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.     

Derivatization of Aminophosphonic Acids for HPLC Analysis

Abstract

A general method for the derivatization of alpha-aminophosphonic acids for HPLC analysis is described. The method involves the conversion of the amino functionality to the corresponding 9-fluorenylmethoxycarbonyl (FMOC) urethane followed by esterification of the phosphonic acid moiety utilizing triethyl orthoformate. The sensitivity is compared to that achievable with a previously described N-trifluoroacetyl diethyl ester procedure on the basis of UV detection. Derivative separation using fluorescence detection is also shown.     

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…     

Aflatoxin Analysis by Reverse Phase HPLC Using Post-Column Derivatization for Enhancement of Fluorescence

Abstract

The application of a technique for the determination of aflatoxins by reverse phase HPLC and fluorescence detection incorporating post-column derivatization with iodine, is described. The procedure proved to be extremely sensitive and reproducible. Chromatograms of extracts from maize, peanut butter, sorghum malt and duckling mash are presented illustrating the value of the procedure for confirming the presence of aflatoxins B₁ and G₁.     

Determination of Triterpene Acids from 37 Different Varieties of Raspberry using Pre-column Derivatization and HPLC Fluorescence Detection

In this work, we have developed an efficient method for the rapid extraction and separation of triterpene acids from 37 different varieties of raspberry via ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME). The triterpene acids were then determined by high-performance liquid chromatography (HPLC) with fluorescence detection using benzimidazo-[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate (BQETS) as the labeling agent. Five triterpene acids, including asiatic acid (AA), maslinic acid (MA), corosolic acid (CA), oleanolic acid (OA) and betulinic acid (BA), were extracted by UA-DLLME using chloroform and acetone as the extracting and dispersing solvents, respectively. After extraction and nitrogen flushing, the extracts were simultaneously characterized by HPLC based on pre-column derivatization using BQETS, a new labeling agent synthesized in our laboratory. Several key parameters affecting the extraction efficiency and derivatization yields were investigated and optimized by response surface methodology (RSM) combined with Box–Behnken design (BBD). The method was further validated for linearity (correlation coefficient R ² > 0.9979), precision (RSD = 0.23–2.45 %), and recovery (RSD = 90–106.5 %). The limits of detection (LODs) and the limits of quantification (LOQs) were determined to be within the range of 1.83–7.69 µg/L and 6.06–25.47 µg/L, respectively. This is the first report of the use of BQETS as a pre-column derivatization agent for the determination of triterpene acids in real samples. The proposed method has been applied to the determination of five triterpene acids in 37 different raspberry varieties with significantly increased sensitivity compared to other methods. The results obtained indicate that the contents of triterpene acids vary significantly across different raspberry varieties.

     

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.     

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.     

N-羟基琥珀酰亚胺-3-吲哚乙酸酯柱前衍生高效液相色谱分离及荧光测定肽及其水解物

本文首次报道了一种新的羧酸活性酯试剂,N-羟基琥珀酰亚胺-3-吲哚乙酸酯作为柱前荧光衍生试剂,流动相为10mmol/L柠檬酸-Na₂HPO₄－pH 3.6的甲醇/水(20/80,V/V)溶液,C₁₈柱,于λ_cx/λ_cm＝278nm/355nm处进行荧光检测,高效液相色谱分离测定了谷胱甘肽、二甘肽、谷氨酸、胱氨酸、甘氨酸.当S/N＝3时,检出限为0.2至2.5Pmol.     

HPLC Assay of Phospholipase A2 Activity Using Low-Temperature Derivatization of Fatty Acids

Abstract

A modified isocratic reverse phase high-performance liquid-chromatographic (RP-HPLC) method for phospholipase (PLA) activity assay is developed. Natural lecithins and synthetic phospholipids are used as substrates, and released fatty acids are analyzed after single-phase derivatization (with p-bromophenacyl bromide) at low temperature. The procedure allows simultaneous determination of the total and specific phospholipase activity. This method was successfully applied to snake venom PLA₂ activity assay using natural (soybean and egg yolk lecithins) and synthetic (dipalmytoylphosphatydylcholine) substrates for quantitative determination of the enzyme activity.     

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.     

