Rapid Analysis of Estrogens in Meat Samples by High Performance Liquid Chromatography with Fluorescence Detection.

A novel reagent named 4-(N-methyl-1,3-dioxo-benzoisoquinolin-6-yl-oxy)benzene sulfonyl chloride (MBIOBS-Cl) for the determination of estrogens in food samples by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Estrogens could be easily labeled by MBIOBS-Cl in Na2CO3-NaHCO3 buffer solution at pH 10.0. The complete labeling reaction for estrogens could be accomplished within five minutes, the corresponding derivatives exhibited strong fluorescence with the maximum excitation and emission wavelengths at 249 nm and 443 nm, respectively. The derivatization conditions, such as the molar ratio of reagent to estrogens, derivatization time, pH, temperature, and buffers were optimized. Derivatives were sufficiently stable to be efficiently analyzed by HPLC with a reversed-phase Agilent ZORBAX 300SB-C18 column with a good baseline resolution. Excellent linear correlations were obtained for all estrogen derivatives with correlation coefficients greater than 0.9998. Ultrasonic-Assisted extraction was used to optimize the extraction of estrogens from meat samples with a recovery higher than 82%. The detection limits (LOD, S/N = 3) of the method ranged from 0.95 to 3.3 µg· kg-1. The established method, which is fast, simple, inexpensive, and environment friendly, can be successfully applied for the detection of four steroidal estrogens from meat samples with little matrix interference.

A novel high-performance liquid chromatography-fluorescence analysis coupled with in situ degradation-derivatization technique for quantitation of organophosphorus thioester pesticide residues in tea.

A novel high-performance liquid chromatography-fluorescence analysis in combination with in situ degradation-derivatization (ISD-D) technique was developed for simultaneous determination of seven organophosphorus thioester pesticides (OPTPs) in tea. The ISD-D technique was based on degradation of OPTPs by a nucleophilic substitution reaction between phenylbutane-1,2,3-trione-2-oxime and OPTPs, which can give thiol degradation products (DPs). The thiol DPs obtained were derivatized with the novel derivatization reagent N-(4-(carbazole-9-yl)-phenyl)-N-maleimide (NCPM) in a syringe. Attractively, NCPM itself did not fluoresce, whereas the derivatives of the thiol DPs fluoresced intensely, with excitation and emission maxima at 290 nm and 368 nm, respectively, which extraordinary reduced the background interference and increased the detection sensitivity for thiol DPs. Excellent linearity (R2 > 0.995) for all OPTPs was achieved, with limits of detection and limits of quantitation ranging from 0.23 to 0.45 µg/kg and from 0.75 to 1.43 µg/kg, respectively. Satisfactory recoveries ranging from 90.5% to 96.0% were obtained for all OPTPs. The ISD-D technique provided a novel and sensitive strategy for quantitation of trace amounts of OPTPs in real samples. Graphical abstract ᅟ.

Photoinduced electron transfer from polymer-templated Ag nanoclusters to G-quadruplex-hemin complexes for the construction of versatile biosensors and logic gate applications.

In this paper, fluorescent Ag nanoclusters (Ag NCs) templated by hyperbranched polyethyleneimine (PEI) are utilized as a versatile probe through the photoinduced electron transfer (PET) between PEI-Ag NCs and G-quadruplex-hemin complexes. In the presence of hemin and target molecule, the specific conjugation with its aptamer induces the conformational change of the DNA sequence, releasing the G-quadruplex sequence part. Once the G-quadruplex-hemin complexes are introduced, electron transfer from the PEI-Ag NCs to G-quadruplex-hemin complexes occurs, resulting in fluorescence quenching. Through changing the sensing DNA sequence, this novel PET system enables the specific detection of target DNA and adenosine triphosphate (ATP) with the wide linear range of 1-200 nM and 5-500 nM, respectively, and the corresponding limit of detection as low as 0.3 nM for target DNA and 1.5 nM for ATP. In addition, the proposed method is successfully applied to the determination of ATP in human serum samples with satisfactory recoveries, and a logic gate is fabricated using target molecules and hemin as inputs and the fluorescence signal of PEI-Ag NCs as an output.

3-(2-Bromoacetamido)-N-(9-ethyl-9H)-carbazol fluorescent probe and its application for the determination of thiophenols in rubber products by HPLC with fluorescence detection and atmospheric chemical ionization mass spectrometry identification.

