上柱前样品萃取与浓缩技术在毛细管电泳法测定微量氨基酸中的应用

目的建立一种上柱前样品萃取与浓缩技术测定生物样品中微量氨基酸的方法.方法氨基酸与2,4-二硝基氟苯衍生化反应完成后,用10 ml叔丁基甲醚抽提2次,每次5 ml,以除去多余衍生化试剂.然后用6 mol/L盐酸2滴酸化,再用6 ml乙酸乙酯萃取两次,每次3 ml.最后合并上层萃取液,50°C水浴吹干,剩下含氨基酸的残留物用水和乙腈重组,用毛细管电泳法分析定量.结果本法具有操作简单、萃取效率高的特点,平均萃取产率可达99.3%.若用水和乙腈重组残留物至原来体积的十分之一,则柱前浓缩效果可达10倍.结论柱前萃取和浓缩技术操作简单,是一种可用于提高毛细管电泳法测定微量氨基酸灵敏度的方法.     

上柱前术品萃取与浓缩技术在毛细管电泳法测定微量氨基酸中的应用

目的：建立一种上柱前样品萃取与浓缩技术测定生物样品中微量氨基酸的方法，方法：氨基酸与2，4－硝基氟苯衍生化反应完成后，用10ml叔丁基甲醚抽提2次，每次5ml，以除去多余衍生化试剂，然后用6mol/L盐酸2滴酸化，再用6ml乙酸乙酯萃取两次，每次3ml。最后合并上层萃取液，50℃水浴吹干，剩下含氨基酸的残留物用水和乙腈重组，用毛细管电泳法分析定量，结果，本法具有操作简单，萃取效率高的特点，平均萃取产率可达99．3％，若用水和乙腈重组残留物至原来体积的十分之一，则柱前浓缩效果可达10倍，结论：柱前萃取和浓缩技术操作简单，是一种可用于提高毛细管电泳法测定微量氨基酸灵敏度的方法。     

《上海医学检验杂志》2002年第17卷主题索引

ＡＡｍｐＣβ内酰胺酶 　 4株～启动子和衰减子突变大肠埃希菌的分析研究 (4 ) :2 0 4癌胚抗原　乳头溢液中～检测在乳腺癌诊断中的价值 (1) :47氨基酸　上柱前样品萃取与浓缩技术在毛细管电泳法测定微量～中的应用 (4 ) :2 16Ｂβ Ｎ 乙酰氨基己糖苷酶 　血清～自动分析法改进 (6) :3 60β 羟丁酸 　血清～测定在小儿Ⅰ型糖尿病并发酮症及酮症酸中毒中的应用 (4 ) :2 19白蛋白　两种染料结合法测定血清～的差异 (4 ) :2 46白细胞介素　腹膜透析患者透出液～ -6和ＩＬ 8的变化 (2 ) :10 6比浊法　尿液和脑脊液蛋白测定自动～的评价 (1) :2 1…     

柱上样本堆积毛细管电泳法同时检测细胞内氨甲蝶呤及其六种代谢产物

目的 建立柱上堆积高效毛细管电泳法同时检测细胞内氨甲蝶呤(MTX)及其6种代谢产物氨甲蝶呤多聚谷氨酸链( MTXPG)的方法.方法 采用高效毛细管电泳技术,以75 mmol/L磷酸盐缓冲液(pH值7.40)作为分离介质,用水和乙腈( V/V=1∶1)作溶剂重组残留样本的方法进行柱上样本堆积,0.5 psi压力进样,进样...     

毛细管电泳测定肝癌肝硬化患者血清氨基酸的临床意义

目的　用毛细管电泳的方法测定肝癌肝硬化患者血清氨基酸变化,并探讨其临床意义。方法　用P/ACEMDQ毛细管电泳仪对2 0例正常人、2 0例肝癌患者及2 0例肝硬化患者的血浆游离氨基酸进行测定。结果　在9min内分离12种氨基酸,线性在2 0～10 0 0 μmol/L范围,回收率为77. 5 %～113 .3% ,批内精密度2 . 6 %～11. 4 % ,批间精密度5 . 8%～19. 7%。对结果进行统计学分析,发现肝硬化组酪氨酸、丝氨酸、色氨酸明显升高,丙氨酸明显下降;肝癌组酪氨酸、半胱氨酸、缬氨酸明显升高,脯氨酸明显下降。结论　毛细管电泳测定血浆游离氨基酸对肝癌肝硬化的诊断和治疗有指导意义。     