Pre-Column Derivatization in HPLC of Amino Acids and Peptides

Abstract

The development of high preformance liquid chromatography with permanently bonded non-polar, so called reversed phase packings has been useful in the separation of a variety of peptides and amino acid derivatives. Limits to the detectability of underivatized substances are on the order of several nanomolar. Sensitivity enhancement became very important for the analysis and determination of peptide hormones, their metabolites and fragments, trace constituents and amino acids existing in very little quantity in biological and clinical samples. Therefore chemical derivatization techniques have been introduced into HPLC, too. By derivatization not only sensitivity enhancement, but selectivity can be achieved.     

高效液相色谱荧光衍生法检测醇和酸的进展

高效液相色谱荧光衍生化醇和酸能提高检测灵敏度,改善分离效果,综述了最近十多年来高效液相色谱荧光衍生法检测醇和酸的进展情况。列出了衍生化试剂的结构,评述了衍生化的反应条件。指出了今后的发展方向。     

2-(11H-苯[a]咔唑)-乙酸在脂肪胺的高效液相色谱分离及质谱鉴定中的应用

采用新型荧光衍生试剂2-(11H-苯[a]咔唑)-乙酸(BCA)进行柱前衍生并经荧光检测对脂肪胺进行高效液相色谱(HPLC)分离和质谱定性.衍生物荧光激发和发射波长为λex=285 nm,λem=384 nm.60 ℃下在乙腈溶剂中用N-乙基-N′-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)作催化剂, 衍生反应15 min后获得稳定的荧光产物.在Hypersil BDS C18 (4.0 mm×200 mm, 10 μm) 色谱柱上, 采用梯度洗脱对12种脂肪胺衍生物进行了优化分离.采用大气压化学电离源(APCI Source)正离子模式进行质谱定性, 实现了各种脂肪胺衍生物的快速、准确测定.脂肪胺的线性回归系数不小于0.999 8 , 检出限(S/N=3)为5.73 ～31.3 fmol.     

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;...     

A Novel Fluorescence Detection Method For Organosilakes

Abstract

Quinones such as phenanthraquinone and anthraquinone react with organosilanes giving persistent, strongly fluorescent radicals. the reactions are photochemically initiated, rapid, and facile; detection of concentrations of silanes in the 10^?7 M range are readily achieved. the starting materials do not fluoresce making the procedure simple, selective and specific.     

新型荧光试剂柱前衍生化高效液相色谱荧光检测法测定醇类化合物

采用新型的紫外、荧光衍生试剂咔唑－9－乙基氯甲酸酯，对15种脂肪醇柱前衍生的反应条件，包括反应温度、催化剂的选择、衍生化时间等，进行了考察。结果表明：催化剂选用吡啶，反应温度80℃，时间15min，衍生化具有最大且恒定的检测响应。利用高效液相色谱对衍生产物进行分析，同时对衍生物的分离进行了优化选择，达到了较好的基线分离。检测限为13.3-93.5nmol/L。     

Fluorescent Labeling of Fatty Acids with 9-Anthryldiazomethane (ADAM) for High Performance Liquid Chromatography

Abstract

A novel method for rapid and simple fluorescent labeling of carboxylic acids with 9-anthryldiazomethane (ADAM) for HPLC is described. This reagent is stable in solution and highly reactive with carboxylic acids. Esterification proceeds at room temperature without catalyst. The labeled compounds are separated using a reversed-phase system and determined with a flow-through flurometer. Picomole level of fatty acids are determined by this method.     

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.     

Determining Hydrogen Cyanamide in Fruit by Derivatization with 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate and HPLC with Fluorescence Detection

Hydrogen cyanamide is a plant-growth regulator used on fruit crops, and has a high toxicity. In this study, a simple and sensitive method for detecting hydrogen cyanamide in fruit was developed. In the proposed method, cyanamide was extracted with water from homogenized fruit samples (grape, kiwi, and peach). The extract was purified through twice liquid–liquid extraction. Then, the purified extract was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Finally, the cyanamide derivative was analyzed using HPLC with fluorescence detection. This method was validated at 2, 0.2, and 0.01 mg kg⁻¹ cyanamide. The recovery rates were between 87.1 and 96.6 %, and the intraday and interday reproducibilities were within 1.8 and 2.2 %, respectively. The limits of detection and quantification were 2.4 and 8 μg kg⁻¹ cyanamide, respectively. This method was applied to explore the residue degradation of cyanamide in the field. The results show that the half-life of cyanamide residues on grapes was <2.0 days. Compared with the previous methods, this simple method is sensitive and can be applied for detecting cyanamide in a wide range of fruits.