Determination of Free Fatty Acids in Bryophyte Plants and Soil by HPLC with Fluorescence Detection and Identification by Online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C₈ column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.     

高效液相色谱-质谱分离鉴定荧光试剂标记的脂肪酸

以2-(2-(10-蒽基)-萘[2,3-d]咪唑)-乙基-对甲苯磺酸酯(ANITS)作为柱前衍生化试剂,在Eclipse XDB-C8色谱柱上,梯度洗脱实现了20种游离脂肪酸(FFA)衍生物的完全基线分离。90℃下在DMF溶剂中以K2CO3作催化剂,选取衍生试剂摩尔数为脂肪酸的7倍,衍生反应40min可获得稳定的荧光产物。激发和发射波长分别为250nm和512nm。采用大气压化学电离源(APCI)正离子模式,实现了油菜蜂花粉中游离脂肪酸的质谱鉴定。所有脂肪酸的线性相关系数均大于0.9999,检出限为24.76~98.79fmol。     

Determination of Fatty Acids in Saliva of Smokers and Nonsmokers by HPLC with Fluorescence Detection Using a Hydrazine-Based Difluoro-boraindacene Reagent

1,3,5,7-Tetramethyl-8-daminozide-difluoroboradiaza-s-indacence (TMBODIPY-H), a pre-column fluorescent derivatization reagent, was applied to the analysis of fatty acid by high-performance liquid chromatography with fluorescence detection. Using this reagent, 12 fatty acids from propionic acid (C3) to stearic acid (C18) can be derivatized at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide within 2 h. The baseline separation of these derivatives can be achieved within 46 min by gradient elution. The detection limits of these fatty acids are in the range of 0.1–1 nM and the linear ranges of most of them are 0.5–100 nM. This was the first application of hydrazine-based difluoro-boraindacene reagent for the analysis of fatty acids, and the proposed method has been successfully used for the determination of saliva samples of smokers and nonsmokers with recoveries of 88–110 %.     

Determination of 30 Free Fatty Acids in Two Famous Tibetan Medicines by HPLC with Fluorescence Detection and Mass Spectrometric Identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 °C with anhydrous K₂CO₃ as catalyst. A mixture of C₁–C₃₀ fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C₈ column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI–MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were >0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were <3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1–38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.     

柱前衍生-反相高效液相色谱荧光测定及质谱鉴定土壤和苔藓中的脂肪酸

脂肪酸广泛分布于动植物体内,对于调节某些代谢作用和生理功能起着重要作用,其含量测定对进一步深入研究生理学和病理生理学以及对临床诊断具有重要意义[1-2]。某些动植物体内富含人体必需的多种脂肪酸,对其进行定量分析,对饮食营养和保健品开发同样具有重要指导意义。脂肪酸在     

Determination of Fatty Acids by High-Performance Liquid Chromatography with Electrochemical Detection Using A Ferrocene Derivatization Reagent

Abstract

New derivatization method using ferrocene reagents has been developed for the determination of fatty acids by high-performance liquid chromatography with electrochemical detection. Condensation of fatty acids with 3-bromoacetyl-1, 1'-dimethyl-ferrocene was effected in the presence of 18-crown-6 and potassium fluoride. The resulting esters showed the satisfactory sensitivity at +0.60 V vs. an Ag/AgCl reference electrode with a detection limit of 0.5 pmole. Also the high selectivity was obtained by using a twin electrode electrochemical detector. The proposed derivatization method was found applicable to the determination of fatty acids in human serum.     

Determination and Identification of Primary Aliphatic Amines Using 4-(1H-Phenanthro[9,10-d]Imidazol-2-yl)Benzoic Acid as Novel Pre-Column Labeling Reagent by LC with Fluorescence Detection and Atmospheric Pressure Chemical Ionization Mass Spectroscopy

4-(1H-Phenanthro[9,10-d]imidazol-2-yl)benzoic acid (PIBA), a new amine-reactive fluorescent probe was synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines from environmetal water samples. Derivatization was successfully carried out by a condensation reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as the dehydrant. Studies on derivatization conditions indicated that the condensation reaction proceeded rapidly and smoothly in the presence of pyridine. Amines were labeled with PIBA to give the corresponding sensitively fluorescent derivatives with an excitation maximum at λ _ex 260 nm and an emission maximum at λ _em 445 nm. Identification of derivatives was carried out by online APCI–MS–MS and showed an intense protonated molecular ion corresponding m/z [M + H]⁺ in the positive ion mode. Excellent linear responses were observed with coefficients of >0.9996. Detection limits were 6.7–27 fmol (at a signal-to-noise ratio of 3). The mean intra- and inter-assay precision for all amine levels were <2.79 and 3.85%, respectively. The established method for the determination of aliphatic amines from real water was satisfactory.     

