脂肪胺荧光衍生物的高效液相色谱分离及质谱鉴定

采用新型荧光衍生试剂2-(9-吖啶酮)-乙酸(AAA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性.衍生物荧光激发和发射波长为λex=404nm,λem=440nm.30℃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)做催化剂,衍生反应20min后获得稳定的荧光产物.在HypersilBDSC18(4.6mm×100mm,5μm)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(APCISource)正离子模式进行在线柱后质谱定性,实现了各种脂肪胺衍生物的快速、准确测定.该方法具有良好的重现性,多数脂肪胺的线性回归系数大于0.9996,检测限为12.09~25.52fmol.     

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.     

柱前衍生-高效液相色谱分离测定及质谱鉴定脂肪胺

采用新型荧光衍生试剂2-(2-苯基-1-氢-菲[9,10-d]咪唑)-乙酸(PPIA)进行柱前衍生,经荧光检测实现了脂肪胺的高效液相色谱(HPLC)分离测定及柱后质谱鉴定。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)做缩合剂,衍生反应15min可获得稳定的荧光产物。脂肪胺衍生物荧光检测波长为380nm(激发波长为260nm)。在EclipseXDB-C8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,测定了造纸厂废水、大鼠端脑和酸奶中脂肪胺的含量。经柱后在线质谱大气压化学电离源(APCISource)正离子模式实现了各种脂肪胺衍生物的质谱鉴定,借助对活性中间体的质谱解析确定了衍生反应的反应机理。该方法具有良好的重现性和回收率,多数脂肪胺的线性回归系数大于0.9996;检出限为3.1~18fmol(S/N=3∶1)。     

氮杂环荧光试剂2-(11H-苯[a]咔唑)乙基氯甲酸酯对胺类化合物标记及质谱鉴定

含氮类荧光试剂2-(11H-苯[a]咔唑)乙基氯甲酸酯(BCEC-Cl)经柱前衍生处理后,采用荧光检测及柱后在线质谱鉴定对食品中胺类化合物进行了测定。标记反应在pH9的硼酸缓冲液中进行,3min后获得稳定的荧光产物。衍生物于λex=279nm激发条件下产生强烈荧光,其发射波长在λem=380nm处。产物的分离在EclipseXDB-C8色谱柱(150mm×4.6mm,5μm)上进行,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(APCISource)正离子模式进行在线的柱后质谱鉴定,实现了多种脂肪胺衍生物的快速、准确测定。建立的方法具有良好的重现性,回归系数大于0.9996,检出限为1.8~14fmol。     

氮杂多环光致发光分子用于食品中脂肪胺的分析及质谱鉴定

对含氮类光致发光分子2-（11H-苯[a]咔唑）-异丙基氯甲酸酯进行柱前衍生处理,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。衍生物于λex=279nm产生强烈荧光,发射波长为λex=380nm。40℃下在pH9．0的硼酸缓冲液中,衍生反应3min后获得稳定的荧光产物。在Eclipse XDB-C8色谱柱（4．6mm×150mm,5μm）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。该方法具有良好的重复性,多数脂肪胺的线性回归系数大于0．9994,检出限在2．73～23．16fmol水平。     

牛血清白蛋白中氨基酸的高效液相色谱荧光测定及质谱鉴定

利用新型荧光试剂1,2苯-并-3,4二-氢咔唑-9乙-基氯甲酸酯(BCEOC)作为柱前衍生化试剂,在Hypersil BDS C18(4.6 mm×200 mm,5μm)反相色谱柱上,采用梯度洗脱对氨基酸衍生物进行了分离检测。检测的激发和发射波长分别为λex=333 nm,λem=390 nm。采用电喷雾离子源(ESI Source),实现了牛血清白蛋白中氨基酸的定性测定。荧光定量检测的线性回归系数均大于0.999;检出限为6.3~117.6 fmo。l     

污水中脂肪胺荧光衍生物的高效液相色谱分离及质谱鉴定

利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-异丙基氯甲酸酯(BCIC)作为柱前衍生化试剂,在乙腈中,以硼酸钠缓冲液(pH 9)为催化剂,40 ℃下衍生反应10 min后获得稳定的荧光产物.在Eclipse XDB-C8色谱柱上,通过梯度洗脱对12种游离脂肪胺进行了分离和在线质谱定性.激发和发射波长分别为λex=333 nm,λem=390 nm.采用大气压化学电离源(APCI)正离子模式,实现了污水中脂肪胺的定性及相应含量测定.脂肪胺的线性回归系数大于0.9996,检出限在10.57～37.83 fmol.     

