RP-HPLC法同时测定骨瓜提取物注射剂中15种氨基酸的含量

目的建立1种同时测定骨瓜提取物注射剂中15种氨基酸含量的RP-HPLC法。方法以异硫氰酸苯酯(PITC)为柱前衍生试剂对样品中15种氨基酸进行衍生化,采用Ocean C18(250 mm×4.6 mm,5μm)色谱柱,流动相A为水-乙腈(体积比1∶4),流动相B为浓度0.1 mol·L-1乙酸钠-乙腈(体积比97∶3),梯度洗脱,流速为1.0 m L·min-1,柱温为25℃,检测波长为254 nm,进样量为10μL。用内标法计算15种氨基酸的含量。结果 15种氨基酸质量浓度在1.24~160.72 mg·L-1内与峰面积呈良好的线性关系(r=0.997 7~1.000 0),15种氨基酸的平均加样回收率在97.7~102.4%内,加样回收率的RSD值在1.7%~3.3%内。结论该方法准确、可靠、灵敏,可以用于同时测定骨瓜提取物注射剂中15种氨基酸的含量。     

DNFB柱前衍生化RP-HPLC法测定氨肽素的氨基酸含量

目的用二硝基氟苯 (DNFB)柱前衍生化RP HPLC法测定氨肽素中氨基酸的含量。方法采用二硝基氟苯为衍生试剂进行衍生 ,ODS(KromasilC18,4 6mm× 2 5 0mm ,5 μm)色谱柱分离 ,以乙腈 0 0 5mol·L-1乙酸钠水溶液为流动相 ,梯度洗脱 ,3 60nm检测 ,在 3 0min内 ,测定氨肽素的氨基酸组成与含量。结果线性范围 (总氨基酸 1 4种 )为 0 1 5～ 0 75 g·L-1;回收率为 94 8%～ 1 0 3 5 % ;RSD为0 2 6%～ 1 47% (n =5 )。结论DNFB柱前衍生化RP HPLC法可有效控制产品质量     

重组人甲状旁腺激素1-34的氨基酸组成分析

分析重组人甲状旁腺激素 1-3 4的氨基酸组成 ,为该品种的结构鉴定提供依据。水解条件含 1%苯酚的6mol L盐酸 ,10 5℃水解 2 4h ;衍生方法异硫氰酸苯酯 (PITC)柱前衍生法 ;氨基酸测定色谱条件色谱柱为PhenomenexprodigyODS 10 0A。 (4 .6mm× 2 5cm ,5 μm) ;流动相A为 0 .1mol L醋酸钠 (pH6.5 ) -乙腈 (93 :7) ,B为水 -乙腈 (1:4) ;二元梯度洗脱 ;检测波长为 2 5 4nm ;流速为 1ml min ;柱温为 40℃。结果 :实验值与理论值一致 ,偏差小于 10 %。本法准确、可靠、重现性好 ,可用于该产品的质量控制。     

阿莫西林分散片含量测定方法的探讨

目的　考察不同样品溶解方法对阿莫西林含量测定结果的影响。方法　分别用水和磷酸盐缓冲液作为溶剂,超声和振摇不同时间。结果　超声溶解明显比振摇溶解效果好。样品在磷酸盐缓冲液(pH5 0)中的溶解性比在水中好,但在 15min以后,两种溶剂的溶解性并无明显差别。结论　采用水作溶剂,对样品超声处理 2 5min,可以达到比较理想的溶解效果。     

2D-HPLC同时测定小鼠胸腺中的酸性氨基酸异构体

目的 研究D-酸性氨基酸的代谢组学,建立同时分离和定量体内样品中酸性氨基酸D、L体的离线二维HPLC法.方法 利用硅胶基质ODS整体柱为第一维分离色谱柱,流动相为乙腈-三氟醋酸-水(9∶0.05∶91);Chiralpak QD-1-AX色谱柱为第二维手性分离柱,流动相为含10 mmol·L-1柠檬酸的甲醇-乙腈(50∶50),荧光法检测,离线手性2D-HPLC分析系统.结果 两对对映体能同时得到良好地分离(Rs＞2.50);各异构体的定量线性范围均为0.005 ～5 pmol(r＞0.9998),检出限和定量限分别为2、5 fmol,回收率均在98％ ～ 105％,日内日间精密度的RSD均＜5％.应用该法测得SAMP1及SAMR1小鼠胸腺组织中D-天门冬氨酸的浓度分别为212.2、163.4 nnmol·g-1(n=6).结论 所用方法具有较高的选择性、灵敏度和准确性,可用于体内样品中酸性氨基酸异构体的同时测定.     

