A novel method has been established for the rapid separation and determination of free fatty acids from 37 different varieties of raspberry. In this study, a new fluorescent labeling reagent for fatty acids, 2-(4-amino)-phenyl-1-hydrogen-phenanthrene [9, 10-d] imidazole (PIA), has been synthesized and successfully applied to the high-performance liquid chromatography (HPLC) determination of fatty acids in raspberry. The novel method has been optimized by HPLC with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS/MS). The 22 main fatty acids (FAs) present in raspberry were derivatized by PIA and separated on a reversed-phase Hypersil GOLD column with gradient elution. The main experimental parameters affecting extraction efficiency and derivatization yield were investigated and optimized by response surface methodology (RSM) combined with Box-Behnken design (BBD). Under the optimum conditions, the method was successfully applied for the analysis of 22 fatty acids in 37 different varieties of raspberry. Good linear correlations were observed for all fatty acids with correlation coefficients of > 0.9978. Limits of detection and quantification (LOD and LOQ) were in the range of 0.12 to 0.49 ng/mL and 1.07 to 2.81 ng/mL, respectively. Furthermore, the results indicated that the raspberries were rich in fatty acids, but the contents of the fatty acids varied among the different varieties.
A rapid, sensitive, and selective precolumn derivatization method for the simultaneous determination of eight thiophenols using 3-(2-bromoacetamido)-<i>N</i>-(9-ethyl-9<i>H</i>)-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 <i>m/z</i> 251.3, 223.3, 210.9, 195.8, and 181.9. At the same time, derivatives exhibited intense fluorescence with an excitation maximum at λ<sub>ex</sub> = 276 nm and an emission maximum at λ<sub>em</sub> = 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 (<i>n</i> = 3) were in the range of 87.21-101.12%.
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.
<br>Display Omitted<br>• 4′-Carbonyl chloride rosamine was synthesized and used for NTs by UHPLC-MS/MS. • <b>In situ</b> UA-DDLLME was reported for the simultaneous determination of AANTs and MANTs. • The method was sensitive, selective, low matrix effect, speedy and eco-friendly. • A new analytical tool in diagnosis of AD-related disease.<br>Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as Alzheimer’s disease (AD). In order to investigate the potential links, a new simple, fast, accurate and sensitive analytical method, based on <b>in situ</b> ultrasound-assisted derivatization dispersive liquid-liquid microextraction (<b>in situ</b> UA-DDLLME) coupled with ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), has been developed and validated. The quantitation of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD rats were performed in this work. The <b>in situ</b> UA-DDLLME procedure involved the rapid injection of the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing the new designed and synthesized 4′-carbonyl chloride rosamine (CCR) as derivatization reagent, into the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and microextraction of analytes were simultaneously completed within 1 min. Good linearity for each analyte (R > 0.992) was observed with low limit of detections (LODs, S/N > 3). Moreover, the proposed method was compared with direct detection or other reported methods, and the results showed that low matrix effects and good recoveries results were obtained in this work. Taken together, <b>in situ</b> UA-DDLLME coupled with UHPLC-MS/MS analysis was demonstrated to be a good method for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be expected to be highly useful in AD diseases’ clinical diagnostics and may have potential value in monitoring the efficacy of treatment.
A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M + H]+ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O]+ for pentose, hexose, and glyceraldehydes, and at m/z [MH-CmH2m-1Om+1-H2O]+ for alduronic acids, such as galacturonic acid and glucuronic acid (m = n - 2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.
Background: Swertia chirayita, has been commonly used under the name "Zang-yin-chen" for the treatment of liver infections, inflammation, abdominal pain, and bacterial infection in traditional Tibetan medicine. However, the bioactive components with anti-inflammatory activities and underlying mechanisms remain poorly evaluated.Study Design/methods: Repeated column chromatography yielded two main xanthones from petroleum ether (PE) and ethyl acetate fractions of whole plants of S. chirayita, and their structures were determined as bellidifolin (1) and swerchirin (2) on the basis of spectroscopic data and literature analysis. The anti-inflammatory activities and mechanisms of anti-inflammation of these two isolated xanthones were determined via enzyme-linked immunosorbent assay (ELISA) and western blot in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages in vitro.Results: Anti-inflammation assay demonstrated that 1 and 2 inhibit the production of the pro-inflammatory cytokines interleukin-6 (IL-6) and TNF-α in LPS-stimulated RAW 264.7 macrophages. Xanthone 1 also potently inhibited the production of prostaglandin E2 (PGE2) by suppressing the protein expression of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. Western blot showed that the phosphorylation of c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs were remarkably attenuated by 1 in a concentration-dependent manner. Particularly, Compound 1 suppressed the phosphorylation of the inhibitor κB kinase-β (IKK-β), Akt, and p65 subunit of nuclear factor-kappaB (NF-κB).Conclusion: The potent suppressive effects of 1 from S. chirayita on inflammatory mediators by blocking the expression of COX-2 and phosphorylation of Akt, IKK-β, MAPK and NF-κB, activation in LPS-stimulated macrophages suggest that 1 can be a preventive therapeutic candidate for the management of inflammatory-mediated immune disorders.
