反相高效液相色谱法直接测定水蛭中14种未衍生氨基酸的含量

建立了一种反相高效液相色谱/蒸发光散射检测器方法,同时测定中药水蛭中14种未衍生氨基酸含量。以异亮氨酸等氨基酸为标准品、PrevailTMC18(250mm×4.6mmi.d.,5μm)为色谱柱、梯度洗脱、漂移管温度115℃、气体流量2.5L/min,在25min内即可将水蛭中14种氨基酸分离测定。氨基酸质量浓度0.072~2.29g/L时,其对数值与峰面积对数值线性关系良好;14种氨基酸的加样回收率为94.8%~104.4%;信噪比为3时,异亮氨酸检出限为20mg/L。该法快速、简便、准确,可作为水蛭中氨基酸直接测定方法,亦可为其它中药中氨基酸分析提供参考。     

高效液相色谱法测定浓缩果汁中5种可溶性糖

建立同时测定浓缩果汁中木糖、果糖、葡萄糖、蔗糖和麦芽糖的高效液相色谱–蒸发光散射检测方法(HPLC–ELSD)。样品采用乙腈–水(50∶50)稀释,用酰胺键合色谱柱BEH Amide分离,蒸发光散射检测器检测,在10 min内完成木糖、果糖、葡萄糖、蔗糖、麦芽糖的分离。浓缩果汁中5种可溶性糖的质量浓度在0.1～2.0 mg/m L与色谱峰面积均呈线性关系,相关系数为0.991 0～0.993 1,检出限为0.23%～0.98%,样品加标回收率为88.6%～104.3%,测定结果的相对标准偏差为2.2%～6.3%(n=6)。该方法快速、准确,可作为浓缩果汁中糖含量的质量控制方法。     

反相高效液相色谱-蒸发光散射检测法同时测定阿胶中的17种未衍生氨基酸

建立了反相高效液相色谱-蒸发光散射检测法(HPLC-ELSD)同时测定中药阿胶中17种未衍生氨基酸含量的方法。采 用PrevailTMC18色谱柱 (250 mm×4.6 mm i.d., 5 μm)，以乙腈-0.7%三氟醋酸溶液(含5.0 mmol/L七氟丁酸）为流 动相进行线性梯度洗脱，流速为0.8 mL/min，在漂移管温度115 ℃、氮气流量2.5 L/min条件下，在25 min内即可完成 对阿胶中17种氨基酸的分离测定。氨基酸质量浓度为0.073～2.327 g/L时,其峰面积的对数值与质量浓度的对数值线性 关系良好；17种氨基酸的加样回收率为93.5%～104.8%；信噪比为3时,测得氨基酸的最低检测限介于18.2 mg/L与54.6 mg/L之间。该法快速、简便、准确,可作为阿胶中氨基酸的直接测定方法,亦为其他药物中氨基酸的分析提供了参考。     

亲水作用色谱-蒸发光散射检测保健食品中三种支链氨基酸的含量

建立了亲水作用色谱-蒸发光散射(HILIC-ELSD)法直接测定保健食品中L-亮氨酸、L-异亮氨酸和L-缬氨酸三种支链氨基酸(BCAA)含量的分析方法。采用Merck ZIC-HILIC色谱柱(150×4.6mm,5μm)。流动相为乙腈-20mmol/L乙酸铵溶液(体积比70∶30),等度洗脱,流速1.2mL/min。在漂移管温度50℃,雾化氮气压力0.3MPa的条件下,20min内可完成三种目标支链氨基酸的分离测定。所测三种支链氨基酸的线性关系良好,平均回收率在96.96%~109.32%之间。该方法不需要衍生,检测过程简单方便,能够准确快速地测定批量样品中支链氨基酸的含量。     

反相离子对色谱-蒸发光闪射法测定α-复方酮酸片中的5种有效成分

建立了反相离子对色谱结合蒸发光散射检测器同时测定α-复方酮酸片中消旋羟蛋氨酸钙(HMACa),酮缬氨酸钙(KVCa),消旋酮异亮氨酸钙(KILCa),酮亮氨酸钙(KLCa)和酮苯丙氨酸钙(KPACa)含量的方法。液相色谱条件:以乙腈-20 mmol/L乙酸铵缓冲溶液(含30 mmol/L正戊胺,pH 7.0)为流动相进行梯度洗脱,流速为0.6 mL/min,柱温为35℃。蒸发光散射检测器(ELSD)漂移管温度为50℃,载气流速为1.0 mL/min。方法的线性范围为20~150 mg/L,5种物质的相关系数(r)均大于0.999,检出限分别为7.05,16.66,8.00,8.00和14.28 mg/L,样品加标回收率在87.5%~109.8%之间,相对标准偏差(RSD)均小于3.2%(n=5)。方法适用于α-复方酮酸片中5种有效成分的同时测定。     

柱前衍生-反相高效液相色谱法测定荔枝果蒂中的氨基酸

建立了AccQ· Tag柱前衍生-高效液相色谱法测定荔枝果蒂中氨基酸含量的方法.荔枝果蒂样品在120℃下真空水解22 h,再与AQC衍生剂进行衍生,并采用反相液相色谱法-荧光检测器进行分析,外标法计算样品中17种氨基酸的含量.17种氨基酸在35 min内可完全分离,荔枝果蒂中氨基酸的含量在2.5～25 μmol/L范围内与色谱峰面积呈良好的线性关系,相关系数不小于0.999.方法的加标回收率在71.2％～92.0％之间,测定结果的相对标准偏差为3.16％～9.94％(n=6),方法的检出限(S/N=3)在0.000 5～0.0012 mg/L之间.     

