高效液相色谱荧光检测法测定尿雌三醇

报告用高效液相色谱测定孕妇尿内总雌三醇的方法，尿标本在酸水解后用乙醚提取Ｅ３，蒸于乙醚，残留物用流动相重组，标本成分用Ｃ８柱分离，以２８５ｎｍ波长激发，在６１０ｎｍ测定Ｅ３的自然荧光。本法批内ＣＶ为１．２％－１．５％批间ＣＶ分别为２．３％和４．０％本法在４．３３－１３８．７１μｍｏｌ／Ｌ（１．２５－４０．００ｍｇ／Ｌ）间呈线性，最低检出１．８μｍｏｌ／Ｌ（０．５２ｍｇ／Ｌ，信号／噪声比为３）。     

荧光光度法测定孕妇尿中雌三醇

荧光光度法测定孕妇尿中雌三醇王丽娟，李琳（成都市妇产科医院检验科，成都６１００１５）关键词雌三醇，尿液，荧光光度法孕妇尿中雌三醇（Ｅ３）含量的测定，是临床上诊断妊娠中毒症和胎盘功能不良等疾病的重要指标。目前实验室测定采用放射免疫法，分光光度法，高效液...     

反相高效液相色谱法测定雌三醇栓中雌三醇含量

本文报道用反相高效液相色谱法测定雌三醇栓中雌三醇的含量。采用ＹＷＧ－Ｃ１８不锈钢分析柱（１０μｍ，２５０×４．６ｍｍ），以甲醇∶水（６０∶４０）为流动相，样品用甲醇溶解后取０．５ｍｌ置５０℃水浴中通空气流挥干，残渣溶于１００μｌ流动相，进样２０μｌ。在２８１ｎｍ处检测药物峰高，按外标法定量。本法具有操作简便、灵敏准确等特点，适合于任何含量的雌三醇制剂中雌三醇含量测定。     

高效液相色谱串联质谱法测定血清非结合雌三醇的前处理研究

目的优化高效液相色谱串联质谱法(LC-MS/MS)测定血清游离雌三醇(uE3)的前处理方法。方法以活性炭吸附血清为研究基质,通过加入已知量的雌三醇(E3)标准品及E3-2-3-4-13 C3内标,在LC-20AD XR高效液相色谱系统和API 5500串联四级杆质谱仪上对E3检测的色谱条件、质谱条件及萃取条件等进行优化。结果色谱条件:选用菲罗门Knietex色谱柱(100.0mm×2.1mm,2.6μm);有机相为甲醇,水相为0.1mmol/L氟化铵水溶液,8min梯度洗脱。质谱条件:选用电喷雾(ESI)负离子模式和多反应监测(MRM)模式分析,选择质荷比(m/z)287→m/z 145作为E3的定量离子对,m/z 287→m/z 171作为定性监测离子对;选择m/z 290→m/z 148作为内标的定量离子对,m/z 290→m/z 174作为定性监测的离子对。萃取条件:萃取剂为正己烷/乙酸乙酯(50/50,v/v),样品与萃取剂的比例为1∶2,萃取率可达85.71%。结论 E3检测的色谱条件、质谱条件、萃取条件已得到全面优化,为后续建立LC-MS/MS测定人血中uE3的参考方法打下良好基础。     

高效液相色谱荧光检测法测定血浆同型半胱氨酸

[目的]建立使用高效液相色谱、等度洗脱、荧光检测法测定血浆同型半胱氨酸的方法。[方法]血浆同型半胱氨酸经Tris(2-Carboxyethyl)phosphate(17CEP)还原后，与7-Fluoro-benzo-2-oxa-1．3-diazole-4-sulohonate(SBD-F)衍生化，以胱氨为内标，C18反相色谱柱分离、等度洗脱，荧光检测器测定，以峰面积比进行定量。[结果]线性范围可达100μmol／L，最低检测限0．3μmol／L，批内不精密度1．2％-2．4％，批间不精密度1．4％-2．5％，平均回收率96．3-103％。[结论]本方法洗脱条件简单，准确性、特异性好，适用于临床实验室测定血浆同型半胱氨酸的含量。     

尿雌三醇(E_3)测定在高危妊娠中的应用

报告用改良Ittrich法测定尿中雌三醇(E_3)的正常值,并以此观察妊娠毒血症、过期妊娠、双胎妊娠,以及妊娠合并心脏病、贫血患者的尿中E_3值的变化,认为它是监护高危妊娠的有效方法。     

高效液相色谱-荧光检测法测定人血浆中洛美沙星的浓度

盐酸洛美沙星为喹诺酮类衍生物,系广谱抗菌药物,国内外已上市多年。高效液相色谱法测定人血浆中洛美沙星浓度的方法多有报道,但均使用紫外检测方法,样品处理过程复杂[1,2]。本文通过探索,发现盐酸洛美沙星在200~400nm之间有荧光吸收,灵敏度可比紫外检测法提高数十倍,检测限能够     