新型荧光试剂用于土壤中脂肪胺的高效液相色谱-质谱测定

利用新型荧光试剂2-(2-(10-蒽基)-苯并咪唑)-乙酸(ABIA)为柱前衍生化试剂,在Akasil-C18色谱柱上,通过梯度洗脱对12种游离脂肪胺进行了分离和在线质谱定性。以乙腈为溶剂,N-乙基-N′-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)为缩合剂,在50℃条件下衍生反应20 min后获得稳定的荧光产物。激发波长和发射波长分别为260 nm和430 nm,采用大气压化学电离源(APCI)的正离子模式,实现了土壤中脂肪胺的定性及其含量的测定。脂肪胺的线性相关系数大于0.9990,检出限为11.72~25.63 fmol。     

土壤和水中脂肪胺的高效液相色谱荧光测定及质谱鉴定

利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基氯甲酸酯(BCEOC)作为柱前衍生化试剂,在EclipseXDB-C8色谱柱上,梯度洗脱对12种游离脂肪胺进行了优化分离。40℃下在乙腈溶剂中以硼酸盐缓冲溶液作催化剂,衍生反应10min后获得稳定的荧光产物。激发和发射波长分别为λex=333nm,λem=390nm。采用大气压化学电离源(APCI)正离子模式,实现了土壤和造纸污水中脂肪胺的定性测定。采用荧光法进行分析物的定量测定。多数脂肪胺的线性回归系数大于0.999,检出限在18.65~38.82×10-15mol。方法具有稳定良好的重现性,对实际样品测定结果满意。     

荧光衍生中性糖的高效液相色谱分析

在HypersilODS2色谱柱上,利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基肼基甲酸酯(BCEC)作柱前衍生化试剂,采用梯度洗脱对5种中性糖荧光衍生物进行了优化分离.65℃下在乙腈溶剂中以冰乙酸作催化剂,衍生反应6.5h后获得稳定的荧光产物,衍生反应完全.激发和发射波长分别为λex=333nm,λem=390nm.线性回归系数均在0.999以上,检测限为24.3～62.1fmol.     

Simultaneous determination of histamine and histidine by liquid chromatography following intramolecular excimer-forming fluorescence derivatization with pyrene-labeling reagent.

A highly sensitive and selective fluorometric method for the determination of histamine and histidine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent followed by reversed-phase liquid chromatography. The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by derivatization. The derivatives afforded intramolecular excimer fluorescence (440 - 540 nm), which can clearly be discriminated from the normal fluorescence (360 - 420 nm) emitted from reagent blanks. The detection limits (signal-to-noise ratio = 3) were femto mole levels.     

Determination of catecholamines and indoleamines in human urine based on intramolecular excimer-forming derivatization and fluorescence detection.

A liquid chromatographic (LC) determination of catecholamines and indoleamines is described. This is based on intramolecular excimer-forming fluorescence derivatization with 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and phenolic hydroxyl moieties in a molecule, were converted to the corresponding polypyrene-labeled derivatives by one-step derivatization. They afforded intramolecular excimer fluorescence, which can clearly be discriminated from the normal fluorescence emitted from reagent blanks. The detection limits (S/N = 3) for catecholamines and indoleamines were femto-mole levels per 20-microL injection. Furthermore, this method was applied to a urine assay.     

Liquid chromatographic determination of dicarboxylic acids based on intramolecular excimer-forming fluorescence derivatization

A highly sensitive and selective fluorimetric determination method for dicarboxylic acids (C5-C12) has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid hydrazide (PBH), followed by reversed-phase liquid chromatography (LC). The carboxylic acids were converted to the corresponding dipyrene-labeled derivatives by reaction with PBH in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acids. The structures of the derivatives and the emission of excimer fluorescence were studied by LC with mass spectrometry and with spectrofluorimetry, respectively. The PBH derivatives of the carboxylic acids could be separated by reversed-phase LC on an ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) were 1.3 fmol to undetectable for a 20-microl injection.     

Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization.

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.     

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C(18) column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2x10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N=3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain.     

Precolumn derivatization of nucleotides based on fluorescent carbamate formation of the sugar moieties in high-performance liquid chromatography.

The fluorescence derivatization of nucleotides with 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo++ furan in the presence of sodium azide and the separation of the derivatives by high-performance liquid chromatography are described. The reagent reacts with 5'-terminal hydroxyl groups of nucleotides to produce the corresponding fluorescent carbamates. The derivatives of mono- and oligonucleotides are separated by chromatography on a reversed phase column (TSKgel ODS-80TM) and the derivatives of octa- and deca-nucleotides on a size exclusion column (TSKgel G3000SWXL). The detection limits (signal-to-noise ratio = 3) are 0.8-6.0 pmol on column. 5'Phosphorylated nucleotide also gives a fluorescent derivative after alkaline phosphatase-mediated dephosphorylation.     

Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group

A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).     

柱前手性衍生化反相高效液相色谱法分离拉贝洛尔对映体

以乙酰葡萄糖异硫氰酸酯（GITC）作柱前手性衍生化试剂,用反相高效液相色谱法成功地分离了拉贝洛尔的两对对映异构体,并以荧光检测和紫外检测作对照,确认了4个衍生物的色谱峰。     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.