液液萃取-HPLC法和在线萃取-HPLC法测定卡马西平血药浓度的比较研究

目的比较卡马西平血药浓度监测的常规液液萃取-HPLC法和在线萃取-HPLC法的优缺点。方法液液萃取-HPLC法：以叔丁基甲醚为萃取溶剂,普萘洛尔为内标,分析柱为UltimateC18（51μm,4．6×250mm2）．流动相为乙腈-含0.3％甲酸的0．3％醋酸铵溶液（34：66）,检测波长为286nm,流速为1．0ml／min：在线萃取-HPLC法：以6％高氯酸溶液为蛋白沉淀剂在线萃取,预柱为VenusilC18（5tzm,4．6×30mm2）,分析柱为VenusilC18（5lxm,4．6×250mm2）,流动相为乙腈-0.3％醋酸铵溶液（40：60）,检测波长为286nm,流速为1．0ml／min。结果卡马西平在3．07—15．05μg／ml范围内线性良好．用液液萃取一HPLC法测定时r=0．9995,最低检测限为100ng／ml,回收率〉92．0％,日内RSD〈2．25％,日间RSD〈2．52％：用在线萃取一HPLC法测定时r=0．9999,最低检测限为30ng／ml,回收率〉103．8％,日内RSD〈1．21％,日间RSD〈1．21％。结论液液萃取-HPLC法灵敏度较低,操作较繁琐,溶剂消耗大,不宜于大批量样品测定;在线萃取-HPLC法灵敏度高,简单快速,稳定性强,易于实现质控．适用于低浓度的大批量样品的测定。     

HPLC法测定不同加工方法金银花中绿原酸和木犀草苷的含量

目的通过采用高效液相色谱法测定不同加工方法所得金银花药材中绿原酸和木犀草苷的含量,考察不同加工方法对金银花质量的影响。方法绿原酸含量测定采用Agilent Zorbax SB-C18色谱柱(4.6 mm×250mm,5μm),流动相:乙腈-0.4%磷酸(15∶85);流速:1.0 mL·min-1;检测波长:327 nm;柱温:室温。木犀草苷含量测定采用Hypersil Phenyl-2色谱柱(4.6 mm×150 mm,5μm),流动相:乙腈-0.5%冰醋酸,二元梯度洗脱,0¹5 min,乙腈10%15 min,乙腈10%²0%,1520%,15³0 min,乙腈20%,3030 min,乙腈20%,30⁴0 min,乙腈20%40 min,乙腈20%³0%;流速:1.0 mL·min-1;检测波长:350 nm;柱温:40℃。结果不同加工方法对金银花药材中绿原酸和木犀草苷的含量有一定影响。结论烘干法和微波法所得样品质量较好,且药材的外观色泽与药材内在质量之间存在较为密切的关系。     

高效液相色谱法测定盐酸雷尼替丁胶囊中有关物质

目的 　建立测定盐酸雷尼替丁及其胶囊中有关物质的方法。 方法 　采用高效液相色谱法 ,色谱柱为 zorbax2 13 -C1 8柱 ( 2 5 0 mm× 46mm,5 mm) ,流动相为 0 .0 5 mol· L- 1柠檬酸溶液 (用三乙胺调节 p H至 3 .5 ) -乙腈 ( 87∶ 13 ) ,检测波长为 3 14 nm。结果 　原料的可分离出 3个杂质峰 ,胶囊剂可分出 4个杂质峰。 结论 　 HPL C法简便、快速、准确 ,可作为盐酸雷尼替丁样品中杂质的检测方法。     

