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。     

柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛（o-phthalaldehyde，OPA）为衍生试剂，建立了柱前衍生-高效液相色谱（HPLC）同时测定血清中氨基酸类神经递质牛磺酸（Tau）、谷氨酸（Glu）、甘氨酸（Gly）、γ-氨基丁酸（γ-GABA）和单胺类神经递质多巴胺（DA）含量的分析方法。血清与乙醇以1：2的体积比混合，进行蛋白质沉淀后离心，取其上清液，氮吹至近干。前处理后的样品与OPA进行柱前衍生，衍生化产物采用Luna 5u C18色谱柱（250 mm×4.6 mm，5 μm）分离，以柠檬酸-乙酸钠缓冲溶液（pH 3.73）为流动相A、乙腈为流动相B进行梯度洗脱，流速为1.0 mL/min，柱温为30℃，检测波长为338 nm。5种神经递质在各自范围内线性关系良好（r²≥0.9866），检出限为0.10~0.40 μmol/L，不同加标水平下目标物的加标回收率为87.57%~115.31%，相对标准偏差均低于7.80%。方法操作简单，灵敏度高，精密度、线性关系和回收率等方法学指标较好，可实现血清中氨基酸类及单胺类神经递质的同时检测。     

基于液相色谱-串联质谱的氨基酸代谢组学方法研究黄芪注射液治疗脑缺血

建立了基于液相色谱-串联质谱技术的氨基酸代谢组学分析方法。选用Diamonsil C18色谱柱,乙腈-0.1%甲酸为流动相,梯度洗脱,三重四级杆质谱检测器,MRM扫描方式,12 min内对血浆中12种氨基酸进行定量分析。应用本方法对脑缺血模型和黄芪注射液的治疗作用做出评价。通过偏最小二乘判别分析统计发现,脑缺血12 h,其血浆中的氨基酸代谢表达存在显著差异,结合载荷图及VIP值确定丝氨酸、天冬氨酸、甘胺酸等7种脑缺血生物标识物;采用黄芪注射液治疗后,从得分图上可以看到氨基酸代谢的轨迹发生了较大变化;与脑缺血模型组比较,丝氨酸、天冬氨酸、甘胺酸等生物标识物均向正常水平趋近。     

Turbo flow在线净化-超高效液相色谱-串联质谱法检测水果中5种咪唑类农药残留

建立了Turbo flow （TF）在线净化-超高效液相色谱-串联质谱同时检测水果中吡咪唑、咪唑烟酸、咪鲜胺、咪唑嗪和咪唑菌酮等5种咪唑类农药的方法。以饱和氯化钠溶液溶解样品,乙腈提取,蒸干,乙腈-水（1：1,v/v）溶液定容后进入Turbo flow-超高效液相色谱-串联质谱系统分析,外标法定量。对影响TF净化的条件如TF色谱柱、流动相、洗脱溶液、洗脱速率等进行了优化。在优化的实验条件下,以Turbo flow C18 （50 mm×1.0 mm）为净化柱,Hypersil GOLD aQ （100 mm×2.1 mm）为分析柱,乙腈和5 mmol/L甲酸铵溶液（含0.1%（v/v）甲酸）为流动相进行梯度洗脱,在电喷雾正离子选择反应监测模式下进行检测。结果表明,各目标化合物在0.0075~0.75 mg/L范围内线性关系良好（相关系数均大于0.99）,定量限为0.005 mg/kg。对实际样品分别加标0.005、0.01、0.05和0.5 mg/kg,回收率为71.2%~122.4%,相对标准偏差（RSD）为0.5%~8.9%。该方法简便、快速,结果准确可靠,适用于水果中5种咪唑类农药残留的检测。     

超高效液相色谱-高分辨质谱法同时测定白酒中19种氨基酸

建立了白酒样品中19种氨基酸的超高效液相色谱-高分辨质谱（UPLC-HRMS）检测方法。样品经烘箱干燥法除乙醇,水定容,使用水相滤膜过滤后上机检测。以甲醇-0.1%甲酸作为流动相,经Hypersil GOLD色谱柱（150 mm×2.1 mm,1.9μm）分离,在正离子扫描模式下,一级母离子全扫描采集化合物信息,外标法定量分析。结果表明,19种氨基酸在其线性范围内线性关系良好,相关系数（r2）不小于0.986 2,方法的检出限为0.003～0.015μmol/L,定量下限为0.008～0.050μmol/L,白酒样品中19种氨基酸的加标回收率为88.2%～119%,相对标准偏差为0.23%～5.6%,适用于白酒样品中氨基酸分析。利用该方法分析了不同轮次酱香酒中氨基酸含量,检出L-丙氨酸、L-精氨酸、L-天冬氨酸、L-谷氨酸、甘氨酸、L-组氨酸、L-异亮氨酸、L-亮氨酸、L-赖氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-酪氨酸、L-缬氨酸和L-瓜氨酸共16种氨基酸。不同轮次酒中氨基酸含量的显著水平P值均小于0.05,表明不同轮次酒中氨基酸含量具有显著性差异。     