A rapid, sensitive, and selective precolumn derivatization method for the simultaneous determination of eight thiophenols using 3-(2-bromoacetamido)-N-(9-ethyl-9H)-carbazol as a labeling reagent by high-performance liquid chromatography with fluorescence detection has been developed. The labeling reagent reacted with thiophenols at 50°C for 50 min in aqueous acetonitrile in the presence of borate buffer (0.10 mol/L, pH 11.2) to give high yields of thiophenol derivatives. The derivatives were identified by online postcolumn mass spectrometry. The collision-induced dissociation spectra for thiophenol derivatives gave the corresponding specific fragment ions at m/z 251.3, 223.3, 210.9, 195.8, and 181.9. At the same time, derivatives exhibited intense fluorescence with an excitation maximum at λex = 276 nm and an emission maximum at λem = 385 nm. Excellent linear responses were observed for all analytes over the range of 0.033-6.66 µmol/L with correlation coefficients of more than 0.9997. Detection limits were in the range of 0.94-5.77 µg/L with relative standard deviations of less than 4.54%. The feasibility of derivatization allowed the development of a rapid and highly sensitive method for the quantitative analysis of trace levels of thiophenols from some rubber products. The average recoveries (n = 3) were in the range of 87.21-101.12%.

Determination of bisphenol A, 4-octylphenol, and 4-nonylphenol in soft drinks and dairy products by ultrasound-assisted dispersive liquid-liquid microextraction combined with derivatization and high-performance liquid chromatography with fluorescence detection.

A novel hyphenated method based on ultrasound-assisted dispersive liquid-liquid microextraction coupled to precolumn derivatization has been established for the simultaneous determination of bisphenol A, 4-octylphenol, and 4-nonylphenol by high-performance liquid chromatography with fluorescence detection. Different parameters that influence microextraction and derivatization have been optimized. The quantitative linear range of analytes is 5.0-400.0 ng/L, and the correlation coefficients are more than 0.9998. Limits of detection for soft drinks and dairy products have been obtained in the range of 0.5-1.2 ng/kg and 0.01-0.04 µg/kg, respectively. Relative standard deviations of intra- and inter-day precision for retention time and peak area are in the range of 0.47-2.31 and 2.76-8.79%, respectively. Accuracy is satisfactory in the range of 81.5-118.7%. Relative standard deviations of repeatability are in the range of 0.35-1.43 and 2.36-4.75% for retention time and peak area, respectively. Enrichment factors for bisphenol A, 4-octylphenol, and 4-nonylphenol are 170.5, 240.3, and 283.2, respectively. The results of recovery and matrix effect are in the range of 82.7-114.9 and 92.0-109.0%, respectively. The proposed method has been applied to the determination of bisphenol A, 4-octylphenol, and 4-nonylphenol in soft drinks and dairy products with much higher sensitivity than many other methods.

Highly selective and sensitive determination of free and total amino acids in Apocynum venetum L. (Luobuma tea) by a developed HPLC-FLD method coupled with pre-column fluorescent labelling.

Amino acids (AA) are important chemical constituents of tea leaves remarkably influencing the quality of tea. In this study, free AA and total AA in Apocynum venetum L. (Luobuma tea) were estimated by HPLC equipped with fluorescent detector using 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEEC) as a fluorescent labelling reagent. Different parameters for derivatization and separation were optimized. AA were rapidly derivatized within 3 min at room temperature with DBCEEC. In conjunction with a gradient elution, a baseline resolution of 20 analytes was achieved on a reversed-phase Hypersil BDS C18 column. LC separation for the derivatized AA showed good reproducibility. Twenty AA were detected and showed significant linear responses with correlation coefficients (>0.9992). This developed method offered the low detection limit of 2.88-23.4 fmol.

A sensitive fluorescence reagent, 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)-ethoxy]ethyl chloroformate, for amino acids determination in Saussurea involucrate and Artemisia capillaris Thunb using high-performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/electrospray ionization source.

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 coefficients > 0.9993. 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 highly sensitive and selective method for determination of phenoxy carboxylic acids from environmental water samples by dispersive solid-phase extraction coupled with ultra high performance liquid chromatography-tandem mass spectrometry.

