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A rapid, accurate and sensitive method, using the stable isotope labeling (SIL), microwave-assisted dispersive liquid-liquid micro extraction (MADLLME) and the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), was developed and validated for the determination of hydroxyl UV Filters in environmental water samples. A pair of new isotopic tags D0-/D3-1-methylindole-3-acetic acid (D0-/D3-MIAA) is synthesized, with which a simple yet efficient pretreatment MADLLME-SIL is developed. Under the optimized conditions (80℃, 240W, 180s), the sample pretreatment including analyte extraction, pre-concentration and isotope labeling can be finished conveniently in only 9min. D0-/D3-MIAA labeling improves the chromatographic retention by strengthening the hydrophobicity and enhances the MS response for 3-4 orders of magnitude. Excellent linearity is established by the H/D ratios of 1/10-10/1 with the correlation coefficients >0.9990. The quite low detection limits (0.54-1.79ng/L) are achieved, ensuring the trace detection. This method is successfully applied to a series of environmental water samples. The recoveries (93.2%~103.5%) are significantly improved and the analysis time is largely reduced (

The presence or absence of acetyl groups in oligosaccharides greatly affects the biological activities of glycoproteins. Consequently, in studies on glycoproteins, sensitive and accurate methods are required for the analysis of acetylated amino sugars (AASs) are required. We report here our newly developed method for the determination of four AASs [N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-galactosamine (GalNAc), N-acetyl neuraminic acid (Neu5Ac), N-glycolyl neuraminic acid (Neu5Gc)]. This method is based on the optimization of the release of AASs from Potentilla anserina L. glycoprotein and involves modification of experimental design and the use of high-performance liquid chromatography coupled with UV detection and mass spectrometry identification. Using our optimized conditions of derivatization and separation, we successfully detected these four AAS derivatives with good precision and accuracy. This is the first report of determination of the four AASs simultaneously on the basis of its release study. The newly developed method exhibits a high potential for the analysis of AASs from various bio-samples and provides a sensitive method for further study of the relationship between AAS composition and their bioactivities in glycoprotein.

A novel, sensitive and convenient method, utilizing ultrasonic-assisted closed in-syringe hydrolysis and derivatization (UCSHD) prior to high performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD) and online mass spectrometry (MS) identification, has been developed for determination of sialic acids. The pivotal parameters affecting the release of sialic acids from serum and the derivatization were investigated using a response surface methodology (RSM). Under optimized conditions, the maximum yield of sialic acids release were achieved and they were labeled successfully in a relatively short time of 72 min (traditional time > 3 h) for the reason of the combination of hydrolysis steps with derivatization in a closed system with the assistance of ultrasonication. Excellent linearity (R2 > 0.9991) in the calibration range of 0.5–16 μmol mL−1 and quite low detection limits (LODs) (0.30 pmol for Neu5Ac and 0.21 pmol for Neu5Gc) were achieved. When the established UCSHD-HPLC-FLD-MS method was applied to the analysis of sialic acids in various human sera, low relative error (RE: −3.4% to 2.5%), high recoveries (90–96%) and intra- and inter-day precisions (RSD, 0.9–2.2% for Neu5Ac and 1.4–2.8% for Neu5Gc) were also obtained, demonstrating obvious advantages for the accurate, sensitive and convenient determination of sialic acids in bio-samples.

High-performance liquid chromatography coupling with atmospheric pressure ionization mass spectrometry (LC-API-MS) has been widely used in the pharmaceutical industry, clinical research, environmental science, metabolomics studies and so on. However, LC-API-MS often suffers from the matrix effect in its applications in complex samples. Matrix effects can heavily affect reproducibility, linearity, and accuracy of the method leading to erroneous quantitation. The use of isotope-labeled analyte as internal standards provides an efficient solution to overcome matrix effects in LC-API-MS analysis. However, the isotope-labeled standards are expensive and not always available, especially in the analysis of large numbers of analytes. In recent years, the stable isotope-coded derivatization reagents, as an alternative way, have been used to quantify small molecules in LC-API-MS analysis of complex samples. In this review, an overview of differential isotope-coded derivatization reagents is presented, focusing on the applications to low molecular weight compounds by LC-API-MS. The recently reported isotope-coded derivatization reagents were summarized in category of their target analytes, specifically peptides, amines, phenols, carboxylic acids, aldehyde or ketone, saccharides and thiols. It indicates that isotope-labeled derivatization reagent would have an increasingly wide range of applications in the LC-API-MS analysis.