柱前衍生高效液相色谱法测定缩宫素的氨基酸组成

目的:建立一种测定缩宫素氨基酸组成的柱前衍生化-HPLC法。方法:缩宫素经酸水解产生的游离氨基酸用异硫氰酸苯酯衍生后,以0.1mo·lL-1醋酸钠溶液-乙腈(97∶3,pH 5.20)和乙腈-水(4∶1)为二元流动相,流速为1.0 mL.min-1,经RP-C18色谱柱梯度洗脱,于254 nm处测定。通过外标法计算样品的氨基酸组成。结果:氨基酸浓度在0.12～0.28μmol.mL-1范围时与峰面积呈良好的线性关系(r均大于0.999);定量限介于4.73～31.7ng;回收率范围为90.4%～101.8%,RSD范围为0.25%～3.2%。结论:本实验建立的分析方法分离效果好、灵敏、可靠,成功用于缩宫素的氨基酸组成测定研究。     

三七与三七药渣中游离氨基酸的含量比较及氨基酸提取工艺的研究

目的：比较三七药材与复方血栓通胶囊三七药渣中十八种氨基酸的含量,探讨三七药渣入药的合理性,并对三七氨基酸提取工艺进行考察。方法：采用高效液相色谱法进行含量测定,色谱柱为岛津Shim-pack XR-ODS色谱柱（3.0mm×75mm,2.2μm）;流动相以乙腈-0.05mol/L醋酸钠溶液梯度洗脱,0～8min,乙腈为5%;8～42min,乙腈为5%→28%;42～45min,乙腈为28%→40%;45～70min,乙腈为40%→60%;70～80min,乙腈为60%→5%;80～90min,乙腈为5%;柱温：室温;检测波长：254nm;流速：1mL×min-1;进样量：5μL。记录时间：70min。提取工艺优选采用单因素试验法考察提取次数、提取时间和溶媒倍量3个影响因素。结果：三七药渣中十八种氨基酸含量为0.092%～0.149%;最佳提取工艺为12倍量的0.1mol·L-1盐酸超声提取3次,每次1h。结论：三七药渣中仍含有大量氨基酸,建议三七原药材直接入药。     

A simpler and faster capillary electrophoresis method for determination of mianserin enantiomers in human serum

A stereospecific sample stacking capillary zone electrophoresis method is described for determination of S(+) and R(-) enantiomers of mianserin (1,2,3,4,10,14b-hexahydro-2-methyldibenzo[c,f]pyrazino[1,2-a]azepine) in human serum. The enantiomers of mianserin were extracted from human serum in one step extraction procedure using the mixture n-heptane:ethyl acetate (80:20, v/v). After separation of layers and freezing at -28 degrees C the organic layer was decanted and evaporated under a stream of nitrogen. The sample was dissolved in the mixture: water:methanol:acetonitrile (2:1:1, v/v/v). Separation was conducted in an aqueous solution of phosphoric acid (0.075M) adjusted to pH = 3.0 with concentrated triethylamine, and 2 mmole/L of 2-hydroxypropyl-beta-cyclodextrin. The analytes were measured by ultraviolet detection at 214 nm after separation on a Fused-Silica eCAP capillary. Clozapine was used as an internal standard. Recovery of the enantiomers from serum ranged from 82.94 to 90.37%. Total time of analysis was 49 minutes, whereas the other methods needed up to 100 minutes.     

Evaluation of solid-phase sorbents for the analysis of ropinirole in whole blood

In this paper, the extraction and analysis of ropinirole from whole blood using solid-phase cartridges is presented. Previously published methods for the analysis of this drug have employed plasma samples using C(18) cartridges. Liquid-liquid extraction has been employed for analysis of postmortem samples. In the method, drug free blood was spiked with ropinirole (0 to 10 ng) and an internal standard (quinidine). The samples were buffered with distilled water and centrifuged. The supernatant liquid was applied to previously conditioned endcapped C(6), C(18), and C(8)/SCX solid-phase extraction columns. The columns were washed, dried, and eluted with various solvents systems. The eluants were collected and evaporated. The residue was dissolved in 100 microL of aqueous 0.1% trifluoroacetic acid and analyzed by liquid chromatography using a C(18) (4.6 x 150 mm, 5-microm particle size) column and monitored at 250 nm, using diode-array detection. A mobile phase consisting of methanol/0.1% TFA in distilled water (22:78 v/v) was employed. The data was collected and appraised. It was found that 3-mL 200-mg CEC06 C6 (Hexyl endcapped) solid-phase columns that had been washed with 3 x 3 mL water and 3 x 3 mL acetonitrile and eluted with a solvent system consisting of 95:5 v/v acetonitrile/ammonia performed best. The linear range for this analysis was found to be from 0 to 10 ng/mL. The limit of detection was determined to be 1 ng/mL with a limit of quantification of 2.5 ng/mL.     

