高效液相色谱法同时测定虫草素和腺苷

本文用微波法提取蛹虫草培养基和子实体中的虫草素及腺苷,并利用高效液相色谱法同时测定虫草素和腺苷.选择Inertsil ODS-SP柱(150×4.6 mm,5 μm)为色谱柱,乙腈-水为流动相,流速1.0 mL/min,柱温35℃,二极管阵列检测器(DAD)检测,检测波长260 nm,进样量10 μL.在2～10 μg...     

高效液相色谱法测定蛹虫草中腺苷和虫草素

在蛹虫草中腺苷和虫草素含量的反相高效液相色谱分析中,所用色谱柱为WatersNOVA-PAK C18(3.9 mm×300 mm,4μm),流动相为0.01 mol.L-1KH2PO4-K2HPO4缓冲溶液(pH 6.86)+1%(体积分数)四氢呋喃,流速为1.0 mL.min-1。紫外检测的波长为260 nm,峰面积与腺苷浓度和虫草素浓度之间的线性范围依次为1.27~50.5 mg.L-1和3.23~129.0 mg.L-1,其检出限依次为0.18 ng和0.25 ng。在腺苷及虫草素的浓度水平依次为5.05 mg.L-1和12.9 mg.L-1时测试方法的精密度,求算得RSD(n=7)值依次为0.094%和0.792%。用标准加入法试验方法的回收率,求得两者的平均回收率依次为103.2%和98.5%。     

HPLC-ELSD法测定蛹虫草中6种游离氨基酸的含量

建立了反相高效液相色谱-蒸发光散射检测(HPLC-ELSD)直接测定6种未衍生游离氨基酸(脯氨酸Pro、缬氨酸Val、蛋氨酸Met、异亮氨酸Ile、亮氨酸Leu、苯丙氨酸Phe)含量的分析方法。采用VenusilMP-C18色谱柱,以甲醇和水为流动相进行线性梯度洗脱,流速为0.8mL·min-1,漂移管温度40℃,氮气流速2.5L·min-1;优化了流动相的洗脱条件。结果表明,在优化实验条件下,6种氨基酸峰面积的对数值与氨基酸质量浓度的对数值呈良好线性,检出限为10.0~15.0mg·mL-1,方法精密度及稳定性良好。采用该法对微波法提取的蛹虫草子实体中的氨基酸进行测定,待检蛹虫草样本中不含缬氨酸(Val),其余5种氨基酸的总量为16.00mg/g,约占蛹虫草总质量的1.60%,其中脯氨酸(Pro)(占0.72%)和苯氨酸(Phe)(占0.56%)的含量较高。已检出的5种Pro、Met、Ile、Leu、Phe含量分别为7.20、0.88、0.69、1.65、5.58mg/g。     

La(NO3)3对蛹虫草活性物质含量的影响EI北大核心CSCD

蛹虫草是一种著名的药用和食用真菌,研究者们常在培养基中添加亚硒酸钠、Fe SO4等无机物来提高蛹虫草活性物质的含量。本文采用高效液相色谱法和紫外分光光度法测定了La(NO3)3处理后蛹虫草活性物质的含量,探究La(NO3)3对蛹虫草活性物质含量的影响。结果表明,在实验浓度范围内,10,50和200 mg·L-1这3个浓度对蛹虫草的活性物质含量影响较大。La(NO3)3浓度为10 mg·L-1时,对喷司他丁、虫草酸和多糖合成具有促进作用,其含量分别为空白组的6.2倍、2.4倍和1.3倍,La(NO3)3对蛹虫草的腺苷和N6-2-羟乙基腺苷合成具有抑制作用,其含量仅为空白组的24.29%和16.87%。La(NO3)3浓度为50 mg·L-1时,对虫草素合成具有显著促进作用,其含量为空白组的8.2倍,La(NO3)3对多糖合成具有抑制作用,其含量仅为空白组的73.14%。La(NO3)3浓度为200 mg·L-1时,对腺苷和N6-2-羟乙基腺苷合成具有促进作用,其含量分别为空白组的1.5倍和2.6倍。La(NO3)3对麦角甾醇和蛋白质的含量未产生显著影响(P>0.05)。     