A method of high performance liquid chromatography-fluorescence detection-mass spectrometry identification (HPLC-FLD-MS) coupled with pre-column derivatization to determine bile acids (BAs) from bio-sample has been developed here. The labeling reagent of 2-(2-(pyren-1-yl)-1H-benzo[d]imidazol-1-yl)-ethyl-4-methyl benzenesulfonate (PBITS) contains six benzene rings and two weak basic nitrogens in the structure, resulting in a higher degree of conjugation and perfect ionization efficiency. The PBITS could easily and quickly label BAs at the optimized derivatization conditions and the labeling yield was close to 100%. Separation of the derivatized BAs exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Hypersil BDS C18 column. Calculated detection limits for HPLC-FLD, at a signal-to-noise ratio of 3, were 10.09–16.33 fmol. Excellent linear responses were observed with correlation coefficients >0.9995. The mean inter-day precision for all standards was

P>In this study, we developed a method of liquid chromatography with fluoresce detector (LC-FLD) and on-line mass spectrometry identification (MSI) using 2-[2-(7H-dibenzo [a, g] carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC-Cl) as pre-column derivatisation reagent for determination of the amino acids (AAs) in Potentilla anserina L. Separation of the derivatised AA exhibited a good baseline resolution in combination with a gradient elution. All AA derivatives give excellent linear responses with correlation coefficients of > 0.9992. The detection limits of each AA were 2.60-24.3 fmol. Eighteen AAs, involving seven essential AAs, were detected in Potentilla anserina L. Quantitative recoveries of the AAs from the Potentilla anserina L. were 84-107%, and the relative standard deviation values were < 1.45%. The established method in this study was sensitive and precision enough to separate and quantify AA composition of Potentilla anserina L. Good compositional data were obtained from the analysis of the AAs obtained from Potentilla anserina L. root.

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. (C) 2011 Elsevier B.V. All rights reserved.

BACKGROUND: Supercritical CO(2) was utilised to extract Chinese star anise seed oil (CSASO), and a three-level Box - Behnken factorial design from response surface methodology was applied to optimise the extraction conditions, including pressure, temperature and amount of modifier (ethanol). The compositional analysis of fatty acids in CSASO was performed by HPLC with fluorescence detection using 2-(11H-benzo[a]carbazol-11-yl)-ethyl-4-methylbenzenesulfonate (BCETS) as labelling reagent. Identification was carried out by online atmospheric chemical ionisation-mass spectrometry. RESULTS: The optimum extraction conditions were as follows: extraction pressure, 27.72 MPa, extraction temperature, 46.22 degrees C, and amount of modifier, 8.58 vol.%. The experimental result showed that the maximum extraction yield was 25.31 +/- 0.22% (w/w) under the conditions proposed. The compositional analysis indicated that CSASO mainly contained C18: 2, C18 : 1, C18 : 3, C20 :4, C16, C18 and C20 fatty acids. CONCLUSION: In this study, a fast, simple and high-efficiency supercritical technique for extracting oil from Chinese star anise seed was developed. Simultaneous determination of fatty acids in CSASO using BCETS as the labelling reagent with HPLC fluorescence detection and online mass spectroscopy identification has been successfully achieved. (C) 2010 Society of Chemical Industry

A sensitive method for the determination of free fatty acids using 1,2-benzo-carbazole-9-ethyl-p-toluenesulfonate (BCETS) as tagging reagent with fluorescence detection has been developed. BCETS could easily and quickly label fatty acids in the presence of the K(2)CO(3) catalyst at 80 A degrees C for 30 min in N,N-dimethylformamide solvent. In this study, fatty acids from the extracted Potentilla anserina L. plant sample were sensitively determined. The corresponding derivatives were separated on a reversed-phase Eclipse XDB-C(8) column by LC in conjunction with gradient elution. The identification was carried out by post-column APCI-MS in positive-ion detection mode. BCETS-fatty acid derivatives gave an intense molecular ion peak at m/z [M+H](+), the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at m/z [M'+CH(2)CH(2)](+), m/z 216.6 and m/z [MH-H(2)O](+) (here, M': corresponding molecular mass of the fatty acids). The fluorescence excitation and emission wavelengths of the derivatives were at lambda (ex) 279 nm and lambda (em) 380 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are more than 0.9994. Detection limits, at a signal-to-noise ratio of 3:1, are 10.79-34.19 fmol for the labeled fatty acids.

