A Closer Examination of 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Amino Acid Derivatization in HPLC with Multiple Detection Modes

Chromatographia - 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate is a well-known reagent used to derivatize amino acids (AAs) into their aminoquinolyl carbamate (AQC) analogs. Originally, AQC...     

Determination of Defibrase in Pharmaceutical Formulation by LC with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate Derivatization and Fluorescence Detection

A sensitive LC method with pre-column fluorescence derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate has been developed for the determination of defibrase from the venom of Agkistrodon acutus (also named defibrase in the Chinese Pharmacopoeia) in pharmaceutical formulation. By a denaturing procedure prior to derivatization, full and homogeneously derivatization of defibrase was obtained and demonstrated using MALDI-TOF-MS. The specificity of the method was validated by assessing interference from the placebo and by forced degradation. The method has demonstrated good linearity (r = 0.9998), accuracy (mean recovery 98.53%) and precision (RSD < 2.5%). Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method was successfully applied to the quantitative determination of defibrase in pharmaceutical formulation.     

The Determination of T-2 Toxins by HPLC with Chemical Derivatization and Fluorescence Detection

Even though many pharmaceuticals show native chromophoric properties, there are also many important compounds, such as anticholinergic drugs, toxins, steroids and anticancer agents, which are not chromophoric and can not be determined by usual spectra analysis. T-2 toxin is one kind of the compounds. The detection of these compounds by HPLC imposes severe limitation on sensitivity and ofen togather with unacceptable constraints on mobile phase selection. Precolumn or postcolumn derivatization technology by using chromophoric group is often an inexpensive but effective way to overcome this problem, which make these compounds chromophoric and then enhance detcetability of them. For trace analysis, there is a balance to be attained between rigour of sample cleanup and selectivity of detection. Derivatization of target compounds of pharmaceuticals interest offers significant advantages, such as improving selectivity and thus reducing the complexity of the sample cleanup required.     

高效液相色谱分离-在线光化学衍生/荧光光谱法测定5种水溶性维生素的研究

建立了高效液相色谱分离-在线光化学衍生/荧光光谱法测定水溶性维生素烟酸(NIA)、烟酸胺(NIC)、B1、B12及B2的新方法.以含有0.018 mol/L三乙胺、0.002 mol/L庚烷磺酸钠的0.05 mol/L磷酸盐缓冲液(A相,pH 5.8)和甲醇(B相)为流动相(85:15),等度洗脱分离5种水溶性维生素;...     

Fast Analysis of Free Amino Acids in Tobacco by HPLC with Fluorescence Detection and Automated Derivatization

A fast, simple, and sensitive HPLC method for the determination of free amino acids in tobacco was described. A fully automated sample processor performed precolumn derivatization of both primary and secondary amino acids with o‐phthalaldehyde/3‐mercaptopropionic acid and 9‐fluorenylmethyl chloroformate (FMOC‐Cl), respectively. All reactions were fully automated by means of an injector programme and accomplished in 10 min. Sample preparation consisted of a single step of extraction with 0.1 mol/L HCl at ambient temperature (assisted by sonication) in 30 min, followed by filtration of an aliquot and derivatization. By optimization of sample preparation and HPLC conditions, separation of 20 amino acids in 30 min was achieved. Detection limits ranged from 0.50 to 1.40 μg/g; coefficients of variation ranged from 1.8% to 3.9%; recoveries ranged from 84.6% to 108.5%. The method was applied to the analysis of amino acids contents of tobacco leaves in different varieties and flue‐curing period.     

Amino Acid Determination in Grape Juices and Wines by HPLC Using a Modification of the 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate (AQC) Method

Summary A novel rigid spherical biporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin (denoted Resin B) has been fabricated by radical suspension copolymerization, with superfine granules of calcium carbonate, and toluene andn-heptane, as porogenic agents, as the basis of a novel porogenic mode, cooperation of solid granules and solvents. The pore structure, static adsorption behavior, and chromatographic properties of Resin B were characterized and compared with those of Resin A (with only solvents as porogens). The results indicated that the biporous resin is a promising chromatographic medium for highspeed protein separation with good mechanical performance and high dynamic adsorption capacity and column efficiency at high flow rates.     

Ultraviolet Photolysis and Ortho-Phthalaldehyde-Mercaptoethanol Derivatization of Pharmaceuticals for Enhanced Fluorescence Detection in HPLC

Abstract

Several classes of nitrogenous pharmaceutical were examined for fluorescence after ultraviolet (UV) radiation induced photolysis followed by reaction with o-phthalaldehyde-2-mercaptoethanol (OPA-MERC), and after UV photolysis alone. Photolyses were examined in water, mixtures of methanol/water (1:1), and acetonitrile/water (1:1). Acetone was assessed as a photosensitizer to enhance photolysis and fluorescence response. Flow injection analysis and high-performance liquid chromatographic techniques were used for several pharmaceuticals. The analytes were subjected to UV photolysis and reaction with OPA-MERC reagent for generation of fluorophores that responded to fluorescence detection. During photolysis, solvent type as well as the presence of photosensitizers seem to play a significant role in the formation of primary amines and fluorophores. Photochemical transformation products of some of the pharmaceutical chemicals are proposed. Analytical figures of merit were determined for some analytes. This fluorescence detection approach is applicable for a number of pharmaceuticals at nanogram level.     