高效液相色谱快速测定尿假尿苷

介绍一种不用苯硼酸亲和柱的高效液相色谱法快速测定尿假尿甙。本法只需简单的标本预处理，使用ＰＨ３．３磷酸盐缓冲液动相和２６８ｎｍ检测，每个标本测定只需１２分钟，批内和批间变异分别为３．２７％和４．８６％，线性范围超过１７００μｍｏｌ／Ｌ，常见的１０种抗肿瘤药物不干扰测定。     

高效液相色谱-荧光检测法测定盐酸格拉司琼在小鼠体内的血药浓度

目的 建立高效液相色谱-荧光检测法测定小鼠全血中格拉司琼浓度的方法,考察方法的专属性、线性关系、准确度、精密度、稳定性、提取回收率等效能指标.方法 采用腈基柱;流动相：磷酸三乙胺缓冲盐溶液-乙腈（85∶15 v/v）;检测波长：λex=302 nm,λem=360 nm;柱温26℃;流速1.0 ml/min;进样量20μl.生物样品预处理采用氢氧化钠碱化,二氯甲烷：乙醚（2：3）萃取.结果 小鼠全血中格拉司琼在0.5～ 50.0 ng/ml浓度范围内线性关系良好,回归方程为Y=5.35＆#215;102X＋ 6.40＆#215;102,r=0.9968.本方法在低、中、高3个浓度质控样品的日内精密度分别为7.32％,4.74％,6.86％,日间精密度分别为11.2％,13.03％,7.68％;提取回收率均在76％以上.结论 高效液相色谱-荧光检测法测定血中格拉司琼浓度简便、快速、准确、专属性强.     

Determination of porphyrins by high performance liquid chromatography: fluorescence detection compared to absorbance detection

A comparison is made between determination of porphyrin methyl esters by high performance liquid chromatography (HPLC) with absorbance detection and with fluorimetric detection. Detection limits with absorbance detection vary from 0.8 pmol for 2-COOH-porphyrins to 5 pmol for 8-COOH-porphyrins. With fluorometric detection the corresponding figures are 0.04 pmol and 0.4 pmol. Fluorimetric detection also has a better reproducibility, and it is more specific than absorbance detection. The use of HPLC with fluorimetric detection thus permits rapid, highly efficient and specific quantitative detection and identification of porphyrins in complex biological samples.     

高效液相色谱-荧光法测定血清犬尿喹啉酸和色氨酸方法的建立

目的建立同时检测血清犬尿喹啉酸（kynurenic acid，KYNA）和色氨酸（tryptophan，Trp）的高效液相色谱-荧光法。方法采用高效液相色谱在线衍生技术，通过对最佳检测波长、流速、流动相中醋酸锌浓度和乙腈比例等因素的探讨，得出测定Trp和KYNA的最优实验方案，并对其进行方法学评价；测定50名正常人血清KYNA和Trp含量。结果KYNA保留时间约为8．1min，线性范围为1．05～2093nmoL／L，最低检出浓度为0．05nmoL／L，平均回收率为101．19％；Trp保留时间约为11．3min，线性范围为0．49～196μmoL／L，最低检出浓度为0．001μmoL／L，平均回收率为104．43％，二者日内、日间测定的变异系数均小于5％，苯丙氨酸、酪氨酸、5-羟色胺和犬尿氨酸等物质对该法均无干扰。健康成人血清KYNA和Trp含量分别为（24．25±9．11）nmol／L和（49．05±11．67）μmol／L。结论建立的方法简便、快速、稳定、可行，适用于临床和科研工作。     

高效液相色谱-荧光检测法在慢性肾功能不全患者血清芳香族氨基酸检测中的临床价值

目的 建立同时测定血清AAA含量的HPLC-FLD法,探讨CRI患者血清AAA含量变化及其临床应用价值。方法血清标本来自于100名健康体检者和80例CRI患者。将CRI患者按2002年美国肾脏基金会(NKF)诊断分期标准进行分期：CKD 2期4例、CKD 3期12例、CKD 4期12例和CKD 5期52例;按CRI不同病因分组：慢性肾炎型32例、糖尿病型36例和高血压型12例。血清经高氯酸去蛋白后,离心取上清液测定,外标法定量。采用Megres C18色谱柱,流动相为乙腈：水（体积比为1∶9）,流速为1.0 ml/min,荧光检测器在不同时间段设定特定波长对血清AAA进行测定。对健康对照组和CRI患者组血清中AAA总量、Tyr、Phe和Trp含量及不同分期和不同病因CRI患者血清Tyr、Phe和Trp含量进行比较,同时评价血清AAA总量诊断CRI的敏感度与特异度。结果Tyr、Phe和Trp线性范围分别为0.550～275.000、3.050 ～1 220.000和0.049～49.000 μmol/L,最低检测限分别为0.014、0.500和0.005 μmol/L,平均回收率分别为100.9％、101.3％和98.5％,日内精密度为2.32％～3.92％（平均为3.13％）,日间精密度为3.18％ ～4.20％（平均为3.58％）。CRI患者组血清AAA总量、Tyr、Trp含量及Tyr/Phe比值分别为(135.74±12.23)、(52.27±8.25)、(21.49±4.25) μmoL/L和[0.87（0.68 ～1.05）],低于健康对照组的(174.47±11.57)、（63.53±4.68）、(44.22±3.67) μmol/L和[0.97（0.94～1.00）],差异均有统计学意义（t=- 14.709、4.452、22.100,U=266.000,P均＜0.05）。不同分期CRI患者Tyr、Phe和Trp含量差异无统计学意义;Tyr含量在慢性肾炎组、高血压组和糖尿病组间差异无统计学意义,Phe在慢性肾炎组与高血压组、慢性肾炎组与糖尿病组间差异有统计学意义（U= 114.00、395.00,P均＜0.05）,Trp在慢性肾炎组与糖尿病组间差异有统计学意义(U=349.00,P＜0.05)。血清AAA总量诊断CRI的敏感度、特异度分别为90％ (72/80)和100％ (100/100)。结论HPLC-FLD法测定血清AAA简便、快速,敏感度高及特异度好,同时测定血清AAA含量对CRI患者的诊断和评价有一定价值。     