DLLME-TLC快速检测血、尿中有机磷农药

目的运用分散液-液微萃取技术，建立快速测定血、尿中敌敌畏、乐果、马拉硫磷、乙基对硫磷等有机磷农药的新方法。方法常温下，于5ml尿、5g血中加入1ml丙酮，80μl 三氯甲烷，萃取2min后，静置1min，吸取萃取剂三氯甲烷用薄层色谱法进行定性。结果在选定条件下，与公安部行业标准GA/101—1995中薄层色谱法比较，样品前处理由原来的至少2h缩短为8min，有机磷农药检出限由200μg降低为尿2μg/ml、血6μg/g。结论该方法操作简单、省时、对环境友好，成本低，可快速测定血、尿中有机磷农药。     

DLLME-TLC快速检测血、尿中有机磷农药

目的 运用分散液-液微萃取技术,建立快速测定血、尿中敌敌畏、乐果、马拉硫磷、乙基对硫磷等有机磷农药的新方法 .方法 常温下,于5ml尿、5g血中加入1ml丙酮,80μl三氯甲烷,萃取2min后,静置1min,吸取萃取剂三氯甲烷用薄层色谱法进行定性.结果 在选定条件下,与公安部行业标准GA/T101-1995中薄层色谱法比较,样品前处理由原来的至少2h缩短为8min,有机磷农药检出限由200gμg降低为尿2μg/ml、血6μg/g.结论 该方法 操作简单、省时、对环境友好,成本低,可快速测定血、尿中有机磷农药.     

胶束动电毛细管电泳法直接测定血清肌酐

目的：建立高效毛细管电泳测定血清肌酐的新方法．方法：选择pH7．2,120mmol／LSDS,30mmol／L磷酸盐缓冲液作胶束电动毛细管电泳运动液,利用外标法定量,检测发长235um,非涂演毛细管21cm×50μm（i．d）,电压10kV,自动进样2s,电泳6min．结果：肌酐的线性范围27．65～7080μmol／L,最低检测限为9．19μmo／L．方法的日内和日间变异系数分别小于3．0％和3．6％,肌酐的回收率为99．8％～105．5％．内生性化合物和10种常用的药物对此方法无干扰．结论：建立了测定血清风牙的毛细管电泳方法．该方法简单、快速,可应用于临床样品检测．     

固相萃取反相高效液相色谱法测定全血环孢素A

目的　建立固相萃取反相高效液相色谱法测定全血环孢素A的方法。方法　全血中的环孢素A采用Wa tersSep PakC18萃取小柱提取浓缩 ,色谱柱WatersNova PakC18;流动相　乙腈∶甲醇∶水 (32 0∶30∶15 0 ,V/V) ;流速 0 6 5ml/min ;检测波长 2 0 6nm ,环孢素D作内标。结果　方法的回收率为 95 8% ,相对标准差 (RSD)为 7 5 % ,线性范围为5 0～ 2 0 0 0 μg/L ,r=0 9999。 结论　该方法特异、敏感、准确 ,可用于全血环孢素A的测定。     

气相色谱法测定消毒剂戊二醛含量的准确性观察

目的建立毛细管气相色谱法对消毒剂戊二醛含量的分析方法。方法采用SGE H2极性大口径毛细管气相色谱仪,确定其对消毒剂戊二醛含量测定参数。结果戊二醛含量在10~1000μg/ml范围内,用该气相色谱法检测线性良好,相关系数大于0.999,回收率92%~104%,相对标准偏差小于3%,最低检出限为0.05 mg/L。结论该方法操作简便,灵敏度高,重现性好,样品不经前处理稀释后可直接进样检测,结果准确可靠。     