HPLC with In-Capillary Optical Fiber Laser-Induced Fluorescence Detection of Picomolar Amounts of Amino Acids by Precolumn Fluorescence Derivatization with Fluorescein Isothiocyanate

In this paper, a fluorescein isothiocyanate (FITC) precolumn derivatization technique in conjunction with an HPLC-in-capillary optical fiber laser-induced fluorescence (HPLC-ICOF-LIF) detection method has been developed for determination of amino acids. The HPLC separation of FITC-labeled amino acids and the ICOF-LIF detection system are studied and optimized. Optimum separation conditions were obtained with a gradient elution program of acetonitrile and phosphate buffer (10 mM, pH 6.8). The ICOF-LIF detection system comprises a 530-??m capillary and a 380-??m optical fiber. The analyses of amino acids display excellent linear relationship between peak area and concentration with correlation coefficients greater than 0.999 and the method also provides good repeatability with RSD < 3%. The detection limits for FITC-tagged amino acids are very low and the lowest LOD for tyrosine is 51 pM. The proposed method has been successfully applied to determination of amino acids in human serum. Our developed HPLC-ICOF-LIF system is cheap, simple, stable, and sensitive which is potentially useful for the formulation analysis and bioanalysis.     

改良QuEChERS/HPLC-光化学在线衍生荧光检测法测定猪肉中18种磺胺类药物残留量

建立了猪肉中磺胺类药物的改良Qu ECh ERS/高效液相色谱-光化学在线衍生荧光检测方法。样品用1%乙酸-乙腈溶液提取,PSA,C18和石墨化碳黑(GCB)混合粉末作为吸附剂,Qu ECh ERS净化后进行HPLC分析,以Platisil ODS色谱柱(4.6 mm×250 mm,5μm)分离,在线光化学衍生后进入荧光检测器检测。选择激发波长为320 nm,发射波长为450 nm,柱温36℃,流动相为0.3%冰乙酸-甲醇,梯度洗脱,可实现18种待测组分的基线分离。在优化实验条件下,18种磺胺类药物的质量浓度在0.05~110.28μg·m L-1范围内与其峰面积呈良好线性,相关系数均大于0.992 0。方法检出限(S/N=3)为1~18μg·kg-1,定量下限(S/N=10)为3~60μg·kg-1。加标水平为0.02~4.49 mg·kg-1时,猪肉中18种磺胺类药物的平均回收率为71.2%~113.4%,绝大部分集中在80%~100%之间,相对标准偏差(RSD)为0.8%~8.7%。该方法前处理快速简便、选择性强、有机溶剂用量少,检测可靠,准确性和灵敏度高,适用于猪肉中磺胺类药物残留的快速检测。     

高效液相色谱柱后衍生化法用于氨基甲酸酯类农药的测定

研究了用高效液相色谱（ＨＰＬＣ）柱后衍生化荧光检测系统测定氨基甲酸酯类农药的方法。用ＲａｄｉａｌＰａｋＣ＿（１８）柱在不同流动相梯度条件下对六种农药和三种代谢产物进行分离,通过柱后衍生化技术提高荧光检测器的灵敏度。结果表明：该方法分离效果良好,最低检测浓度为２μｇ／Ｌ,最小检出量为２ｎｇ。     

高效液相色谱柱后衍生法测定谷物中9种氨基甲酸酯类农药及3种代谢物残留量

研究了用ＷａｔｅｒｓＣａｒｂａｍａｔｅ分析系统柱后衍生化荧光检测器测定谷物中氨基甲酸酯类农药残留量的方法。用ＷａｔｅｒｓＣａｒｂａｍａｔｅＡｎａｌｙｓｉｓＣｏｌｕｍｎ对９种农药和３种代谢产物进行分离,碱液水解,ＯＰＡ柱后衍生,有很好的选择性、重现性和灵敏度,最低检出限为５μｇ／ｋｇ。     

过氧化氢柱前荧光衍生化液相色谱法测定麻痹性贝毒的研究

液相色谱-荧光检测法(LC-FLD)测定贝类样品中石房蛤毒素(STX)和decarbamoylsaxitoxin(dcSTX)。样品经30 mmol/L HAc超声提取,C18固相萃取柱净化,2%碱性H2O2荧光衍生,C18色谱柱(4.6 mm×250 mm,5μm)分离,以乙腈-0.1 mol/L甲酸铵溶液(5∶95,V/V)作流动相,流速1.0 mL/m in。结果表明,STX和dcSTX衍生物在7m in内获得完全分离。在空白样品中添加标准品使浓度0.01~2.0μg/g,得到峰面积与浓度呈良好线性,线性相关系数>0.998。添加浓度在0.1、0.8和1.6μg/g的回收率为87%~97%(n=8);相对标准偏差为8%~13%。方法检出限(S/N=3)分别为STX 1.0 ng/g和dcSTX 0.3 ng/g。另外,采用四极杆-飞行时间质谱(Q-TOF-MS)对STX和dcSTX衍生物进行了结构解析。     

在线衍生微流控芯片电泳和激光诱导荧光法测定单细胞中谷胱甘肽

微流控分析芯片的网络结构和微米通道尺寸适合于单细胞进样、控制和分离分析^[1~4].在测定细胞内容物时,大多采用柱前细胞内衍生法^[1,2,4],但操作复杂,需多次离心分离,且能透过细胞膜标记胞内组分的荧光试剂较少.     