Using three different derivatization methods to mark the amine compounds, we compared the difference of derivatization efficiency and gave the optimal method. 5-(2-Hydroxyethyl)benzoacridine reacts with coupling agent N,N′-carbonyldiimidazole (CDI) to form an activated amide intermediate 2-(benzoacridin)ethyl-imidazole-1-carboxylate (BAEIC). BAEIC, which is dual-sensitive probe, reacts preferably with amino compounds at 80 °C in the presence of 4-dimethylaminopyridine (DMAP) catalyst in N,N-dimethylformamide (DMF) solvent to give the corresponding sensitively fluorescent derivatives with an excitation maximum at 280 nm of λex and an emission maximum at 510 nm of λem. BAEIC-amine derivatives simultaneously exhibited high-ionization potential with percent ionization δ changing from 5.62% to 58.08% in aqueous acetonitrile and from 2.14% to 56.58% in aqueous methanol. Derivatives are not only sensitive to fluorescence but also to MS ionizable potential. The fluorescence detection limits are 0.12–0.59 ng mL？1 (at a signal-to-noise ratio of 3). The online Atmospheric Pressure Chemical Ionization Mass Spectrometry (APCI-MS) detection limits are 1.89–14.12 ng mL？1 (at a signal-to-noise ratio of 5).

Dual-sensitive probe of 1-imidazole-2-(5-benzoacridine)-ethanone (IBAE) for the determination of free amines with fluorescence detection and online highly sensitive atmospheric chemical ionization-mass spectrometry identification (APCI-MS) has been developed. 2-(Benzoacridine)-5-acetic acid (BAAA) reacts with coupling agent N,N'-carbonyldiimidazole (CDI) to form a highly activated amide intermediate 1-imidazole-2-(5-benzoacridine)-ethanone (IBAE), which is dual-sensitive probe. The amide intermediate (IBAE) reacts preferably with amines in dimethylformamide (DMF) solvent to give the high yields of derivatives. IBAE-amine derivatives are not only sensitive to fluorescence but also to MS ionizable efficiency. The percent ionization 6 values change from 0 to 57.32% in aqueous acetonitrile and from 0 to 62.14% in aqueous methanol. The relative standard deviations of the peak areas with fluorescence detection for each amine are

This paper, for the first time, reported a speedy hyphenated technique of low toxic dual ultrasonic-assisted dispersive liquid-liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) for the simultaneous determination of 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT). The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. A mass spectrometry sensitizing reagent, 4'-carboxy-substituted rosamine (CSR) with high reaction activity and ionization efficiency was synthesized and firstly used as derivatization reagent. Parameters of dual-UADLLME, MAD and UHPLC-MS/MS conditions were all optimized in detail. Low toxic brominated solvents were used as extractant instead of traditional chlorinated solvents. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.010 and 0.015ng/mL for PPD and PPT, respectively) were achieved. The main advantages were rapid, sensitive and environmentally friendly, and exhibited high selectivity, accuracy and good matrix effect results. The proposed method was successfully applied to pharmacokinetics of PPD and PPT in rat plasma.

A new labeling reagent for carboxylic acids, 2-(2-(anthracen-10-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)ethyl 4-methylbenzenesulfonate (APIETS) has been designed and synthesized. It was used to label eight fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linoleic acid and linolenic acid) and four hydroxy pentacyclic triterpene acids (oleanolic acid, ursolic acid, betulinic acid and maslinic acid), successfully. APIETS could easily and quickly label carboxylic acids in the presence of K(2)CO(3) catalyst at 85 degrees C for 35 min in N,N-dimethylformamide solvent. The carboxylic acids derivatives were separated on a C(8) reversed-phase column with gradient elution and fluorescence detection at lambda(ex)/lambda(em) = 315/435 nm. Identification of these derivatives was carried out by online mass spectrometry with atmospheric pressure chemical ionization in positive ion mode. The detection limits obtained were 13.37-30.26 fmol (signal-to-noise ratio of 3). The proposed method has been applied to the quantification of carboxylic acids in sultana raisin (Thompson seedless), hawthorn flake (Crataegus pinnatifida Bge.), Lycium barba rum seed oil and Microula sikkimensis seed oil with recoveries over 95.3%. It has been demonstrated that APIETS is a prominent labeling reagent for determining carboxylic acids with high performance liquid chromatography. (C) 2011 Elsevier B.V. All rights reserved.