This study presents an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid-liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe-emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high-speed counter-current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe-emodin, physcione, and chrysophanol.
In this paper, an efficient method was successfully established by the combination of macroporous resin (MR) and high-speed counter-current chromatography (HSCCC) for rapid enrichment and separation of aloe-emodin 8-O-β-D-glucoside, emodin 1-O-β-D-glucoside, emodin 8-O-β-D-glucoside and piceatannol 4'-O-β-D-(6″-O-gallate)-glucoside. Six kinds of macroporous resins were investigated in the first step and X-5 macroporous resin was selected for the enrichment of the target compounds. The recoveries of the target compounds reached 89.0, 85.9, 82.3 and 84.9% respectively after 40% ethanol elution. In the second step, the target compounds were separated by HSCCC with a two-phase solvent system composed of chloroform/ethyl acetate/methanol/water (8:1:6:5, v/v). The established method will be helpful for further characterization and utilization of Rheum tanguticum. The results demonstrate that MR coupled with HSCCC is a powerful technique for separation of bioactive compounds from natural products.
The present work is to study the chemical constituents from petroleum ether fraction of Tibetan medicine Swertia chirayita by column chromatography and recrystallization. The structures were identified by physical and chemical properties and spectral data as swerchirin (1), decussatin (2), 1,8-dihydroxy-3,5,7-trimethoxyxanthone (3), 1-hydroxy-3,5,7,8-tetramethoxyxanthone (4), bellidifolin (5), 1-hydroxy-3, 7-dimethoxyxanthone (6), methylswertianin (7), 1-hydroxy-3,5-dimethoxyxanthone (8), erythrodiol (9), oleanolic acid (10), gnetiolactone (11), scopoletin (12), sinapaldehyde (13), syringaldehyde (14), and β-sitosterol (15). Compounds 3, 4, 9, 11-14 were isolated from S. chirayita for the first time. Compounds 9 and 12 were firstly isolated from the genus Swertia. The cytotoxic activities of compounds 1, 2, 5, 7 and 8 against human pancreatic cancer cell lines SW1990 and BxPC-3,and the protective effects of these compounds against hydrogen peroxide (H2O2)-induced oxidative stress in human endothelium-derived EA.hy926 were investigated in vitro. The results showed no obvious effect at the high concentration of 50 μmol•L⁻¹.
The comparative study of bloodletting therapy between traditional Chinese medicine and Tibetan medicine in view of history development, theoretic basis, bloodletting location, bloodletting tool, operation method, bloodletting amount, indications, contraindications and the others are conducted in this paper. It is pointed out that the bloodletting therapy could be better carried forward and developed through the interaction and integration of bloodletting therapy between traditional Chinese medicine and Tibetan medicine in term of the theoretic, practical and development patterns under the guidance of these two different medical theoretical systems.
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 <i>Potentilla anserina</i> L<i>.</i> 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 <i>Potentilla anserina</i> L<i>.</i> Quantitative recoveries of the AAs from the <i>Potentilla anserina</i> L<i>.</i> 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 <i>Potentilla anserina</i> L. Good compositional data were obtained from the analysis of the AAs obtained from <i>Potentilla anserina</i> L<i>.</i> root.
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.
A new fluorescent labeling reagent, benzimidazo[2,1-<i>b</i>]quinazolin-12(6<i>H</i>)-one-5-ethyl-<i>p</i>-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 K<sub>2</sub>CO<sub>3</sub> 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<sup>−1</sup> 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 <i>Coriandrum sativum</i> L. from different habitats in China.
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-(<i>p</i>-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-<i>f</i>]-9,10-phenanthrene (TSPP) in <i>N</i>,<i>N</i>-dimethylformamide (DMF) at 90 °C with anhydrous K<sub>2</sub>CO<sub>3</sub> as catalyst. A mixture of C<sub>1</sub>-C<sub>30</sub> fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C<sub>8</sub> 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 <i>Gentiana straminea</i> and <i>G. dahurica</i> 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, <i>n</i> = 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.