高效液相色谱法测定地顶孢霉培养物中的腺苷

建立测定虫草源饲料添加剂地顶孢霉培养物中腺苷含量的高效液相色谱方法。样品经过研碎、超声处理,离心、过滤后上机测定。腺苷的最佳提取条件:以超纯水为浸提液,用超声波浸提,浸提温度为40℃,浸提时间为55 min。使用Waters Spherisorb ODS2柱(150 mm×3.9 mm,5μm),以甲醇-0.01 mol/L磷酸二氢钾混合液(10∶90)为流动相,流量为1.0 mL/min,进样体积为20μL,检测波长为254 nm。腺苷的质量浓度在0.5~100μg/mL范围内与色谱峰面积成良好的线性关系,相关系数为0.9990。样品测定结果的相对标准偏差为1.65%(n=6),3水平加标的平均回收率为98.19%。该方法简便、快捷,可准确测定虫草饲料添加剂地顶孢霉培养物中腺苷的含量。     

离子交换色谱分离-紫外检测法测定吡啶离子液体阳离子

建立了用离子交换色谱分离-紫外检测法测定N-乙基吡啶、N-丁基吡啶和N-丁基四甲基吡啶3种吡啶离子液体阳离子的方法。采用磺酸型阳离子交换柱,以乙二胺-柠檬酸-乙腈为流动相,研究了流动相和色谱柱温度对离子保留行为的影响和规律。实验发现,吡啶阳离子的保留过程是放热过程。优化后的色谱条件:流动相为乙二胺(0.2 mmol//L)-柠檬酸(0.3 mmol//L)-乙腈(0.5%,v/v,pH=4.2),流动相流速为1.0mL/min,色谱柱为Shim-pack IC-C1阳离子交换柱,色谱柱温度为30℃。在此条件下3种吡啶阳离子可以达到基线分离。所测阳离子的检出限(S/N=3)分别为0.01、0.01、0.02mg/L,峰面积的相对标准偏差(n=5)小于0.8%。紫外检测法测定化学实验室合成的吡啶离子液体样品,样品加标后测得的加标回收率在96.3%~104%。方法准确、可靠、快速,具有较好的实用价值。     

基于UPLC-Q-TOF-MS法分析凉山虫草化学成分

应用超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q-TOF-MS)对凉山虫草提取物的主要化学成分进行定性分析。采用Agilent SB-C_(18)(50 mm×4.6 mm,1.8μm)色谱柱,以0.1%甲酸水-乙腈溶液为流动相,梯度洗脱,流速0.25 m L/min,柱温30℃;质谱分析采用信息关联模式(IDA)正、负离子分别采集。应用Peak View软件的Formula Finder等功能、各色谱峰的质谱数据与数据库匹配结合各色谱峰的二级碎片裂解规律,共鉴定出27个化合物,其中8个为虫草中未曾报道物质;主要化学成分包括生物碱、糖苷、核苷、氨基酸等成分。该方法精确、可靠、高效,适用于凉山虫草成分的快速鉴定,为凉山虫草的开发利用以及阐明其药效物质基础提供了参考。     

高效液相色谱法定量测定中药千层塔提取物中的石杉碱甲

建立了高效液相色谱快速定量测定中药千层塔提取物中石杉碱甲含量的分析方法。千层塔提取物经甲醇/水/甲酸(10/90/0.2, v/v/v)提取并定容后,过滤膜后直接分析。色谱分离选用XCharge C18色谱柱(150 mm×4.6 mm, 5 μm),以水(含0.1%三氟乙酸)和乙腈(含0.09%三氟乙酸)为流动相进行梯度洗脱,流速为2 mL/min,于310 nm波长下检测,可在10 min内完成石杉碱甲的快速分离分析。结果表明,石杉碱甲在2.12～106 mg/L范围内线性关系良好(相关系数为0.9999);平均加标回收率为102.34%,相对标准偏差(RSD)为0.46%;日内及日间精密度均小于2%,满足定量要求。该方法简便、快速,结果可靠,重现性好,可作为千层塔提取物质量评价的依据。     