场增强样品堆积-毛细管电泳法测定山楂粉中的氨基酸

采用场增强样品堆积-毛细管电泳法建立了在线富集氨基酸的分析方法,用于中药山楂中水解氨基酸的检测,并进行了回收率实验. 采用富集电压为-20 kV,进样压力为3 psi,进样时间为50 s,紫外检测波长为214 nm,运行缓冲溶液为270 mmol/L乙酸-270 mmol/L乙酸钠（pH=4.15）-6%（V/V）乙腈溶液,分离电压为17 kV,组氨酸、精氨酸、色氨酸、酪氨酸、缬氨酸、苯丙氨酸、亮氨酸、苏氨酸、丙氨酸、甘氨酸、谷氨酸和门冬氨酸等12种氨基酸在50 min内达到分离,检出限在0.000 3~0.08 μg/mL之间.     

高效液相色谱对液态奶中三聚氰胺的快速测定

建立了有机溶剂提取和离子对色谱相结合的三聚氰胺快速检测方法.样品中加入有机溶剂振荡提取,取上清液过滤进行高效液相色谱(HPLC)分析.对丙酮、乙腈、乙醇和异丙醇的提取物分别在甲醇-离子对试剂流动相体系和乙腈-离子对试剂流动相体系中进行测定和比较.结果显示,选择合适的流动相,使用丙酮、乙醇或异丙醇为提取剂可以获得较好的提取效果.在甲醇-离子对试剂流动相体系中,三聚氰胺的质量浓度在1.0 ～100.0 mg/L范围内与色谱峰面积呈良好的线性关系(r=0.999 98),检出限为0.1 mg/kg,采用丙酮为提取剂,加标回收率为97% ～103%,相对标准偏差(RSD)小于5%.     

超高压液相色谱-串联四极杆质谱法对牛奶中24种磺胺类药物残留的检测

利用超高压液相色谱-电喷雾串联四极杆质谱（UPLC-MS/MS）联用技术,建立了一种能在10 min内快速分离和测定牛奶中24种磺胺类药物残留的方法。样品经匀浆、超声、乙腈重复提取、氮吹浓缩,流动相溶解,饱和正己烷脱脂。采用ACQUITY UPLCTMBEHC18柱（100 mm×2.1 mm i.d.,1.7μm）,以乙腈-0.2%乙酸水溶液（体积比1∶9）为流动相,梯度洗脱,目标分析物使用超高压液相色谱-电喷雾串联质谱进行测定;以保留时间和离子对进行定性和定量,在ESI（＋）和MRM监测模式下进行样品分析。该方法检出限（LOD）为0.04～1.35μg/kg;在1～200μg/L范围内线性关系良好,回收率为61%～117%,相对标准偏差为2.92%～18.98%。该法样品前处理简单、分析速度快、回收率和灵敏度高、检出限低,可以满足各国对牛奶中24种磺胺类药物的检测要求。     

高效液相色谱-串联质谱法测定动物源性食品中矮壮素残留

建立了高效液相色谱-串联质谱测定动物源性食品中矮壮素残留的分析方法。样品经含1%（v/v）乙酸的乙腈溶液提取、正己烷脱脂、阳离子固相萃取柱（PCX）净化,采用Venusil MP C18（2）色谱柱（150 mm×2.1 mm,3 μm）分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,采用电喷雾电离、正离子模式扫描,多反应监测模式（MRM）检测,基质匹配标准曲线内标法定量。结果表明：矮壮素在0.200~500 μg/L范围内呈良好线性,相关系数（r²）均不低于0.9993,方法的定量限为0.500 μg/kg;以猪肉、牛肉、羊肉、鸡肉、鸡蛋、猪肾、牛肝、羊肾、鸡肝、牛奶为基质,矮壮素的平均加标回收率为93.4%~101%,相对标准偏差为2.3%~8.0%。该方法基质干扰小,灵敏度高,准确可靠,适用于动物源性食品中矮壮素残留的定量检测。     