高效毛细管电泳分离氨基酸部分影响因素的探讨

目的探讨高效毛细管电泳分离氨基酸的有关影响因素。方法用毛细管电泳法对20种常见氨基酸的2,4—二硝基氟苯(DNFB)衍生物进行了分离研究,分析缓冲液浓度、pH值、有机溶剂及表面活性剂对分离的影响。结果缓冲液浓度、pH值及有机溶剂对氨基酸的DNFB衍生物分离有很大的影响,而表面活性剂基本对其无影响。在优化条件下,17种氨基酸除亮氨酸、异亮氨酸外均达到基线分离。结论以浓度为30 mmol/L、pH值9.9的硼砂溶液含15%异丙醇为运行缓冲液,分离氨基酸DNFB衍生物效果最佳,分离时间30 m in,出峰17个。     

毛细管电泳-激光诱导荧光法分离四种神经递质类氨基酸的条件优化

目的探讨毛细管电泳分离神经递质类氨基酸的影响因素。方法采用毛细管电泳法分离4种神经递质类氨基酸的荧光素异硫氰酸酯(FITC-AAs)衍生物,分析电泳缓冲液、表面活性剂以及衍生条件对分离的影响。结果电泳缓冲液的组成、浓度、pH值、表面活性剂以及衍生条件对4种FITC-AA衍生物分离有很大影响,在以75 mm ol/L,pH 9.42硼酸盐含100 mm ol/LSDS为电泳缓冲液,50 mmol/LpH 9.8硼酸盐为衍生缓冲液,4种FITC-AA衍生物20 min内得到很好分离。结论通过条件优化,毛细管电泳-激光诱导荧光技术分离神经递质类氨基酸的分离效果能得到很大提高。     

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.     

Determination of amino acids by ion-exchange chromatography on filter paper spotted blood samples stored at different temperatures and for different periods: comparison with capillary and venous blood

OBJECTIVE: To determine whether the well-established filter paper spotted blood method used for the determination of some amino acids could be reliably used to measure all amino acids and whether amino acid results thus obtained are reproducible and comparable to the results obtained by measuring plasma amino acids in either capillary or venous blood. METHODS: This is a prospective study in which blood samples from a finger-prick were collected in capillary tubes and at the same time blotted on filter papers; another sample was taken from a vein, from 19 healthy volunteers aged between 18 and 24 yr after a strict 12-h overnight fast. Another 9 healthy adult volunteers provided blood samples on filter papers for the storage study; 9 samples were analyzed immediately; 9, 8 and 4 samples were stored at -20 degrees C, -4 degrees C and room temperature respectively and analyzed after 14 days; 8 samples stored at -20 degrees C were analyzed after 4 weeks. RESULTS: Intra-sample reproducibility in the filter paper blood from the same individual was found to be mostly less than 20%, while for the capillary blood was less than 5%. The greatest variability was in cystine and methionine. There was no significant difference between results obtained from capillary blood and from venous blood, but there was a significant difference between amino acid concentrations in venous and capillary blood on the one hand and filter paper blood on the other. Storage at different temperatures and for a varied period of time showed little change except in serine, glutamate, ornithine, histidine, cystine and methionine. There was a 30% decrease in concentrations of most amino acids in filter paper blood when compared to capillary or venous blood probably because of loss in the extraction process. CONCLUSION: A new set of values for amino acids in filter paper blood in normal individuals is presented. Blood spotted filter paper could be used to screen practically all inborn errors of amino acid metabolism.     

Rapid analysis of parathion in biological samples using headspace solid-phase micro-extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS).

A simple and rapid method for the analysis of parathion in biological samples is presented. The method consists of the extraction of parathion from blood samples by headspace solid-phase micro-extraction (SPME), followed by capillary gas chromatography and mass spectrometry detection. The recoveries in the blood samples after addition of ammonium sulphate and sulphuric acid were between 85% and 89% compared to samples prepared in water. Linearity was established over a concentration range of 0.1-5 microg/g blood with acceptable coefficients of correlation and limits of detection reached 0.02-0.05 microg/g. The time for an analysis is 57 minutes for one sample, including the extraction step. In conclusion, HS-SPME in combination with GC/MS is an effective method for the determination and quantification of parathion-ethyl and parathion-methyl in biological material.     

