Cuihua Song

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.

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.

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.

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.

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.

An improved reagent for determination of aliphatic amines with fluorescence and online atmospheric chemical ionization－mass spectrometry identification

An improved reagent named 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC-Cl) for the determination of aliphatic amines by high-performance liquid chromatography (HPLC) with fluorescence detection and post-column online atmospheric chemical ionization-mass spectrometry (APCI-MS) identification has been developed. DBCEC-Cl could easily and quickly label aliphatic amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H](+) under APCI-MS in positive-ion mode. The ratios for fluorescence responses were I(DBCEC-amine)/I(BCEC-amine)=1.02-1.60; I(DBCEC-amine)/I(BCEOC-amine)=1.30-2.57; and I(DBCEC-amine)/I(FMOC-amine)=2.20-4.12 (here, I was relative fluorescence intensity). The ratios for MS responses were IC(DBCEC-amine)/IC(BCEC-amine)=4.16-29.31 and IC(DBCEC-amine)/IC(BCEOC-amine)=1.23-2.47 (Here, IC: APCI-MS ion current intensity). Detection limits calculated from 0.0244 pmol injection, at a signal-to-noise ratio of 3, were 0.3-3.0 fmol. The relative standard deviations for within-day determination (n=6) were 0.045-0.081% for retention time and 0.86-1.03% for peak area for the tested aliphatic amines. The mean intra- and inter-assay precision for all amine levels were <3.64% and 4.67%, respectively. The mean recoveries ranged from 96.9% to 104.7% with their standard deviations in the range of 1.80-2.70 (RSDs%). Excellent linear responses were observed with coefficients of >0.9991.

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.

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.

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.

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.