A rapid and efficient method for extraction of 12 phenoxy carboxylic acids (PCAs) in environmental water samples was established based on metal-organic framework MIL-101 assisted dispersive solid phase extraction (DSPE). 12 PCAs were labeled by d0-10-methyl-2-(piperazin-1-ylsulfonyl)anthracen-9(10H)-one(d0-MASPz) and d3-10-methyl-2-(piperazin-1-ylsulfonyl)anthracen-9(10H)-one(d3-MASPz), allowing each analyte to have an isotope internal standard. A stable isotope-coded strategy for the detection of 12 PCAs was developed under optimized extraction conditions and UHPLC-MS/MS conditions. All PCAs analytes were in good linearity in the concentration range of 5-1000 ng/L, and the calibration function was verified by the Mandel fitting test with a 95% confidence level. The LODs and LOQs were estimated by the IUPAC's recommendations. The LODs ranged from 0.18 to 0.88 ng/L, and LOQs ranged from 0.59 to 2.90 ng/L. The intra-day and inter-day precision in three spiked levels (5, 50 and 100 ng/L) were in the range of (1.40 ±â€¯0.14) %- (2.76 ±â€¯0.12) % and (2.60 ±â€¯0.26) %- (3.83 ±â€¯0.32) %, respectively. The developed method has been successfully applied to the determination of PCAs in environmental water samples with recoveries ranging from 95.3% to 105.5%. All of the precision and recovery analyses were done in triplicate.

A colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA based on silver nanoclusters and unmodified gold nanoparticles.

Human telomere DNA plays a vital role in genome integrity control and carcinogenesis as an indication for extensive cell proliferation. Herein, silver nanoclusters (Ag NCs) templated by polymer and unmodified gold nanoparticles (Au NPs) are designed as a new colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA. Ag NCs can produce the aggregation of Au NPs, so the color of Au NPs changes to blue and the absorption peak moves to 700nm. While the telomere DNA can protect Au NPs from aggregation, the color turns to red again and the absorption band blue shift. Benefiting from the obvious color change, we can differentiate the length of telomere DNA by naked eyes. As the length of telomere DNA is longer, the variation of color becomes more noticeable. The detection limits of telomere DNA containing 10, 22, 40, 64 bases are estimated to be 1.41, 1.21, 0.23 and 0.22nM, respectively. On the other hand, when telomere DNA forms G-quadruplex in the presence of K+, or dsDNA with complementary sequence, both G-quadruplex and dsDNA can protect Au NPs better than the unfolded telomere DNA. Hence, a new colorimetric platform for monitoring structure conversion of DNA is established by Ag NCs-Au NPs system, and to prove this type of application, a selective K+ sensor is developed.

Sensitive determination of thiols in wine samples by a stable isotope-coded derivatization reagent d0/d4-acridone-10-ethyl-N-maleimide coupled with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis.

As the key aroma compounds, varietal thiols are the crucial odorants responsible for the flavor of wines. Quantitative analysis of thiols can provide crucial information for the aroma profiles of different wine styles. In this study, a rapid and sensitive method for the simultaneous determination of six thiols in wine using d0/d4-acridone-10-ethyl-N-maleimide (d0/d4-AENM) as stable isotope-coded derivatization reagent (SICD) by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. Quantification of thiols was performed by using d4-AENM labeled thiols as the internal standards (IS), followed by stable isotope dilution HPLC-ESI-MS/MS analysis. The AENM derivatization combined with multiple reactions monitoring (MRM) not only allowed trace analysis of thiols due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the fluctuation in MS/MS signal intensity due to instrument. The obtained internal standard calibration curves for six thiols were linear over the range of 25-10,000pmol/L (R2≥0.9961). Detection limits (LODs) for most of analytes were below 6.3pmol/L. The proposed method was successfully applied for the simultaneous determination of six kinds of thiols in wine samples with precisions ≤3.5% and recoveries ≥78.1%. In conclusion, the developed method is expected to be a promising tool for detection of trace thiols in wine and also in other complex matrix.

Sensitive and background-free determination of thiols from wastewater samples by MOF-5 extraction coupled with high-performance liquid chromatography with fluorescence detection using a novel fluorescence probe of carbazole-9-ethyl-2-maleimide.