高效液相色谱-紫外/荧光检测方法测定河豚毒素

河豚毒素（TTX）的准确定性及定量测定一直是人们研究的重点,其检测方法有多种。小鼠法是最常用和最简便的方法,但具有定量不准确且重复性差等缺点。由于河豚毒素在NaOH溶液中加热具有产生荧光的特性,可以利用荧光检测器测定TTX的含量,因此,本文利用反相高效液相色谱法（RP-HPLC）,采用紫外和荧光两套检测系统对TTX的准确定量进行了初步探索。     

衍生化高效液相色谱法测定高分子受阻胺光稳定剂

以对硝基苯甲酰氯为酰基化试剂,通过与含胺基的高分子受阻胺光稳定剂944在四氢呋喃中发生酰胺化反应形成沉淀,改变反应配比确定对硝基苯甲酰氯与高分子受阻胺光稳定剂944反应比例为5∶1。利用傅立叶红外光谱分析反应沉淀来实现高分子受阻胺光稳定剂944的定性分析,利用高效液相色谱分析反应上层清液,实现定量分析,紫外可见检测器检测波长为360 nm,反应测试结果回收率可达到90%以上。     

Novel Method for HPLC Analysis of Triterpenic Acids Using 9-Anthryldiazomethane Derivatization and Fluorescence Detection

Triterpenic acids are a group of secondary plant metabolites which are part of the cuticular waxes covering fruits, leaves, and flowers. To date, quantitative analysis of these compounds has often been conducted using high-performance liquid chromatography coupled with spectrophotometric detection or mass spectrometry; however, these methods have some major drawbacks. This paper reports a new method of analysis implementing derivatization with 9-anthryldiazomethane and fluorescence detection. The method consists of the extraction of analytes from a matrix, purification with anion exchanging SPE columns, and an optional step of the alkaline hydrolysis of triterpenic acid esters. The paper also describes a fast and easy method for the synthesis of the derivatization agent. The detection limits of the method presented are approximately 100-fold lower than in a similar method using ultraviolet spectrophotometry as the mode of detection. The recovery and repeatability of the method are at satisfactory levels.     

Determination of Vitamin B6 Compounds in Foods Using Liquid Chromatography with Post-Column Derivatization Fluorescence Detection

The liquid chromatographic determination of six vitamin B₆-related compounds, the three B₆ vitamers, their corresponding phosphorylated esters, and an excretion product, is optimized using the reversed-phase technique with a stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl. The isocratic mobile phase consisted of a phosphate buffer, and fluorescence detection involved a post-column derivatization reaction using sodium hydrogensulphite to enhance the fluorescence of the phosphate ester. Peaks were identified by the retention characteristics and fluorescence spectra. Detection limits ranged from 1–25 ng mL^–1. Two extraction procedures using acid hydrolysis and enzymatic hydrolysis were compared. The method was applied to the determination of B₆ vitamin derivatives in different types of food including beef liver, egg yolk, baby food cereals and honey. The natural free vitamers appeared in honey and baby food cereals, while the phosphorylated esters were found in the foods of animal origin. An assay using two certified reference materials gave results within the certified range.     

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

Atpresentseparationsandquantitativedeterminationsofaminoacidsbymeansofnewfluorescencereagentsforpre-columnorpost-columnderivatizationinRP-HPLCarestillanactivefiled,developmentshavingbeensummarizedbySnyder'.MostaminoacidsdonotshowUVabsorptionin220-254urn,henceinordertoincreasedetectionsensitivityandimproveselectivity,generallyderivatizationreagentsareemployed.Phenylisothiocyanate(PITC)',OPAand3,5-dinitrobenzoylchloride3arewellknownderivatizationreagefltsforthedeterminationofaminocompounds…     

Determination of Gatifloxacin in Semen by HPLC with Diode-Array and Fluorescence Detection

The objective of this work was to develop an analytical HPLC method, using DAD and fluorescence detection, for determination of gatifloxacin in semen. A reversed-phase column was used with 90:10 water-acetonitrile, containing 10 mM TBA and 25 mM citric acid, as mobile phase. Semen was deproteinized with acetonitrile. Recovery was 95 ± 10%. The limits of quantification by DAD and fluorescence were 2.3 and 0.03 μg.mL^?1 respectively, with RSD of 3.4% for DAD and 2.8% for fluorescence. The method with fluorescence detection was used for quantification of gatifloxacin in the semen of patients under treatment.