高效液相色谱荧光测定及质谱鉴定土壤和苔藓中的脂肪酸

以吖啶酮-9-乙基对甲苯磺酸酯(AETS)作荧光衍生化试剂,建立了灵敏、简单的游离脂肪酸反相高效液相色谱测定方法。在Ec lipse XDB-C8色谱柱上,实现了19种游离脂肪酸(FFA)衍生物的完全基线分离。选取AETS摩尔数为脂肪酸的6倍,以DMF作溶剂,在85℃条件下以K2CO3作催化剂可获得稳定的荧光产物,条件温和、衍生产率高。利用柱后在线串联质谱以APC I大气压化学电离源正离子模式实现了各组分的质谱定性。对土壤和3种苔藓植物中(树藓、狭叶绢藓、曲尾藓)FFA组分定量结果表明,苔藓植物从土壤中富集了大量的游离脂肪酸。荧光检测的激发和发射波长分别为404 nm和440 nm。绝大多数脂肪酸的线性相关系数大于0.9996,检出限为12.3~43.7 fmol。本方法具有良好的重现性,用于实际样品测定,结果满意。     

Determination of Glyphosate and Aminomethylphosphonic Acid in Water by LC Using a New Labeling Reagent, 4-Methoxybenzenesulfonyl Fluoride

A rapid, low-cost, and highly sensitive analytical method to detect glyphosate and its major metabolite, aminomethylphosphonic acid (AMPA), in water samples has been developed, involving a derivatization with a new labeling reagent, 4-methoxybenzenesulfonyl fluoride, followed by reverse phase liquid chromatography with ultraviolet detection. The limits of detection of both of glyphosate and AMPA in real water samples are 0.1 μg L^?1 with simple pre-concentration. This method has proven to be sensitive and reliable for determination of glyphosate and AMPA in water samples.     

血清中游离脂肪酸的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 乙基对甲苯磺酸酯(BDETS)对19种游离脂肪酸(FFAs)进行柱前衍生,在EclipseXDB C8反相色谱柱上,采用梯度洗脱优化分离.90℃下在DMF溶剂中以K2CO3作催化剂,衍生反应30min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)正离子模式进行柱后在线质谱定性.多数脂肪酸的线性回归系数大于0.9989,检测限为24.80～80.37fmol.实现了人体血清中长链脂肪酸的定性及相应含量测定.     

LC–ESI–MS Determination of 20 Free Amino Acids in Tibetan Medicine Gentiana dahurica with Pre-Column Fluorescence Derivatization

Concentrations of 20 free amino aicds (FAAs) in a famous Tibetan medicine Gentiana dahurica was first investigated using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as the pre-column fluorescence derivatization reagent by reversed-phase high performance liquid chromatography (RP-LC). 20 amino acid derivatives (AAD) were separated on a Hypersil BDS C₁₈ column with a good baseline resolution within 65 min. Identification of 20 AAD was by online post-column mass spectrometry with an electrospray ionization (ESI) source. The validation of the method was examined by linearity, repeatability, and detection limits. Most linear correlation coefficients for AAD were >0.9990, and detection limits (at signal-to-noise of 3:1) were 6.5–178.2 fmol. There were 18 FAAs found in G. dahurica, of which seven FAAs were necessary to the people’s health and related to the treatment of liver and gall disease. Variation of concentrations of the 20 FAAs showed geographical distribution difference among populations. Meanwhile a stable genetic diversity of FAAs composition of G. dahurica was also revealed at the species level. Results of the present study proved that the established method was rapid and reproducible for further separation and determination of FAAs in more medicinal plants.

     

A New Reagent for Determination of Isocyanates in Working Atmospheres by HPLC Using UV or Fluorescence Detection

Abstract

A new liquid chromatographic method with increased sensitivity has been developed for the determination of isocyanates common in industrial environments. The isocyanates are converted to stable urea derivatives by reaction with 9-(N-methylaminomethyl)-anthracene. These derivatives were analyzed using high performance liquid chromatography on a bonded octadecylsilyl phase using isocratic elution with acetonitrile/water and detected either by a UV or a fluorescence detector. The method was applied to toluene 2, 4- and 2, 6-diisocyanate (2, 4- and 2, 6-TDI), hexamethylene diisocyanate (HDI) and 4, 4-diphenylmethane diisocyanate (MDI).

The influence of various salts on the retention of the reagent amine was studied, as well as the separation of the urea derivatives on different C₁₈-phases. The detection limit is about 1 · 10^?4 mg/m³ for the isocyanates investigated, using either UV or fluorescence detection. This means that the new method is ten to twenty times more sensitive than the previously described reversed phase LC method, which utilized N-4-nitrobenzyl-N-n-propylamine as reagent.     