A sensitive and background-free pre-column derivatization method for the determination of thiol compounds using metal-organic framework material (MOF-5) as dispersive solid-phase extraction (DSPE) adsorbent followed by high-performance liquid chromatography fluorescence detection (HPLC-FLD) has been developed. In this paper, a novel labeling reagent, carbazole-9-ethyl-2-maleimide(CAEM), was synthesized and reacted with thiols at 40°C for 10min in the presence of PBS buffer (0.02mol/L, pH 7.5). Interestingly, CAEM itself had no fluorescence, while its derivatives exhibited intense fluorescence with an excitation maximum at λex 274nm and an emission maximum at λem 363nm, which greatly reduced the background interference and improved the sensitivity of the method. Furthermore, the MOF-5 was prepared and used as DSPE adsorbent for the selective adsorption of thiols from wastewater sample. Under the optimized experimental conditions, an excellent linearity for all analytes over their concentration ranges of 0.01-1.0µmol/L (R2>0.9986)were obtained with the limit of detection (LOD) ranging from 8 to 17.1pmol/L for nine tested thiols. The feasibility of this method for the determination of thiols in wastewater samples had been evaluated and satisfactory average recoveries (n=3) were achieved with the range of 86.6-98.5%.

Accurate Determination of Amino Acids in Serum Samples by Liquid Chromatography-Tandem Mass Spectrometry Using a Stable Isotope Labeling Strategy.

An accurate and sensitive liquid chromatography-tandem mass spectrometry method was developed for the analysis of amino acids (isoleucine, leucine, valine, tyrosine, phenylalanine and tryptophan) in serum samples using a stable isotope labeling strategy. Amino acid samples and standards were, respectively, derivatized by 10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC to form isotopic pairs which co-eluted and were detected by an MS detector at the same time. Accurate internal standard-based quantification was thereby achieved without the use of any internal standard analogy. The labeling reaction of MASC with amino acids is fast, simple and robust. Besides, derivatization increased the molecular weight of amino acids, and therefore they were shifted out of the background noise which was often observed in low mass region. The instrument LODs were in the range of 1.0-2.5 nmol/L. Linearities calculated by comparing theoretical peak area ratios of d0-/d3-MASC derivatives with the experimental peak area ratios were excellent with correlation coefficients of >0.995. The proposed method was successfully applied to the analysis of amino acids in serum samples with high sensitivity and accuracy.

Rapid and sensitive ultrasonic-assisted derivatisation microextraction (UDME) technique for bitter taste-free amino acids (FAA) study by HPLC-FLD.

Amino acids, as the main contributors to taste, are usually found in relatively high levels in bitter foods. In this work, we focused on seeking a rapid, sensitive and simple method to determine FAA for large batches of micro-samples and to explore the relationship between FAA and bitterness. Overall condition optimisation indicated that the new UDME technique offered higher derivatisation yields and extraction efficiencies than traditional methods. Only 35min was needed in the whole operation process. Very low LLOQ (Lower limit of quantification: 0.21-5.43nmol/L) for FAA in twelve bitter foods was obtained, with which BTT (bitter taste thresholds) and CABT (content of FAA at BTT level) were newly determined. The ratio of CABT to BTT increased with decreasing of BTT. This work provided powerful potential for the high-throughput trace analysis of micro-sample and also a methodology to study the relationship between the chemical constituents and the taste.

Sensitive, accurate and rapid detection of trace aliphatic amines in environmental samples with ultrasonic-assisted derivatization microextraction using a new fluorescent reagent for high performance liquid chromatography.