High-Pressure Liquid Chromatographic Assay of Netilmicin in Plasma

A high-pressure liquid chromatographic method for the quantitative determination of netilmicin in plasma was developed. The procedures involve acetonitrile protein precipitation, methylene chloride extraction, and dansylation to form the fluorescent dansyl derivative of netilmicin, which is extracted into ethyl acetate and chromatographed on a reverse-phase column with aqueous acetonitrile as the mobile phase. A good linear relationship between peak height measurements and netilmicin concentrations was found. This method is sensitive and reproducible; a netilmicin concentration as low as 0.5 μg/ml can be measured with only 0.1 ml of plasma sample. The results of assays of plasma or serum samples by this high-pressure liquid chromatographic method correlate well with those obtained by microbiological assays.     

Development of a high-performance liquid chromatographic assay for G418 sulfate (Geneticin).

We have developed a chromatographic assay with high sensitivity and specificity to quantify G418 sulfate (Geneticin), an antibiotic used routinely in molecular genetics experiments for selecting eukaryotic transformants. With this method, G418 in tissues and plasma samples can be quantitated without the confounding factors often associated with biological assays. After removal of proteins in homogenized tissue or plasma samples with methanol (2:1, vol/vol), the amino group of G418 was derivatized with 1-fluoro-2,4-dinitrobenzene (DNFB) to form the UV-visible G418-DNFB product. The DNFB-derivatized G418 was separated on a reversed-phase C18 column with an acetonitrile and water gradient as the mobile phase. Under these assay conditions, the detection limit for G418 sulfate in buffer, plasma, and tissues was recorded at 78 ng/ml and the linearity was recorded for concentrations up to 100 micrograms/ml. The data obtained from this analysis indicate that this assay can be used for the quantitative determination of G418 sulfate in plasma and tissue samples.     

UiO-66-based metal–organic framework for dispersive solid-phase extraction of vanillylmandelic acid from urine before analysis by capillary electrophoresis

Dispersive solid-phase extraction (DSPE) was developed for the extraction of vanillylmandelic acid (VMA) in urine samples prior to capillary electrophoresis with diode array detection (CE-DAD). Extraction of VMA by DSPE was carried out by direct addition of 7.5 mg of synthesized amino-functionalized UiO-66 (Zr) metal–organic framework adsorbent into the 5 mL sample solution (pH 4.0), followed by sonication and centrifugation. The supernatant layer was discarded, then the sedimented adsorbent was eluted using borate buffer (75 mM, pH 10). Effective extraction parameters including the amount of adsorbent, sample pH, adsorption and desorption time, type, volume and pH of eluent, and type of adsorbent dispersion method were systematically investigated. Under the optimized conditions, linearity of the method was from 40 to 2000 μg L⁻¹ with a correlation coefficient over 0.9948. The method detection and quantification limits were 12 and 40 μg L⁻¹, respectively. The relative standard deviations for intra-and inter-day precision were 2.4 and 2.8% (n = 5), respectively. The extraction recovery and enrichment factor values were 90% and 9.0 respectively.

Dispersive solid-phase extraction (DSPE) was developed for the extraction of vanillylmandelic acid (VMA) in urine samples prior to capillary electrophoresis with diode array detection (CE-DAD).     

Iohexol in serum determined by capillary electrophoresis.

Iohexol, a nonionic compound used as a contrast medium for angiography and as a measure of the glomerular filtration rate, was quantified in serum by capillary electrophoresis. Comparable results were obtained for serum samples deproteinized with acetonitrile or analyzed directly after 50-fold dilution with borate buffer. Serum samples were electrophoresed for 2.6 min at 12 kV in a borate buffer with detection at 254 nm and with 3-isobutyl-1-methylxanthine as internal standard. Acetonitrile deproteinization gave a greater sensitivity than did sample dilution. Between-run CVs were between 4.7% and 6.7%, and within-run CVs were between 2.5 and 3.2%. Analytical recoveries were 95-105%. Results of the method compared well with those by high-performance liquid chromatography (slope 0.96, intercept 0.005 g/L). This method demonstrates the potential of capillary electrophoresis for rapid and simple quantification of small molecules.