高效液相色谱-蒸发光散射法测定牛黄中多种有效成分含量及其质量相关性研究

建立高效液相色谱-蒸发光散射法(HPLC-ELSD)同时快速测定牛黄中7种组分的含量的方法。色谱柱为UltimateAQ-C18柱,流动相为乙腈-0.2%甲酸水溶液,梯度洗脱,流速1mL/min;蒸发光散射检测器参数:漂移管温度100℃,载气(N2)流速1.9L/min。在选定色谱条件下,胆固醇、各种胆汁酸及其钠盐峰形良好且能达到很好地分离。各组分色谱峰峰面积的对数与浓度的对数呈良好线形关系(r0.9972);标准加入回收率为98.8%～112.7%;7种组分的最低检出限为0.001～0.007g/L。本方法可简便、快速地同时检测不同牛黄样品中7种组分的含量并可表征不同牛黄样品的质量,从而为该名贵药材的质量控制提供更全面的依据和参考。     

高效阴离子交换色谱积分脉冲安培检测法分析注射液中的氨基酸

建立了高效阴离子交换色谱积分脉冲安培检测器同时分离并测定注射液中18种常见氨基酸、氨基己酸和牛磺酸含量的方法. 注射液用20 mg/L的NaN3溶液稀释1000倍, 经0.22 μm尼龙膜过滤后直接进样分析. 以一定浓度的NaOH和NaAc溶液为淋洗液, 选择合适的梯度淋洗条件, 20种氨基酸在AminoPac PA10阴离子交换色谱柱上在30min内很好地分离, 并用脉冲安培检测器进行了测定. 氨基酸的检出限在0.14～3.81 pmol (25 μL进样, 峰面积定量).     

超高效液相色谱-蒸发光散射法测定功能性饮料中牛磺酸

建立了超高效液相色谱-蒸发光散射法(UPLC-ELSD)测定功能性饮料中牛磺酸含量的方法。样品直接过膜或适当稀释后过膜,经酰胺基色谱柱分离,以乙腈-0.1%甲酸水为流动相,梯度洗脱,蒸发光检测器测定,外标法定量。牛磺酸含量在0.10~2.0 mg/m L范围内线性关系良好,相关系数为0.9992,加标回收率为93.8%~102.5%,相对标准偏差(RSD)为0.62%~2.6%,定量限为0.10 mg/m L。与国标法对比测定了市售功能性饮料中牛磺酸的含量,结果一致。     

微流蒸发光散射检测器的研制及评价

构建了适用于纳升级到微升级流量的毛细管分离体系的微流蒸发光散射检测器(μELSD),实现了其与毛细管液相色谱(eLC)的联用.对雾化器孔径和雾化毛细管内径、蒸发管内径和长度、光散射池尺寸、雾化毛细管位置和辅助载气流量等参数进行了优化.在最优条件下,微流蒸发光散射检测器检出限为直接进样葡萄糖1 ng(S/N＞ 10),线性范围0.01～1.0 μg,重复性好,峰面积RSD(n=6)为0.4％,峰高RSD(n=6)为0.3％.本检测器已成功应用cLC-μELSD平台,使用C18毛细管色谱柱(内径250 μm),0.1％甲酸铵溶液(pH 4.5)-甲醇(60∶40,V/V)为流动相,分离检测了3种常用甜味剂,表明本研究构建的系统可以应用于实际分离检测中,具有分析时间快、溶剂消耗量少、样品需求量小的优点.     

HPLC assay of underivatized free amino acids with column switching and evaporative light-scattering detection

In this communication the authors report the HPLC SEPARATION OF 20 amino acids without derivatization. Separation was achieved by column swithing using a C-8 reversed-phase column and a cationic ion-exchange column. Detection of the underivatized amino acids was accomplished by means of an evaporative laser light-scattering (ELS) detector. The influence of the temperature of the diffusion tube and nebuolzation gas pressure on detector response is reported. The relationship of peak areas vs. mass show a good log-log linearity within a 1:10 range. Detechtion limit is in the order of 200 pmol for most amino acids under the conditions used.     