Plant hormone determination in food matrices has attracted more and more attention because of their potential risks to human health. However, analytical methods for the analysis of multiple plant hormones remain poorly investigated. In the present study, a convenient, selective, and ultrasensitive high-performance liquid chromatography method for the simultaneous determination of multiple classes of plant hormones has been developed successfully using dispersive liquid-liquid microextraction followed by precolumn fluorescent labeling. Eight plant hormones in fruits including jasmonic acid, 12-oxo-phytodienoic acid, indole-3-acetic acid, 3-indolybutyric acid, 3-indolepropionic acid, gibberellin A₃, 1-naphthylacetic acid, and 2-naphthaleneacetic acid were analyzed by this method. The conditions employed for dispersive liquid-liquid microextraction were optimized systematically. The linearity for all plant hormones was found to be >0.9993 (<i>R</i>² values). This method offered low detection limits of 0.19-0.44 ng/mL (at a signal-to-noise ratio of 3), and method accuracies were in the range of 92.32-103.10%. The proposed method was applied to determine plant hormones in five kinds of food samples, and this method can achieve a short analysis time, low threshold levels of detection, and a high specificity for the analysis of targeted plant hormones present at trace level concentrations in complex matrices.
Abstract This presented study describes a method based on high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) using N-(2-iodoacetyl)-1-pyrenemethylamine (NIPA) as a novel fluorescence labeling reagent for the determination of thyreostats in bovine milk. Five thyreostats, belonging to the group of imidazole and thiouracil, were investigated in this work: tapazole (TAP), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracial (PhTU). Thyreostats were specifically purified by a silver ion solid phase extraction (Ag-SPE) cartridge and then labeled using NIPA. The labeled derivatives showed excellent fluorescence property with maximum excitation and emission wavelengths of 330 nm and 375 nm, respectively. The labeled derivatives were separated on a reversed-phase Eclipse SB-C18 column within 12 min. Excellent linearity (R2 > 0.995) of all thyreostats was achieved with the limits of detection (LODs) and the limits of quantitation (LOQs) in the low micrograms per liter range of 0.21–0.30 μg/L and 0.70–1.00 μg/L, respectively. Satisfactory recoveries in the range of 93.5–98.0% were obtained for all thyreostats. The developed method has been successfully applied to analyze thyreostats in bovine milk with good applicability. Thirty bovine milk samples have been investigated, and varying levels of thiouracil were detected in thirteen of these samples. The highest level in the raw milk reached a value of 4.5 μg/L. To our best knowledge, this study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis. Highlights • A pre-column derivatization HPLC-FLD method was developed for the determination of thyreostats in milk samples. • LOD was in the low micrograms per liter range of 0.21–0.30 μg·L−1. • The proposed method was successfully applied to the determination of thyreostats in milk sample. • This study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis.
Chemical isotope labelling in combination with high-performance liquid chromatography-tandem mass spectrometry (CIL-HPLC-MS/MS) is a powerful method for quantitative profiling of targeted molecules. In the current work, we successfully developed a novel CIL-HPLC-MS/MS method for quantitative profiling of residual organophosphorus thioester pesticides (OPTPs) in agricultural products through the determination of the cleavage products of thiol (CP-thiol) compounds. In this method, we synthesized a novel pair of CIL reagents, i.e., N-(4-(carbazole-9-yl)-phenyl)-N-maleimide (NCPM-d0) and its deuterated analogue NCPM-d2, both of which contain a maleimide moiety as the reactive group and an isotope tag to sensitively label CP-thiol compounds. NCPM-d0 was used to label CP-thiol compounds cleaved from OPTPs in the investigated agricultural product samples, and NCPM-d2 was used to label CP-thiol compounds cleaved from OPTPs in the standard substance-spiked organic agricultural product samples. The heavily labelled derivatives were used as the internal standards (ISs) to compensate for the matrix effects during MS analysis. The NCPM-d0- and NCPM-d2-labelled derivatives generated two characteristic product ions (PIs) at m/z 372.5 and 374.5 under collision induced dissociation, respectively, which are used to establish the multiple reaction monitoring (MRM) mode-based detection. The precursor ions of NCPM-d0 and NCPM-d2 labelled derivatives of CP-thiol compounds were deduced according to the structures of the OPTPs. The peak pairs with a fixed mass difference and similar retention times were assigned as potential CP-thiol candidates for the identification of the corresponding OPTPs. Using the proposed method, we successfully determined seven residual OPTPs in agricultural product samples. Taken together, the presented method was demonstrated to be a promising new technique in the quantitation of OPTPs in agricultural product samples with high reliability.