以离子液体作流动相添加剂的高效液相色谱法测定复方苦参注射液中4种生物碱

将离子液体1-丁基-3-甲基咪唑四氟硼酸盐(［BMIM］BF4)作为流动相添加剂建立了同时测定复方苦参注射液中4种主要生物碱的HPLC分析方法。以Agilent TC-C18柱(250 mm×4.6 mm, 5 μm)为分离柱,甲醇-0.1%磷酸水溶液(含2.2×10-4mol/L ［BMIM］BF4)(5:95, v/v)为流动相,流速1.0 mL/min,柱温30 ℃,进样量20 μL,在205 nm下检测。结果表明,苦参碱、槐定碱、氧化槐果碱以及氧化苦参碱的质量浓度分别在25.8～155.0 mg/L, 40.0～240.0 mg/L, 21.7～130.0 mg/L和37.5～225.0 mg/L范围内线性关系良好,相关系数均大于0.9990,平均回收率(n=9)在96.2%和98.9%之间。离子液体的加入能明显改善C18柱分离生物碱的色谱峰形并提高分离度。本法简便、快速、重复性好,可用于复方苦参注射液中生物碱的分离与测定。     

高效液相色谱法测定营养品中的虫草素

采用高效液相色谱法测定营养品中的虫草素。样品10.00g加水至50.00g溶解后,取溶液2.50g,加水30mL混匀后,加入30.0g·L~(-1)偏磷酸溶液1.0mL沉淀干扰物质,上清液在Phenomenex Gemini C_(18)色谱柱(250mm×4.6mm,5μm)上分离,以乙腈和水为流动相进行梯度洗脱,紫外检测波长为260nm。虫草素的质量浓度在0.05~1.00mg·L~(-1)内与峰面积呈线性关系,测定下限(10S/N)为5.0mg·kg~(-1)。加标回收率在100%~102%之间,测定值的相对标准偏差(n=6)小于5.0%。     

Simple HPLC-UV determination of nucleosides and its application to the authentication of Cordyceps and its allies

A simple HPLC-UV method combined with a simple extraction procedure of nucleosides (adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine) was developed and applied to the authentication of Cordyceps and its allies. The separation was performed on a C(18) column by isocratic elution with acetonitrile-water, and UV detection at 260 nm. The amounts of adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine in Cordyceps were 0.28-14.15, 0.006-6.36, 0.01-0.14, 0.68-14.79 and 0.19-20.29 mg/g, respectively. Among the nucleosides studied, cordycepin was characteristically included in Cordyceps militaris (L.) Link. (CM), which is one of key Cordyceps allies, and might be a good marker for authenticating CM. The ratio of nucleosides to adenosine contents in Cordyceps seemed to be a useful marker for authentication and quality control of Cordyceps.     

Quality evaluation of Cordyceps through simultaneous determination of eleven nucleosides and bases by RP-HPLC

A new RP-HPLC method has been developed for the simultaneous determination of 11 nucleosides and bases, including adenosine, cordycepin, cytidine, guanosine, inosine, thymidine, uridine, cytosine, guanine, thymine, and uracil in Cordyceps. Determination was achieved on a Zorbax 300SB C18 analytical column (4.6 x 250 mm id, 5 mm) using gradient elution with diode-array detection. All calibration curves showed good linearity (r2 > 0.9995) within the test ranges. The developed method was simple, rapid, and accurate, and showed good reproducibility for the quantification of 11 nucleosides and bases in natural and cultured Cordyceps with both intra- and inter-day variations of less than 1.8%. Furthermore, hierarchical clustering analysis based on the typical peaks of adenosine, cordycepin, and inosine in HPLC profiles from the 11 tested samples showed that natural and cultured Cordyceps were in different clusters, which could provide a means of discriminating between Cordyceps of different origins. Thus, adenosine, cordycepin, and inosine could be used as markers for quality control of Cordyceps.     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

A solid-phase extraction method for high-performance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study

A sensitive solid-phase extraction/high-performance liquid chromatographic method with ultraviolet detection was established for the analysis of salvianolic acid B in rabbit plasma. The analyte was separated on a reversed-phase column with trifluoroacetic acid-methanol-acetonitrile (70:10:20, v/v/v) as mobile phase at a flow rate of 1 mL/min, and ultraviolet detection at 315 nm. The calibration curve for salvianolic acid B was linear over the range 35-1400 microg/L with coefficients of correlation >0.999. The inter-day and intra-day precisions of analysis were <15%, and assay accuracy ranged from 95.3 to 109.1%. This method is suitable for determining salvianolic acid B in plasma and thus investigating the pharmacokinetics of salvianolic acid B.     