A new fluorescent reagent, 1-(1H-imidazol-1-yl)-2-(2-phenyl-1H-phenanthro[9,10-d]imidazol-1-yl)ethanone (IPPIE), is synthesized, and a simple pretreatment based on ultrasonic-assisted derivatization microextraction (UDME) with IPPIE is proposed for the selective derivatization of 12 aliphatic amines (C1: methylamine-C12: dodecylamine) in complex matrix samples (irrigation water, river water, waste water, cultivated soil, riverbank soil and riverbed soil). Under the optimal experimental conditions (solvent: ACN-HCl, catalyst: none, molar ratio: 4.3, time: 8 min and temperature: 80°C), micro amount of sample (40 µL; 5mg) can be pretreated in only 10 min, with no preconcentration, evaporation or other additional manual operations required. The interfering substances (aromatic amines, aliphatic alcohols and phenols) get the derivatization yields of <5%, causing insignificant matrix effects (<4%). IPPIE-analyte derivatives are separated by high performance liquid chromatography (HPLC) and quantified by fluorescence detection (FD). The very low instrumental detection limits (IDL: 0.66-4.02 ng/L) and method detection limits (MDL: 0.04-0.33 ng/g; 5.96-45.61 ng/L) are achieved. Analytes are further identified from adjacent peaks by on-line ion trap mass spectrometry (MS), thereby avoiding additional operations for impurities. With this UDME-HPLC-FD-MS method, the accuracy (-0.73-2.12%), precision (intra-day: 0.87-3.39%; inter-day: 0.16-4.12%), recovery (97.01-104.10%) and sensitivity were significantly improved. Successful applications in environmental samples demonstrate the superiority of this method in the sensitive, accurate and rapid determination of trace aliphatic amines in micro amount of complex samples.

Development of ultrasonic-assisted closed in-syringe extraction and derivatization for the determination of labile abietic acid and dehydroabietic acid in cosmetics.

Two resin acids, abietic acid (AA) and dehydroabietic acid (DHAA), in cosmetics may cause allergy or toxicoderma, but remain inaccurately investigated due to their lability. In this work, an accurate, sensitive, efficient and convenient method, utilizing the ultrasonic-assisted closed in-syringe extraction and derivatization (UCSED) prior to high performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD) and on-line tandem mass spectra (MS/MS), has been developed. Analytes are extracted by acetonitrile (10/1, v/m) in a sealed syringe under safe condition (60°C; 15 min; nitrogen atmosphere) and then in-syringe derivatized by 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-d]imidazol-1-yl) ethyl-p-toluenesulfonate (ANITS) (8-fold, 93°C, 30 min, DMF as co-solvent, K2CO3 as catalyst). In UCSED, derivatization contributes to increase both analytical sensitivity and stability of analytes. Excellent linearity (r2≥0.9991) is achieved in wide range (75-3000 ng/mL (AA); 150-4500 ng/mL (DHAA)). Quite low detection limits (AA: 8.2-10.8 ng/mL; DHAA: 19.4-24.3 ng/mL) and limits of analyte concentration (LOAC) (AA: 30.0-44.5 ng/mL; DHAA: 70.9-86.7 ng/mL) ensure the trace analysis. This method is applied to the analysis of cosmetic samples, including depilatory wax strip, liquid foundation, mascara, eyeliner, eyebrow pencil and lip balm. No additional purification is required and no matrix effect is observed, demonstrating obvious advantages over conventional pretreatment such as solid phase extraction (SPE). Accuracy (RE: -3.2% to 2.51%), precision (RSD: 1.29-2.84%), recovery (95.20-103.63%; 95.51-104.22%) and repeatability (<0.23%; <2.87%) are significantly improved. Furthermore, this work plays a guiding role in developing a reasonable method for labile analytes.

Analysis of estrogenic compounds in environmental and biological samples by liquid chromatography-tandem mass spectrometry with stable isotope-coded ionization-enhancing reagent.

A sensitive and reliable stable isotope labeling technology was developed for the determination of estrogenic compounds in environmental and biological samples based on the derivatization of estrogenic compounds with 10-methyl-acridone-2-sulfonyl chloride (d(0)-MASC) and its deuterated counterpart d(3)-MASC. The labeling reaction of MASC with estrogenic compounds is simple and robust and can be carried out under mild conditions within 5 min. Internal standard-based quantification was achieved by this labeling strategy without the need of using expensive internal standard analogy to every analyte of interest. Meanwhile, the sensitivity obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was enhanced by 2-3 orders of magnitude compared to the underivatized counterparts. Application of the stable isotope labeling technology in relative and absolute quantification of estrogenic compounds in complicated samples indicated that the labeling strategy was effective in overcoming matrix effects. The proposed method was successfully applied to the analysis estrogenic compounds in different environmental and biological samples with high sensitivity and accuracy.

A sensitive and efficient method for simultaneous trace detection and identification of triterpene acids and its application to pharmacokinetic study.