Determination of urinary norepinephrine and epinephrine by liquid chromatography with fluorescence detection and pre-column derivatization

We assayed norepinephrine and epinephrine by utilizing solvent extraction, fluorescence derivatization, and "high-performance" liquid chromatography. A 100-microL aliquot of urine was extracted twice according to Smedes et al. (J Chromatogr 1982;231:25-39) and subsequently incubated with 1,2-diphenylethylenediamine. A 100-microL aliquot of the resulting mixture was injected into a reversed-phase column, and norepinephrine and epinephrine were detected fluorometrically. The within-day CV was 3.0-4.0% and the between-day CV 3.4-7.1% for normal and high concentrations of both analytes. At low concentrations (15-40 nmol/L) these CVs were 4.2-6.8% and 6.8-9.2%, respectively. The detection limit of the assay was less than 0.4 nmol/L for each analyte. We discuss the critical steps for extraction, derivatization, and chromatography. The present method combines the qualities of high precision, specificity, and sensitivity.     

Determination of urinary cortisol and 6 beta-hydroxycortisol by high performance liquid chromatography

A method using high performance liquid chromatography for the quantitative determination of cortisol and 6 beta-hydroxycortisol in urine is described. Urine extracts were fractionated by high performance liquid chromatography. The peak of 6 beta-hydroxycortisol was identified on the basis of retention time (24.4 min) and its area was measured. The peak of cortisol could not be measured simultaneously with the same column because of interference by other absorbing materials. The cortisol and dexamethasone peaks were collected (Rt values 9.6 and 8.1 min, respectively), rechromatographed on another column system, and the concentration was determined. Precision and accuracy of the present method were within the range commonly achieved by other methods established for both steroids. Clinical utility of the present method was evaluated by measuring urinary cortisol and 6 beta-hydroxycortisol in normal subjects, in Cushing's syndrome and disease patients, and in patients receiving cortisol therapy.     

Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection

Objectives

An HPLC method was developed to quantify serum concentrations of phenylalanine and tyrosine simultaneously using fluorescence detection without derivatization.

Methods

Serum protein is precipitated with trichloroacetic acid, 0.015 mM dihydrogen-phosphate solution is used for separation on reversed-phase C₁₈ material, and acetonitrile is avoided. Both amino acids are monitored utilizing their natural fluorescence at 210 nm excitation and 302 nm emission wavelengths.

Results

One analytical run is completed within 7 min. Lower detection limit for Phe and Tyr is 0.3 μM. Comparison of the new method with a classical HPLC method for total amino acids and using UV-absorption detection reveals a highly significant relationship for Phe and Tyr.

Conclusion

The new HPLC method allows rapid and very sensitive measurement of phenylalanine and tyrosine concentrations.     

高效液相色谱法测定人血清甜菜碱方法的建立

目的 采用柱前衍生高效液相色谱法-紫外检测法建立一种测定人血清甜菜碱浓度的方法.方法 以对溴苯乙酰溴和18-冠醚-6溶于乙腈制成衍生液,将其与甜菜碱反应形成的衍生物用Supelcosil LC-SCX色谱柱进行分离.流动相为乙腈∶水体积比90∶10,含16 mmol/L的氯化胆碱,等度洗脱,流速为0.8 ml/min,检测波长为259 nm,利用甜菜碱标准品绘制标准曲线,外标法定量检测20名健康大学生志愿者血清甜菜碱.结果 甜菜碱的测定线性范围为6.25～200.00 μmol/L,回归方程Y=1 568.1X-2 747.5,R2=0.999 8.最低检测限为3.0 μmol/L.批内不精密度为1.88%～3.79%(平均3.24%),批间不精密度为3.14%～6.76%(平均4.39%).方法 回收率为95.89%～102.86%(平均99.16%).结论 成功建立一种检测人血清甜菜碱浓度的方法,适用于实验室及临床常规检测.     

Diagnosis of GM1 gangliosidosis based on detection of urinary oligosaccharides with high performance liquid chromatography

An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.