溶液捕集-高效液相色谱法测定卷烟主流烟气中主要羰基化合物

卷烟主流烟气中的羰基化合物用含2,4-二硝基苯肼的乙腈-磷酸溶液为吸收液进行捕集,并在此溶液中使羰基化合物衍生化,所得衍生化产物供高效液相色谱分析。采用Merck LichrosphereRP C₁₈色谱柱进行分离,并用不同配比混合的水-乙腈-四氢呋喃-异丙醇（59+30+10+1）、水-乙腈-四氢呋喃-异丙醇（33+65+1+1）和乙腈的混合液作为流动相进行梯度淋洗,用紫外检测器于波长365 nm处测定。方法的检出限（3S/N）在0.04～0.12μg·g^-1之间。方法用于卷烟主流烟气的分析,回收率在75.0%～96.0%之间,测定值的相对标准偏差（n=5）在2.0%～9.3%之间。     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study

The pentapeptide thymopentin (Arg‐Lys‐Asp‐Val‐Tyr, RKDVY) corresponds to amino acids 32–36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope‐labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES‐C₁₈ column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5–800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin.     

天花粉蛋白的化学 III:溴化氰降解片段CBa的氨基酸顺序

The results from the study on the separation, purification, amino acid composition and amino acid sequence of CBa, one of the four CNBr degradation fragments of crystalline trichosanthin, are presented. Its amino acid composition is: Asp3, Thr2, Ser2, Hse1, Glu2, Gly2, Ala6, Val1, Tyr3, Phe3, Lys2, Arg1. The sequence of the CBa is Gly-Tyr-Arg-Ala-Gly-Asp-Thr-Ser- Tyr-Phe-Phe-Asn-Glu-Ala-Ser-Ala-Thr-Glu-Ala-Ala-Lys-Tyr-Val- Phe-Lys-Asp-Ala-Hso.     

Value of high-performance liquid chromatographic analysis of amino acids in the determination of Panax ginseng radix extract effect in cultured neurons

The present research describes a reversed-phase high-performance liquid chromatographic (RP-HPLC) method that allows the determination of several amino acids in primary cultured cortical neurons of rats. The concentration of amino acids was determined by using pre-column derivatization with dansyl chloride and UV-diode array detection. Data show that Panax ginseng radix extract (GS) can modulate amino acid release in neurons. The levels of glutamate (Glu), aspartate (Asp), gamma-aminobutyric acid (GABA) and glycine (Gly) in the GS-treated groups were higher than in the non-treated groups dose-dependentwise. In this case, Glu and GABA were the most released amino acids (74.43% +/- 0.97 and 88.41% +/- 4.12 at ginseng dose 0.01 mg/ml after 1h from treatment, respectively). The values obtained in the determination of the analytical parameters (linearity, precision, limit of detection and accuracy) confirm the quality of the method. The average recoveries for intra and inter-day assay (n = 5) were 101.18 and 102.38 for Asp, 99.35 and 98.44 for Glu, 99.59 and 99.66 for Gly, and 100.06 and 100.37 for GABA. These data proved that the method yields accurate results, with RSD lower than 2.2%. The precision of the method was estimated on the basis of RSD of six injections at two different concentrations of amino acids. This technique is useful in studying the GS-mediated modulation of the dynamic equilibrium of amino acids and neurotransmission in neurons.     

Synthesis and screening of libraries of synthetic tripodal receptor molecules with three different amino acid or peptide arms: identification of iron binders

A novel selectively deprotectable triazacyclophane scaffold was used for the design and split-mix synthesis of two libraries of solid-phase bound tripodal synthetic receptors possessing three different amino acid or peptidic arms. In the synthesis of the first library, the two outer arms consisted of amino acid Ala, Arg, Asp, Gln, Gly, Lys, Phe, Ser, Tyr, or Val and the middle arm consisted of amino acid Asn, Glu, His, Leu, or Pro. The second library contained amino acid and/or (di)peptide arms. The arms were different in all library members. The first outer arm consisted of amino acid(s) Ala, Arg, Gln, Phe, or Ser, the second outer arm consisted of amino acid(s) Asp, Gly, Lys, Tyr, or Val, and the middle arm consisted of amino acid(s) Asn, Glu, His, Leu, or Pro, leading to a 27 000 member library of synthetic tripodal receptor molecules. In on-bead screening experiments, a remarkable selectivity of some library members for Fe(3+) was observed and decoding of their structures by Edman degradation revealed consensus sequences with structural resemblance to non-heme iron proteins.     

