1％，RSD＜4．63％。　用该分析方法测定了家兔口服尼群地平2h后在主要脏器体液中的分布。     

流动注射-化学发光法测定氢化可的松

目的确定以高锰酸钾-亚硫酸钠体系测定氢化可的松的流动注射-化学发光分析方法。方法在酸性条件下,氢化可的松对高锰酸钾-亚硫酸钠体系发光反应具有明显的增敏作用。据此,建立了流动注射化学发光测定氢化可的松的分析方法。结果在优化的实验条件下,氢化可的松质量浓度在1.0×10-9-1.0×10-6g/mL范围内与发光强度呈良好的线性关系,检出限(3R)为4.0×10-10g/mL,对氢化可的松进行11次平行测定,其相对标准偏差为2.2%。结论本方法应用于注射液中氢化可的松含量的测定,快速、准确、简便,灵敏度高、线性范围宽。     

Analysis of ganciclovir and its related substances using high performance liquid chromatography and liquid chromatography-mass spectrometry methods

Objective High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) methods were developed for the determination of ganciclovir and its related substances. Methods A Hypersil ODS2 column (4.6mm×250mm, 5μm) was used with a mobile phase of 0.02M potassium 1.0mL/min, and UV detector set at 254nm was used for monitoring the eluents. Results The method was simple, rapid, selective and capable of separating all related substances at trace level with a detection limit of 0.04μg/mL. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 10.2-153.0μg/mL with r=0.9998. The percentage recoveries ranged from 96.7% to 101.6%, and RSD was 1.24%-1.96% (n=5). Conclusion The method was found to be suitable not only for monitoring the reactions during the process development but also for quality control of ganciclovir. For identification of related substances, LC/MS was used. The mainly related substances of ganciclovir active pharmaceutical ingredients (API) were determined as guanine, (1, 3-dioxolan-4-yl) methyl acetate, and diacetyl guanine.     

Study on chromatographic fingerprint of sarcandra glabra （Thunb.） by microwave-assisted extraction coupled to HPLC/DAD

Microwave-assisted extraction (MAE) was used for extraction of effective components of sarcandra glabra (Thunb.), and then chromatographic fingerprint of sarcandra glabra (Thunb.) was studied by high performance liquid chromatography/diode array detector (HPLC/DAD). The conditions of MAE were optimized by an orthogonal experiment, and then the authentication and validation of the chromatographic fingerprint were conducted. Nine peaks were identified as common peaks in the fingerprint chromatograms, and isofraxidin was considered as a reference compound and quantified. Relative standard deviations of retention time and peak area of each component were less than 3% and 8%, respectively. Similarity and difference analysis were conducted by use of PCA and relation coefficient. Twenty batches of sarcandra glabra (Thunb.) samples from two different producing areas could be classified into two different groups in the PCA model. The results showed that MAE-HPLC/DAD method was simple, efficient and stable for the study of complex chromatographic fingerprint of sarcandra glabra (Thunb.), which could provide more reliable and precise information for quality evaluation.