采用新型固相萃取柱快速测定食用植物油中苯并［a］芘

研究了Bond Elut ENV新型固相萃取柱在食用植物油中苯并[a]芘快速检测中的应用,建立了快速测定食用植物油样品中苯并[a]芘残留量的固相萃取/液相色谱/荧光检测法。样品用正己烷溶解,固相萃取净化,SUPELCOSILTMLC-PAH(25 cm×4.6 mm,5μm)色谱柱分离,以乙腈-水(95∶5)为流动相,荧光检测(λex=297 nm,λem=408 nm),外标法定量。苯并[a]芘的检出限为0.3μg/kg,在1.0～50.0μg/L范围内线性关系良好,相关系数为0.999 6,方法的回收率为79%～102%,相对标准偏差不高于9.4%。该方法准确、实用、简便、快速,在食用植物油的苯并[a]芘残留量检测方面有广泛的应用前景。     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

Validation of a new HPLC‐UV method for determination of the antibiotic linezolid in human plasma and in bronchoalveolar lavage

A rapid and selective HPLC‐UV method was developed for the quantification of linezolid (LNZ) in human plasma and bronchoalveolar lavage (BAL) at the concentrations associated with therapy. Plasma samples were extracted by solid‐phase extraction followed by evaporation to dryness and reconstitution in mobile phase solution. The chromatographic separation was carried out on a C₁₈ column with an isocratic mobile phase consisting of dihydrogen phosphate buffer 50 mm (pH 3.5) and acetonitrile (60:40 v/v). The detection was performed using a photodiode array. Under these conditions, a single chromatographic run could be completed within 12 min. The method was validated by estimating the precision and the accuracy for inter‐ and intra‐day analysis in the concentration range of 25–25600 ng/mL. The method was linear over the investigated range with all the correlation coefficients R > 0.999. The intra‐ and inter‐day precision was within 8.90% and the accuracy ranged from ?4.76 to +5.20%. This rapid and sensitive method was fully validated and could be applied to pharmacokinetic study for the determination of LNZ levels in human plasma and BAL samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of adenosine by RP-HPLC method and its application to the study of adenosine kinase kinetics

An RP-HPLC method for the analysis of adenosine (ADO) has been developed and validated. In the present study, we report an RP-HPLC-based method with modifications of mobile phase and shorter retention time that substantially improved the efficiency of ADO analysis. The HPLC separation of the ADO was achieved on a C18 column, using a mobile phase consisting of water, containing 7% v/v ACN, at a flow rate of 0.8 mL/min. The column effluent was monitored by UV detection at 260 nm. A linear response was achieved over the concentration range of 0.25-100.00 micromol/L. The analytical method inter- and intra-run accuracy and precision were better than +/- 15%. The LOQ was 0.25 micromol/L, with ADO detection in the range of 6.25 pmol per sample. The method has been applied to the study of adenosine kinase (AK) kinetics.     

Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

固相萃取-超高效液相色谱联用测定水果及果酱中的展青霉素

以硅酸镁、硅胶、硅藻土、硫酸钙为原料,加入乙醇研磨成匀浆,干燥,填充于聚丙烯柱管中,制备成新型固相萃取小柱。样品经果胶酶酶解,乙腈提取,固相萃取净化,以C18色谱柱(100 mm×2.1 mm,1.8μm)为分离色谱柱进行定性、定量分析。流动相为0.8%(体积分数)四氢呋喃水溶液,流速为0.5 mL/min,以276 nm波长进行检测。考察了果胶酶对萃取效果的影响、固相萃取小柱的净化效果及最佳色谱分析条件。在0.1~10 mg/L范围内,展青霉素峰高与质量浓度呈良好的线性关系,相关系数(R~2)为1,方法检出限为10.22μg/kg,样品的加标回收率为86.58%~94.84%,相对标准偏差(RSD)为1.45%~2.28%。实验结果表明,自制固相萃取小柱净化效果好,超高效液相色谱分离效能高,样品测定操作方法简单,结果准确,对水果制品的质量安全控制具有重要的意义。