Determination and Application of Nineteen Monoamines in the Gut Microbiota Targeting Phenylalanine,Tryptophan, and Glutamic Acid Metabolic Pathways

It has been reported that monoamine neurotransmitters can be produced by gut microbiota, and that several related metabolites of amino acids in these pathways are associated with nervous system (NVS) diseases. Herein, we focused on three pathways, namely, phenylalanine (Phe), tryptophan (Trp), and glutamic acid (Glu), and established an underivatized liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of nineteen monoamine neurotransmitters and related metabolites in the gut microbiota. The neurotransmitters and related metabolites included Phe, tyrosine (Tyr), l-dopa (Dopa), dopamine (DA), 3-methoxytyramine, Trp, hydroxytryptophan, 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), kynurenine (KN), kynurenic acid (KYNA), melatonin, tryptamine (TA), indole-3-lactic acid (ILA), indole-3-acetic acid (IAA), indolyl-3-propionic acid (IPA), Glu, gamma-aminobutyric acid (GABA), and acetylcholine (Ach). A fluoro-phenyl bonded column was used for separation, and the mobile phase consisted of methanol:acetonitrile (1:1) and water, with 0.2% formic acid in both phases. The compounds exhibited symmetric peak shapes and sufficient sensitivity under a total analysis time of 8.5 min. The method was fully validated with acceptable linearity, accuracy, precision, matrix effect, extraction recovery, and stability. The results showed that neurotransmitters, such as Dopa, DA, 5-HT, GABA, and Ach, were present in the gut microbiota. The metabolic pathway of Trp was disordered under depression, with lower levels of 5-HT, 5-HIAA, KN, KYNA, TA, ILA, IAA, IPA, and Glu, and a higher ratio of KYNA/KN. In addition, some first-line NVS drugs, such as sertraline, imipramine, and chlorpromazine, showed regulatory potential on these pathways in the gut microbiota.     

Fast quantification of amino acids by microchip electrophoresis–mass spectrometry

A fast microchip electrophoresis–nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 μM. Limits of detection were found to be 0.37 μM for Asp and 0.33 μM for Glu (S/N?=?3). Assay repeatability (relative standard deviation, n?=?6) was 4.2 and 4.5 %, for Asp and Glu at 5.0 μM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.     

The mechanism of bacteriorhodopsin functioning. I. The light-induced proton throw-over by retinal

The path of proton motion in protein bacteriorhodopsin is constructed: Tyr 43 → Tyr 26 → Asp 212 → Tyr 83 → Tyr 57 → Tyr 79 → Glu 204 → Tyr 64 → Glu 74. From the position of quantum theory the light-induced isomerization of retinal, the process carrying out the proton and throw-over from the inlet H-bond chain (Tyr 43 → Tyr 26 → Lys 216) to the outlet one (Lys 216 → Asp 212 → … → Glu 74) are investigated. The kinetics of the transition of the excitation from the π-electron subsystem to the vibrational one is studied; the function of the nonequilibrial excitation distribution of the collective intramolecular retinal vibrations is found. The mechanism of deprotonation of Schiff base (transmission of proton to the outlet channel) is considered. The correspondence between the theoretically estimated states and spectroscopically identified forms of bacteriorhodosin (initial form bR, intermediates J, K) is proposed.     

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid,gabapentin, pregabalin,and vigabatrin in human plasma

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988–0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%–94.5%, 90.0%–90.9%, 90.0%–92.1%, and 88.0%–92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.     

Development of a New Pre-Derivatization LC Method for Analysis of Branched-Chain and Aromatic Amino Acids in Rat Plasma and to Monitor their Dynamic Variation as a Result of Acute Hepatic Injury

9,10-Anthraquinone-2-sulfonyl chloride, a new derivatization reagent with strong UV absorbance, has been used to develop a reversed-phase liquid chromatographic method for analysis of branched-chain and aromatic amino acids in rat plasma. Because the reagent has a sulfonyl chloride group it is highly reactive with primary and secondary amino groups and reaction is complete within 5 min at room temperature in phosphate–borate buffer (pH 8.41)–acetonitrile. The resulting adducts were separated on a C₁₈ column at 30 °C and detected at 254 nm. Good linearity was achieved over the concentration range 10–1,000 μmol L^?1 for each amino acid. Intra-day precision (relative standard deviation; RSD, %) and accuracy (bias, %) for all quality-control concentrations, were ≤8.9% and 16.0%, respectively. Inter-day precision and accuracy were ≤9.5% and 11.7%, respectively. The method was used for analysis of branched-chain and aromatic amino acids in the plasma of rats with hepatic injury induced by d-galactosamine.