Pre-column Labeling Reagent Research Articles

Rapid Determination of Amino Acids of Nitraria tangutorum Bobr. from the Qinghai-Tibet Plateau Using HPLC-FLD-MS/MS and a Highly Selective and Sensitive Pre-Column Derivatization Method.

Amino acids are indispensable components of living organisms. The high amino acid content in Nitraria tangutorum Bobr. fruit distinguishes it from other berry plants and is of great significance to its nutritional value. Herein, using 10-ethyl-acridine-3-sulfonyl chloride as a fluorescent pre-column labeling reagent, a method for the efficient and rapid determination of amino acid content in N. tangutorum by pre-column fluorescence derivatization and on-line mass spectrometry was established and further validated. The limits of detection (signal-to-noise ratio = 3) were between 0.13 and 1.13 nmol/L, with a linear coefficient greater than 0.997 and a relative standard deviation between 1.37% and 2.64%. In addition, the method required a short analysis time, separating 19 amino acids within 20 min. Subsequently, the method was used to analyze the amino acid content of Nitraria tangutorum Bobr. from tissues retrieved from seven regions of the Qinghai-Tibet Plateau. Nitraria tangutorum Bobr. was shown to contain a large amount of amino acids, with the total content and main amino acid varying between the different tissues. This research supports the nutritional evaluation, quality control, and development and utilization of Nitraria tangutorum Bobr.

Determination of Free Fatty Acids of Chinese Coriandrum sativum L. Using Benzimidazo[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate as Precolumn Labeling Reagent by LC with Fluorescence Detection

A new fluorescent labeling reagent, benzimidazo[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate (BQETS) was designed and synthesized, and it was successfully applied to the determination of fatty acids with liquid chromatography. BQETS can easily and quickly label fatty acids within 20 min at 90 °C in dimethylformamide with K2CO3 as catalyst. The derivatives exhibit high stability and strong fluorescence with excitation and emission wavelengths of 247 and 401 nm, respectively. The 24 derivatives of fatty acids were completely separated by gradient elution on a Hypersil GOLD C18 column. Excellent linear responses for all fatty acids were observed with correlation coefficients of >0.9991. The method also showed good sensitivity and precision, with limits of detection in the 0.0024–0.0206 μg g−1 range and relative standard deviations ≤9.6 %. This is the first time that BQETS fluorescent probe and its applications for the determination of fatty acids have been reported. Moreover, this is the first report on the comparison of free fatty acids composition in the above-ground part of Coriandrum sativum L. from different habitats in China.

A sensitive high‐performance liquid chromatography method with fluorescence detection for the determination of fatty acids as exemplified for Dendrobium species

A novel high‐performance liquid chromatography (HPLC) method using 2‐(9‐oxoacridin‐10(9H)‐yl) acetohydrazide (OAAH) as pre‐column labeling reagent was applied to the analysis of fatty acids in four kinds of Dendrobium species for the first time. The reaction of OAAH with fatty acids could proceed easily and quickly in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC) as condensing reagent within 20 min. The derivatives exhibit excellent fluorescence property with excitation and emission wavelengths of 255 and 420 nm, respectively. The 20 fatty acid derivatives were separated on a SB C18 reversed‐phase column with gradient elution. Good linear correlations were observed for all fatty acids with correlation coefficients of >0.996. When 50 mg of sample was used for analysis, the detection limits at a signal‐to‐noise ratio of 3 were in the range of 0.10–0.42 µg/g. Free fatty acids and total fatty acids in four kinds of Dendrobium species were analyzed by the developed method. The results indicated that the main fatty acids in Dendrobium species were unsaturated linoleic acid (C18:2n‐6) and saturated hexadecanoic acid. This work provides a useful tool for the safety assessment and quality control of Dendrobium species.Practical applications: Dendrobium species, the stems of which can be used for medical purpose or as health foods, are precious plants in China. Their significant medical effects have recently attracted increasing attention. However, there is little research on the fatty acid composition of Dendrobium species. The proposed method provided a sensitive method for the HPLC analysis of FFAs and TFAs in four kinds of Dendrobium species. This is the first report of the fatty acid compositions of Dendrobium species. It would be helpful for the comprehensive safety or quality assessment of Dendrobium species.

Determination of Bisphenol A and Alkylphenols in Soft Drinks by High-Performance Liquid Chromatography with Fluorescence Detection

A highly sensitive and selective method was developed for the purification and determination of bisphenol A and alkylphenols in soft drinks by using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as pre-column labeling reagent followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC sensitivity of bisphenol A and alkylphenols was greatly enhanced through the introduction of BCEC-Cl moiety with excellent fluorescence property into the target molecules. Meanwhile, the introduction of highly hydrophobic BCEC-Cl moiety into the analytes also greatly increased the hydrophobicity of the target compounds and distinguished them from hydrophilic matrices. Therefore, little interference was observed. Solid-phase extraction with C18 cartridges was applied to sample purification procedure with recoveries of higher than 82 %. When 20 mL of sample was used for analysis, the limits of quantifications of the analytes were between 0.06 and 0.1 mu g L-1. The proposed method was successfully applied to the determination of the target compounds in soft drink samples with a much higher sensitivity than traditional HPLC methods.