Determination of Free and Total Underivatized Amino Acids Including L-Canavanine in Bitter Vetch Seeds Using Hydrophilic Interaction Liquid Chromatography

A new hydrophilic interaction liquid chromatography method coupled with diode-array detector was developed for the determination of 17 underivatized amino acids including L-canavanine in bitter vetch [Vicia ervilia (L.) Willd.] seeds. Amino acids were extracted as free as well as total extracts after acid hydrolysis, followed by chromatographic separation on a Zorbax Rx-SIL column with a mobile phase of acetonitrile/potassium phosphate buffer (12.5?mM; pH 3.0) using gradient elution and detection at 190?nm. The method is characterized by a wide linear range (0.01–200?µg/mL, r?>?0.9987), sufficient accuracy (relative error 86.3–109.1%), and suitable precision for the results (relative standard deviation <4.9% in the case of intra-day and <9.8% in the case of inter-day precision). The limits of detection and quantification for free amino acids ranged from 0.01 to 0.24?mg/g and 0.03 to 0.72?mg/g, respectively, whereas the total amino acids ranged from 0.02 to 0.47?mg/g and 0.07 to 1.43?mg/g, respectively. The mean recoveries of free and total amino acids in spiked samples exceeded 70.3% for most amino acids. The mean total content of free and total amino acids in bitter vetch seeds was 1.71 and 14.88?g/100?g seed, whereas the corresponding values for canavanine were 0.07 and 0.19?g/100?g seed, respectively.     

Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry.

A qualitative determination of 20 underivatized proteinic amino acids by LC-MS is reported. The need for chromatographic separation before mass spectrometry determination is demonstrated based on the study of several amino acid pairs which have some similar characteristics. Two suitable LC-MS systems are proposed for amino acid analysis. A preliminary optimization of these systems has been investigated using evaporative light scattering detection as these two detection modes have the same chromatographic requirements. The amino acid separation was achieved on a Purospher RP-18e or a Supelcosil ABZ+Plus column with tridecafluoroheptanoic acid or pentadecafluorooctanoic acid as volatile ion-pairing reagent in an acetonitrile-water mobile phase. In order to elute the most retained amino acids, an elution gradient based on simultaneously increasing the concentration of acetonitrile and decreasing the concentration of the ion-pairing reagent was used. The detection limits of the present work (without specialized optimization) varied from 0.5 to 1 mg 1(-1).     

液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸

建立了液相色谱-电喷雾离子阱串联质谱(LC-ESI-IT-MS/MS)同时分析烟草中20种游离氨基酸的方法。烟草样品经萃取后过滤直接进样,无需进行衍生和固相萃取等其他前处理步骤。液相色谱采用HyPURITY C18反相色谱柱(200 mm×2.1 mm, 5 μm),采用1%(体积分数,下同)乙腈水溶液(含0.1%九氟戊酸)和90%乙腈水溶液(含0.1%九氟戊酸)为流动相进行梯度洗脱。结果表明,20种氨基酸的检出限(LOD)为0.01～0.05 μmol/L (S/N=3),线性相关系数均大于0.9977,峰面积测定的相对标准偏差(RSD)为0.78%～4.93%。该方法分析效率、灵敏度和选择性高,已成功应用于多种烟草样品中氨基酸的分析测定。     

高效液相色谱法测定非天然氨基酸的光学纯度

 以（1- 氟-2,4-二硝基苯基）-5-L-缬氨 酰胺为手性试剂、反相高效液相色谱法测定非天然氨基酸的光学 纯度。梯度洗脱,流动相A：含体积分数为0.1％的三氟乙酸的乙腈,流动相B：体积分 数为0.1％的三氟乙酸水溶液。L-和D-对映体得到良好分离。准确测定了25种非天然 氨基酸L-和D-对映体的光学纯度。     

Enantiomeric separations of amino acids with inductively coupled plasma carbon emission detection

A chiral crown ether column with a pH 1.9 perchloric acid buffered aqueous mobile phase is used to separate amino acid enantiomers by high performance liquid chromatography. An inductively coupled plasma atomic emission spectrometer coupled to the chromatographic system is used as a detector by monitoring the carbon atomic emission line at 193.09 nm. Seven underivatized amino acids are separated and detected resulting in an average mass detection limit of 5 ng (2.5 ng carbon). The chiral crown ether column resolves compounds with a primary amino group near the chiral center by forming a complex between the crown ether and an ammonium ion moiety from the sample. The -form amino acid always elutes faster than its antipode. The carbon emission detector provides nearly identical sensitivities and similar detection limits for any compounds with comparable mass percents of carbon. Quantification is performed on unknown ratios of amino acids using an internal standard without the need for a calibration curve. Summing the calculated amounts of and amino acid and comparing to the known mixture quantity results in an average error of 1.0% for the seven amino acids separated.     

Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.     

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.     

柱前衍生化-超高效液相色谱法快速测定酱油中的18种氨基酸

建立了一种6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,超高效液相色谱(UPLC)对酱油中18种氨基酸进行快速分离检测的方法。采用BEH C18色谱柱分离,在260 nm波长下检测,以乙酸铵-乙酸-乙腈-水和乙腈-乙酸为流动相,将流动相梯度和流速梯度相结合,在12 min内实现了18种氨基酸衍生物的分离。方法的线性回归系数(r2)均大于0.999,检出限为0.032～0.12 mg/L,日间相对标准偏差(RSD)为0.72%～4.05%,在酱油中18种氨基酸的加标回收率为90.2%～103.7%。该方法前处理过程简单,分离时间短,是检测酱油中氨基酸的有效手段,可用于酱油的质量评定。