脂肪胺的高效液相色谱分离及质谱鉴定

 采用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙酸(BCAA)为柱前衍生化试剂,在Hypersil BDS-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,1-乙基-3-(3-二甲氨基丙基)环己碳二亚胺(EDAC)为缩合剂,在50 ℃条件下衍生反应15 min后获得稳定的荧光产物。激发波长和发射波长分别为333 nm和390 nm。采用大气压化学电离源(APCI)的正离子模式,实现了土壤和污水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0.9993,检测限为12~28 fmol。     

反相高效液相色谱柱前衍生荧光检测法测定游离多胺

合成新型荧光试剂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Eclipse XDB-C8色谱柱上,通过梯度洗脱对5种多胺进行了分离和在线质谱定性。在乙腈中,以硼酸钠缓冲液(pH9)为催化剂,40℃下衍生反应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,实现了人尿中游离多胺的定性及定量测定。多胺的线性相关系数大于0.9996,检出限为5.3～9.9fmol。     

Determination of Penciclovir in Human Plasma Using LC with Fluorescence Detection: Application to a Bioequivalence Study

A novel HPLC method with fluorescence detection and one step sample preparation was developed for the determination of penciclovir in human plasma. Plasma samples (200 μL) were deproteinized by precipitation with 100 μL of perchloric acid and centrifuged. Separation was on an Inertsil ODS-3 column with a mixture of methanol–0.1% orthophosphoric acid as mobile phase. The fluorescence detector was set at Ex 270 nm, Em 375 nm. The assay was selective and linear with a limit of quantification of 0.025 mg L^?1. The mean absolute recovery was 98.1%, while the intra- and inter-day coefficients of variation and percent error values of the assay method were all less than 10%. The assay was successfully applied in a randomized cross-over bioequivalence study of three pharmaceutical products containing 250 mg famciclovir (an oral prodrug of penciclovir) in 18 healthy volunteers.     

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。     

Rapid Determination of Free Amino Acids in Milk by Microchip Electrophoresis Coupled with Laser‐induced Fluorescence Detection

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis (MCE) coupled with laser‐induced fluorescence (LIF) detection was developed. Seven kinds of standard amino acids were derivated with sulfoindocyanine succinimidyl ester (Cy5) and then perfectly measured by MCE‐LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions: 100 mmol·L^?1 sodium borate solution (pH 10.0) as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 µmol·L^?1 to 1.0 µmol·L^?1 and the detection limit was as low as about 1.0 nmol·L^?1. This MCE‐LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.     

LC-Fluorescence Detection Analysis of Amino Acids from Stellera chamaejasme L. Using 2-[2-(Dibenzocarbazole)-ethoxy] Ethyl Chloroformate as Labeling Reagent

A pre-column derivatization method for the sensitive determination of amino acids using the tagging reagent 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC) followed by liquid chromatography with fluorescence detection has been developed. Identification of DBCEC-amino acids derivatives was by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS–MS). DBCEC can easily and quickly label amino acids, and derivatives are stable enough to be efficiently analyzed by LC. Separation of the derivatized amino acids had been optimized on Hypersil BDS C₁₈ column. A perfect baseline separation for 20 amino acid derivatives was achieved with a ternary gradient elution program. The chromophore of dibenzocarbazole group, which comprise a large rigid planar structure with p–π conjugation system, resulted in a sensitive fluorescence detection for amino acid derivatives. The derivatized amino acids were detected with fluorescence detector with excitation maximum and emission maximum at 300 and 390 nm, respectively. Excellent linear responses were observed with coefficients of >0.9993, and detection limits were in the range of 0.78–5.13 fmol (signal-to-noise ratio of 3). The mean accuracy ranged from 83.4 to 98.7% for fluorescence detection. The mean inter-day precision for all standards was <4.2% of the expected concentration. Therefore, the proposed method was a highly sensitive and specific method for the quantitative analysis of amino acids from biological and natural environmental samples.     

2-(苯并吖啶酮)-乙基咪唑酸酯探针对荧光和质谱的双增敏作用

采用3种不同衍生方法对胺类化合物进行标记,比较衍生效率的差别,给出最优方法:5-(2-羟乙基)-(苯并吖啶酮与N,N-羰基双咪唑(CDI)缩合形成的双敏感探针2-(苯并吖啶酮)-乙基咪唑酸酯(BAEIC)与胺的衍生效率最高,产物稳定。BAEIC在N,N-二甲基甲酰胺(DMF)溶剂中以4-二甲氨基吡啶(DMAP)为催化剂,在80℃条件下与胺反应生成的亲核取代物,不仅表现出强烈的荧光,同时具有很强的质谱离子化能力。估算了衍生物在乙腈和甲醇水溶液中百分离子化值δ在5.62%～58.08%和2.14%～56.58%范围内。本方法具有良好的重现性,激发和发射波长为λex/λem=280/510nm,荧光检出限为0.12～0.59μg/L(8.6～79fmol);在线APCI-MS检出限为1.9～14μg/L(544～825fmol)。