Supercritical CO(2) was utilised to extract Chinese star anise seed oil (CSASO), and a three-level Box-Behnken factorial design from response surface methodology was applied to optimise the extraction conditions, including pressure, temperature and amount of modifier (ethanol). The compositional analysis of fatty acids in CSASO was performed by HPLC with fluorescence detection using 2-(11H-benzo[a]carbazol-11-yl)-ethyl-4-methylbenzenesulfonate (BCETS) as labelling reagent. Identification was carried out by online atmospheric chemical ionisation-mass spectrometry.The optimum extraction conditions were as follows: extraction pressure, 27.72 MPa, extraction temperature, 46.22 degrees C, and amount of modifier, 8.58 vol.%. The experimental result showed that the maximum extraction yield was 25.31 +/- 0.22% (w/w) under the conditions proposed. The compositional analysis indicated that CSASO mainly contained C18:2, C18:1, C18:3, C20:4, C16, C18 and C20 fatty acids.In this study, a fast, simple and high-efficiency supercritical technique for extracting oil from Chinese star anise seed was developed. Simultaneous determination of fatty acids in CSASO using BCETS as the labelling reagent with HPLC fluorescence detection and online mass spectroscopy identification has been successfully achieved.

Recent researches shows that amino acids (AA) are not only cell signaling molecules but are also regulators of gene expression and the protein phosphorylation cascade. More precise analysis of AA composition is reckoned to be one of the most important applications in the biomedical and pharmaceutical fields. In this paper, we develop a sample, sensitive and mild method using 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)-ethoxy]ethyl chloroformate (DBCEC) as A labeling reagent for AA determination by high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) and identification with mass spectroscopy. The maximum excitation and emission wavelengths for DBCEC-AA derivatives were 300 and 395 nm, respectively. This method, in conjunction with a gradient elution, offered a baseline resolution of 20 AA on a reversed-phase Hypersil BDS C(18) column. LC separation for the derivatized AA showed good reproducibility, and all AA were found to give excellent linear responses with correlation coefficients0.9993. The calculated detection limits with a 25.0 fmol injection of each AA (at a signal-to-noise ratio of 3:1) ranged from 2.62 to 22.6 fmol. This method was applied to determine the AA composition in Saussurea involucrate and Artemisia capillaris Thunb. Meanwhile, this method exhibits a powerful potential for trace analysis of AA from biomedicine, foodstuff and other complex samples.

A new fluorescent derivatization reagent, 10-ethyl-9-oxo-9, 10-dihydroacridine-2-sulfonyl chloride (EASC), was synthesized. A pre-column derivatization with EASC and reversed-phase high performance liquid chromatographic (RP-HPLC) method with fluorescence (FL) detection and mass spectrometry (MS) identification was performed for the trace analysis of oestradiol (E2) and oestriol (E3) in urine. This reagent shows higher sensitivities in ultraviolet (UV), FL and MS detection than those of dansyl chloride (DNS-Cl), and the fluorescence intensity of EASC is 1000 times higher than that of DNS-Cl. The results of derivatization indic ted that the derivatives can be obtained by the labeling reaction of EASC with estradiol (E2 and estriol (E3) in the presence of NaHCO3 buffer (pH 10.5) at 60 degrees C for 3 min. The excitation wavelength (lamda ex) and emission wavelength (lamda em) were 270 nm and 430 nm, respectively. The established method exhibited excellent reproducibility and recovery. The calibration curve were linear with regression coefficients over 0.9990, and the detection limits (S/N = 3) w 40, 31 fmol for the studied compounds. The practical applicability of the method was demonstrated by analyzing trace of free oestradiol and oestriol in the urine of root voles.

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 were3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of0.9995.

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxyl ethyl carbonylhydrazine (DBCEEC) followed by high-performance liquid chromatography with fluorescence detection and APCI-MS identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxyl ethyl functional group, which resulted in a sensitive fluorescence tagging reagent DBCEEC. DBCEEC Could easily and quickly labeled aldehydes. The maximum excitation (300 nm) and emission (400 nm) wavelengths did not essentially change for all the aldehyde derivatives. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH(2))(n)](+) in positive-ion mode (M: molecular weight of DBCEEC, n: corresponding aldehyde carbon atom numbers). The collision-induced dissociation of protonated molecular ion formed fragment ions at m/z 294.6, m/z 338.6 and m/z 356.5. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10 to 15-fold molar reagent excess. Separation of the derivatized aldehydes had been optimized on ZORBAX Eclipse XDB-C(8) column with aqueous acetonitrile as mobile phase in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.01-10 nmol mL(-1) with coefficients of >0.9991. Detection limits obtained by the analysis of a derivatized standard containing 0.01 nmol mL(-1) of each aldehyde, were from 0.2 to 1.78 nmol L(-1) (at a signal-to-noise ratio of 3). (C) 2009 Elsevier B.V. All rights reserved.