The measurement of propofol in human blood samples by liquid chromatography

A simple, sensitive, reliable and rapid column liquid chromatographic method was developed for the measurement of propofol, a new anesthetic agent, in human maternal and placental blood. The assay involved a single extraction of the drug and internal standard, thymol, from blood buffered with 0.1 M sodium dihydrogen phosphate buffer into cyclohexane. The organic extract, basified with tetramethylammonium hydroxide, in evaporation tube was evaporated to dryness at 30 degrees C under nitrogen. The residue was redissolved in 80 microliters acetonitrile and an aliquot (25-50 microliters) of the concentrate was injected into a C18 reversed-phase column (4 microns, Nova-Pak) linked to a C18 pre-column. The mobile phase consisted of 60% (v/v) acetonitrile in distilled water containing 1% v/v glacial acetic acid and was eluted at 2.5 ml min-1. The components of the column effluent was monitored by a fluorometric detector with excitation and emission wavelengths set at 276 nm and 310 nm, respectively. The method has been used to measure propofol concentrations in blood from maternal veins and placental arteries and veins during cesarean section.     

SPE—HPLC法测定环孢素A全血中药物浓度

目的 建立全血中环孢素A药物浓度的HPLC测定方法。方法 以Nucleodur C18（150mm×4．6mm，5μm）为分析柱，乙腈-水（71：29，v／v）为流动相，柱温为65℃，检测波长为210nm。先以80％乙腈沉淀蛋白，沉淀后的上清液过SPE小柱处理后进样分析。结果 环孢素A在40．0～1600.0 ng·mL^-1线性关系良好（r＝0．9998），在3个浓度下的平均回收率〉95．0％，日内、日间RSD＜10．0％，环孢素A平均萃取回收率〉86.0％，环孢素D平均萃取回收率为91．2％。结论本方法稳定、准确，能用于测定环孢素A血药浓度。     

High-performance liquid chromatographic analysis of nitrendipine in human plasma using ultraviolet detection and single-step solid-phase sample preparation

A high-performance liquid chromatographic (HPLC) method was developed for the determination of nitrendipine in human plasma using solid-phase extraction (SPE) and ultraviolet detection. A 30-microl aliquot of methanol (containing 2 microg/ml of the internal standard, nimodipine) was added to a 1-ml aliquot of biological sample. After vortex-mixing, the mixture was loaded on C(18) SPE cartridge which was conditioned with methanol and distilled water. After washing with distilled water, the SPE cartridge was eluted with 1-ml aliquot of diethyl ether. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100 microl aliquot of mobile phase, and a 50 microl aliquot was injected onto the C(18) reverse-phased column. The mobile phase, 10 mM phosphate buffer (pH 4.5):acetonitrile (50:50, v/v), was run at a flow rate of 1.2 ml/min. The column effluent was monitored using ultraviolet detector at 238 nm. The retention times for nitrendipine and the internal standard were approximately 10.1 and 12.6 min, respectively. The detection limit of nitrendipine in human plasma was 2.0 ng/ml. The coefficients of variation (CV) of the assay were below 16.5% for human plasma, and no interferences from endogenous substances were found. This specific, accurate and precise assay was useful in the study for the pharmacokinetic characteristics of nitrendipine.     