A sensitive and efficient method for simultaneous trace detection of seven triterpene acids was developed and validated for analysis of rat plasma samples. The required micro-sampling of only 20 µL blood reduced the difficulty in blood collection and the injury to animal. The whole pretreatment procedure was more conveniently finished within 26 min through the application of the semi-automated derivatization extraction method to biological samples. Seven analytes were rapidly separated within 30 min on reversed-phase Akasil-C18 column and quantified by fluorescence detector. Online ion trap MS with atmospheric pressure chemical ionization (APCI) source was used for further identification. The novel application of artificial neural network (ANN) combined with genetic algorithm (GA) to optimization of derivatization was performed and compared with the classical response surface methodology (RSM). Optimal derivatization condition was validated by multi-criteria and nonparametric tests and used successfully to achieve the rather high sensitivity (limit of detection: 0.67-1.08 ng/mL). The limit of reactant concentration (LORC) special for derivatization was studied and the lower values (2.53-4.03 ng/mL) ensured the trace detection. Results of validation demonstrated the advantages for pharmacokinetic study, such as higher sensitivity, better accuracy, easier pretreatment and shorter run-time. Pharmacokinetic study of triterpene acids after oral administration of Salvia miltiorrhiza extract to mice was conducted for the first time. The present method provided more sensitive and efficient alternative for the medical detection of bioactive constituents from herbal extract in the biological liquid.

A sensitive and efficient method to systematically detect two biophenols in medicinal herb, herbal products and rat plasma based on thorough study of derivatization and its convenient application to pharmacokinetics with semi-automated device.

A sensitive and efficient method using a semi-automated pretreatment device, pre-column derivatization, multivariate optimization and high performance liquid chromatography with fluorescence and mass spectrometric detection was developed and validated for the systematic determination of two biophenols in four herb-related samples (medicinal herb; herbal products in tablet, capsule and oral liquid forms) and plasma samples after oral administration to rat. Only micro-sampling of 20 µL blood was needed for the analysis, and the pretreatment procedure including blood collection, derivatization by 10-ethyl-acridine-3-sulfonyl chloride (EASC) and injection to the sampling vials was efficiently finished in 10 min with no cumbersome and complicated operation. The novel application of artificial neural network (ANN) coupled with genetic algorithm (GA) to optimization of derivatization condition was executed and compared with the classical response surface methodology (RSM). The optimal condition for derivatization was validated by multi-criteria and nonparametric tests and used successfully to achieve the higher sensitivity (limit of detection: 0.6 and 0.8 ng/mL). The limit of reactant concentration (LORC) was put forward for derivatization method for the first time, and the lower values (2.0-2.7 ng/mL) provided the guarantee for the trace detection with the micro samples (<50 µL) required. The results of validation including selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect and stability demonstrated the advantages of this method. The pharmacokinetic study of major bioactive components salidroside and p-tyrosol in herb Rhodiola crenulata and its products was more conveniently performed in 25 min. The established method could be the sensitive and efficient alternative method for the systematic detection of bioactive components in series of drug carriers from raw herb to herbal products and to blood in medical research. And the approaches of the thorough study played the guiding role in seeking a novel analytical method.

Identification and determination of carboxylic acids in food samples using 2-(2-(anthracen-10-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)ethyl 4-methylbenzenesulfonate (APIETS) as labeling reagent by HPLC with FLD and APCI/MS.

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°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 λ(ex)/λ(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 13.37-30.26fmol (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 barbarum 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.

Development of a new HPLC method with precolumn fluorescent derivatization for rapid, selective and sensitive detection of triterpenic acids in fruits.

Triterpenic acids are widespread in plants and have multiplicity of biological properties. Unfortunately the method for accurate analysis of these compounds remains poorly investigated. This study proposed a highly sensitive and selective precolumn derivatization method for accurate determination of five triterpenic acids (betulinic acid, betulonic acid, maslinic acid, ursolic acid and oleanolic acid) in fruits using acridone-9-ethyl-p-toluenesulfonate (AETS) as fluorescent labeling reagent by HPLC with fluorescence detection (FLD). Response surface methodology was employed to optimize the derivatization reaction, ensuring the sufficient labeling of the analyzed components. The rapid separation of five triterpenic acids could be achieved in as little as 16 min. This developed method offered the exciting detection limits of 1.68-2.04 ng/mL. When applied to several popular fruits in China, it revealed satisfactory applicability and reproducibility. This developed method also exhibits powerful potential for accurate detection of triterpenic acids from other foodstuffs and nature products.