A sensitive method for extraction and determination of endocrine-disrupting compounds from wastewater using 10-ethyl-acridone-2-sulfonyl chloride as pre-column labeling reagent by high-performance liquid chromatography with fluorescence detection

A sensitive pre-column derivatization method using 10-ethyl-acridone-2-sulfonyl chloride (EASC) as pre-column labeling reagent followed by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the determination of eight endocrine-disrupting compounds: 4-octylphenol, 4-nonylphenol, bisphenol A, diethylstilbestrol, estrone, 17α-ethynylestradiol, 17β-estradiol and estriol in wastewater samples. Solid phase extraction (SPE) with ODS C18 cartridges was used for the extraction and purification. Derivatizing parameters including pH value, temperature and concentration of EASC, as well as types of SPE cartridges and eluents were investigated in detail. Under optimal conditions, the quantification limits for the desired compounds ranging from 1.0 to 2.0ngL−1 were obtained. The recoveries were higher than 80.4% with a noticeable improvement for 4-octylphenol and 4-nonylphenol, whose recoveries were usually lower than 50% during the SPE process. The proposed method was successfully applied to the determination of the target compounds in wastewater samples with a much higher sensitivity than traditional HPLC method.

Purification and determination of bisphenol A and alkylphenol in river sediments by high performance liquid chromatography with fluorescence detection

A sensitive method was developed for the purification and determination of bisphenol A and alkylphenols in river sediments by using 2-(9H-carbazol-9-yl) ethyl carbonochloridate (CEOC) as pre-column labeling reagent followed by high performance liquid chromatography (HPLC) with fluorescence detection. Solid phase extraction with Bond Elute Carbon cartridges was applied to sample purification procedure and proved to be simple and effective. The average recoveries of the three target compounds were higher than 80%, with relative standard deviation (RSD) of less than 10%. The sensitivity of the proposed method was about two orders of magnitude higher than traditional HPLC method. When 5.0 g soil sample was used for analysis, the limits of quantification of the analytes were in the range of 0.3-0.6 mu g kg(-1). Investigation of the concentrations in sediment samples indicated that all three analytes were found at levels ranging from 1.1 to 6.1 mu g kg(-1).

Highly sensitive and selective pre-column derivatization high-performance liquid chromatography approach for rapid determination of triterpenes oleanolic and ursolic acids and application to Swertia species: Optimization of triterpenic acids extraction and pre-column derivatization using response surface methodology

Oleanolic acid (OA) and ursolic acid (UA) are the ubiquitous triterpenic acids in plant kingdom and have multiple biological effects. In the present study, a highly sensitive and selective method using a dual-sensitive probe 2-(5-benzoacridine)ethyl-p-toluenesulfonate (BAETS) as pre-column labeling reagent has been developed for rapid determination of the triterpenes OA and UA by HPLC with fluorescence detection (FLD) and online mass spectrometry identification. Response surface methodology as an efficient tool was employed to optimize the ultrasonic-assisted extraction of triterpenic acids from Swertia plants and the pre-column derivatization reaction, respectively, which ensured the highest triterpenic acids recoveries within the shortest extraction time and the sufficient labeling of the analyzed components. Fast separation of the isomers OA and UA could be achieved on a Hypersil BDS C8 column within 7 min. Both of OA and UA gave the good correlation coefficients of 0.9999. This developed method offered the satisfactory detection limits of 1.10 and 1.30 ng mL −1 for UA and OA, respectively. When applied to Swertia species, it showed good reproducibility.

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 λex 260 nm and an emission maximum at λem 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.

Application of 10‐ethyl‐acridine‐3‐sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI‐MS

A simple, sensitive method for the determination of aliphatic amines based on a sulfonylation reaction using 10-ethyl-acridine-3-sulfonyl chloride (EASC) as pre-column labeling reagent with fluorescence detection and APCI-MS identification has been developed. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by HPLC with an excitation maximum at lambda(ex) 270 nm and an emission maximum at lambda(em) 430 nm. Identification of derivatives was carried out by online post-column MS in positive-ion mode. Comparing with the widely used 5-dimethylaminonaphthalene-1-sulfonylchloride (Dansyl-Cl), EASC-amine derivatives not only exhibited high fluorescence but also exhibited excellent MS ionizable potential. Detection limits obtained from 0.10 pmol injection, at a S/N of 3, were 4.0-12.7 fmol. The mean intra- and inter-assay precision for all aliphatic amine levels were <3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of >0.9995.

Determination of 17-hydroxycorticosteroids in urine by fluorescence high-performance liquid chromatography using Dns-hydrazine as a pre-column labeling reagent

A method is described for the determination of urinary 17-hydroxycorticosteroids using fluorescence high-performance liquid chromatography. After enzymatic hydrolysis, 17-hydroxycorticosteroids were extracted using an Extrelut column and then labeled with Dns-hydrazine in hydrochloric acid—ethanol solution. The labeled steroids were chromatographed on a microparticulate silica gel column, the mobile phase was dichloromethane—ethanol—water (900:60:40). The eluate was monitored on a fluorophotometer at 365 nm (excitation) and 505 nm (emission). Linearity of the fluorescence intensities (peak heights) of various 17-hydroxycorticosteroids were obtained between 60 pg and 20 ng. The assay was sensitive, precise and accurate. Comparison with the results obtained by radioimmunoassay gave correlation coefficients of 0.932 for tetrahydrocortisol and 0.930 for tetrahydrocortisone. The proposed method is clinically useful for the routine analysis of urinary 17-hydroxycorticosteroids.