Quantitative analysis of lincomycin in animal tissues and bovine milk by liquid chromatography electrospray ionization tandem mass spectrometry

A sensitive method for determining lincomycin in bovine milk, animal muscles and organs using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) is presented. Milk and homogenized animal tissues were extracted with acetonitrile twice after addition of an appropriate amount of clindamycin, a lincosamide analogue as the internal standard. The combined extracts were finally made up to 10 ml with distilled water and partitioned with hexane to remove the animal fats prior to analysis. Analytes in the extracts were separated on a reversed phase C18 column (250 mm x 2.1 mm, 5 microm) using a mobile phase of a 3:7 (v/v) mixture of 0.1% formic acid in acetonitrile and an ammonium formate buffer (ammonium formate:formic acid:acetonitrile:water, 1:5:50:950, v/v/v/v) running at a flow rate of 0.2 ml min(-1). Presence of lincomycin was confirmed by the presence of two characteristic product ions at m/z 126.1 and 359.2 within a defined retention time window from the precursor ion at m/z 407.2, whilst quantification was based on the relative ratio of the sum of the peak areas at m/z 126.1 and 359.2 for lincomycin to that of the internal standard (peaks at m/z 126.1 and 377.2) with reference to the respective ratios of the calibration standards. The validated method that was found to have linear responses in the calibration range from 25 to 3000 microg kg(-1) and satisfactory intra-day and inter-day accuracy (94.4-107.8%) and precision (1.3-7.8%) at concentrations ranging from 100 to 1500 microg kg(-1) has been applied to real samples and matrix spiked samples. It is considered robust and suitable for analysis of lincomycin in milk and animal tissues.     

Determination of a new phosphodiesterase V inhibitor,DA-8159, in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.     

Multi-syringe chromatography (MSC) system for the on-line solid-phase extraction and determination of hydrochlorothiazide and losartan potassium in superficial water, groundwater and wastewater outlet samples

In this paper, a combination of multi-syringe chromatography analysis technique with extraction disks sorbents for the pre-concentration and determination of hydrochlorothiazide and losartan potassium in superficial water, groundwater and wastewater outlet samples has been developed. The system developed was proved for the determination of hydrochlorothiazide and losartan potassium in spiked water samples with recoveries ranging from 95 to 118%. The method involves the on-line enrichment of the targeted analytes from spiked water samples onto a Cation-SR sorbent material. The analytes are subsequently eluted and transported to the monolithic column, Chromolith Flash RP-18e column (25mmx4.6mm i.d.). The mobile phase used was 10mM potassium dihydrogen phosphate (pH 3.0):acetonitrile:methanol (60:30:10 v/v/v), flow-rate 0.8mLmin(-1). UV detection is carried out at 226nm. Under the optimized chemical and physical variables, the detection limit for hydrochlorothiazide and losartan potassium calculated as 3Syx/b was 0.07 and 0.09mgL(-1), respectively, for a sample loading volume of 1.0mL.     

HPLC with ultraviolet detection for the determination of chloroquine and desethylchloroquine in whole blood and finger-prick capillary blood dried on filter paper

A simple, sensitive, selective and reproducible method based on liquid chromatography was developed for the determination of chloroquine (CQ) and its active plasma metabolite desethylchloroquine (DECQ) in finger-pricked capillary blood spot onto filter paper (DBS) and whole blood samples. Both were separated from the internal standard quinine on a reversed phase C18 column, with the mobile phase consisting of a mixture of 1% diethylamine, acetonitrile and methanol (20:55:25, v:v:v) running at a flow rate of 1.0ml/min. Retention times of QN, DECQ and CQ were 4.5, 5.7 and 6.4min, respectively. Ultraviolet detection was at the wavelength 256nm. Sample preparation was done by extraction with hexane and tert-butyl methyl ether (1:1, v:v). Good precision and accuracy were obtained for both within-day repeatability and day-to-day reproducibility. Limit of quantification (LOQ) for both CQ and DECQ was accepted as 50ng/ml using 80μl DBS sample and 25ng/ml using 150μl whole blood sample. The mean recoveries for CQ, DECQ and internal standard for both whole blood and DBS were between 74 and 87%. The method was successfully applied for a pharmacokinetic study of CQ and DECQ in patients with Plasmodium vivax. Excellence correlation (r=0.997) was observed between the analysis of both CQ and DECQ in paired whole blood and DBS samples.     

Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.