A pair of stable isotope labeling (SIL) reagents, <b>N</b>-(4-(carbazole-9-yl)-phenyl)-<b>N</b>-maleimide (NCPM-d0) and its heavy analogue NCPM-d2, were used for labeling thiol-containing drugs. On basis of SIL, a global isotope internal standard quantitative method for the detection of five thiol-containing drugs by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The NCPM-d0 and NCPM-d2 can easily label thiol-containing drugs under mild conditions within 10 min at 40 °C. The NCPM-d0 and NCPM-d2 labeled thiol-containing drugs can generate two characteristic product ions (<b>m</b>/<b>z</b> at 372.5 and 374.5) under collision induced dissociation, respectively, which is used to establish the multiple reaction monitoring (MRM) based detection. The NCPM labeling combined with MRM analysis not only allowed trace detection of thiol-containing drugs due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the instrument fluctuation in the MS/MS signal intensity. The detection sensitivities of thiol-containing drugs improved by 14.5-650.5-fold due to NCPM-labeling, while the matrix and ion suppression effects were markedly minimized by the SIL strategy. The limits of detection (LODs) and the limits of quantitation (LOQs) were in the range 10.0-15.0 ng·mL−1 and 31.0-50.0 ng·mL−1, respectively. The proposed method was used for the simultaneous determination of five thiol-containing drugs in plasma samples with satisfactory recoveries in the range of 95.0-97.5%.<br>• A stable isotope labeling strategy for analyzing thiol-containing drugs has been developed. • A pair of SIL reagents NCPM-d0 and NCPM-d2 were used to label thiol-containing drugs. • The detection sensitivities of thiol-containing drugs improved by 14.5-650.5-fold. • The proposed method was successfully applied to pharmacokinetic study of captopril.
A simple, sensitive, and selective high-performance liquid chromatography (HPLC) method using 9-(2-iodoethyl)acridone (IEA) as a novel fluorescence derivatizing agent for the simultaneous determination of six thiophenols has been developed. An efficient Pb<sup>2+</sup>-modified OASIS-MCX cartridge was used and could get good recoveries. IEA was successfully used to label thiophenols with high sensitivity and excellent selectivity. The effects of different solvents, pH, and surfactants on fluorescence properties of derivatives were investigated. To obtain the best labeling efficiency, derivatizing parameters including pH value, temperature, and concentration of IEA, as well as types of catalysts were also evaluated in detail. Under the optimal conditions, the separation could be achieved within 12 min with limits of detection (LODs) in the range of 0.6-5.8 μg L<sup>−1</sup> and relative standard deviations (RSDs) < 3.9 %. This is the first time that IEA was applied to the analysis of thiophenols, and the established method has been successfully applied to the trace level detection of thiophenols in industrial wastewater samples.
In this work, we have developed an efficient method for the rapid extraction and separation of triterpene acids from 37 different varieties of raspberry via ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME). The triterpene acids were then determined by high-performance liquid chromatography (HPLC) with fluorescence detection using benzimidazo-[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate (BQETS) as the labeling agent. Five triterpene acids, including asiatic acid (AA), maslinic acid (MA), corosolic acid (CA), oleanolic acid (OA) and betulinic acid (BA), were extracted by UA-DLLME using chloroform and acetone as the extracting and dispersing solvents, respectively. After extraction and nitrogen flushing, the extracts were simultaneously characterized by HPLC based on pre-column derivatization using BQETS, a new labeling agent synthesized in our laboratory. Several key parameters affecting the extraction efficiency and derivatization yields were investigated and optimized by response surface methodology (RSM) combined with Box-Behnken design (BBD). The method was further validated for linearity (correlation coefficient <i>R</i> <sup>2</sup> > 0.9979), precision (RSD = 0.23-2.45 %), and recovery (RSD = 90-106.5 %). The limits of detection (LODs) and the limits of quantification (LOQs) were determined to be within the range of 1.83-7.69 µg/L and 6.06-25.47 µg/L, respectively. This is the first report of the use of BQETS as a pre-column derivatization agent for the determination of triterpene acids in real samples. The proposed method has been applied to the determination of five triterpene acids in 37 different raspberry varieties with significantly increased sensitivity compared to other methods. The results obtained indicate that the contents of triterpene acids vary significantly across different raspberry varieties.
A novel and interesting pre-column derivatisation method was developed for the analysis of triterpenic acids by high-performance liquid chromatography (HPLC) with fluorescence detection. Each triterpenic acid produced two HPLC peaks with similar peak areas after derivatising with chiral 1-(9H-carbazol-9-yl) propan-2-yl-methanesulfonate (CPMS), while the fatty acid derivative of CPMS had only one peak. This phenomenon greatly increased the confidence in analyte confirmation. Compound with only one peak or two peaks differing greatly in their peak areas could be excluded from the target compound list. CPMS was compared with five other derivatising reagents, four of which produced only one peak for one triterpenic acid, to study the possible mechanism. Analytes with different behaviours were also studied to better interpret the mechanism. The proposed method also showed the merits of high sensitivity and less sample consumption. It was successfully applied to the analysis of triterpenic acids in fruit peels and flesh. There is no prior report on the two peak phenomenon of triterpenic acids. The information provided in this study will be helpful for those who are also engaged in derivatisation study.