Cellulose tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase for the enantioseparation of drugs in supercritical fluid chromatography: comparison with HPLC

A cellulose tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase was studied as a tool for the enantioselective separation of 21 selected analytes with different pharmaceutical and physicochemical properties. The enantioseparations were performed using supercritical fluid chromatography. The effect of the mobile phase composition was studied. Four different additives (diethylamine, triethylamine, isopropylamine, and trifluoroacetic acid) and isopropylamine combined with trifluoroacetic acid were tested and their influence on enantioseparation was compared. The influence of two different mobile phase co‐solvents (methanol and propan‐2‐ol) combined with all the additives was also evaluated. The best mobile phase compositions for the separation of the majority of enantiomers were CO₂/methanol/isopropylamine 80:20:0.1 v/v/v or CO₂/propan‐2‐ol/isopropylamine/trifluoroacetic acid 80:20:0.05:0.05 v/v/v/v. The best results were obtained from the group of basic β‐blockers. A high‐performance liquid chromatography separation system composed of the same stationary phase and mobile phase of similar properties prepared as a mixture of hexane/propan‐2‐ol/additive 80:20:0.1 v/v/v was considered for comparison. Supercritical fluid chromatography was found to yield better results, i.e. better enantioresolution for shorter analysis times than high‐performance liquid chromatography. However, examples of enantiomers better resolved under the optimized conditions in high‐performance liquid chromatography were also found.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

Qualitative and quantitative analysis of cinobufacini injection using rapid separation liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and HPLC‐photodiode array detection,a feasible strategy for the quality control of Chinese medicine injections

Cinobufacini injection, prepared from the skin of Bufo bufo gargarizans Cantor, has presented its significant effects on the treatment of hepatitis and various cancers in the clinic. However, as an unclear complex chemical system, the optimization of its quality control markers has been a long‐term challenge. In present study, a feasible strategy integrated markers screening, determination, and statistical analysis was efficiently proposed, especially for the undefined Chinese medicine injections. First, rapid separation LC‐quadrupole‐TOF‐MS method was applied in the identification of 19 major compounds in the cinobufacini injection for the first time. Further, nine high‐level contents active compounds were selected as quality control markers for the quantification analysis. An acceptable and validated determination method was established in 17 batches of cinobufacini injection by HPLC‐photodiode array detection method, including linear regression relationship (r², 0.9996–1), precisions (RSD, 0.02–1.35%), repeatability (RSD, 0.05–1.97%), stability (RSD, 0.1–3.85%), and recovery (95.88–104.89%). Each analyte was detected at its maximum ultraviolet spectra wavelength. Finally, based on the quantification results, principal component analysis was performed on the quality assessment of cinobufacini injections. This three‐step strategy provides a newly feasible solution for the quality control of Chinese medicine injections.     

Analysis of phenolic acids and flavonoids in leaves of Lycium barbarum from different habitats by ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

The leaves of Lycium barbarum (LLB) have been utilized as crude drugs and functional tea for human health in China and Southeast Asia for thousands of years. To control its quality, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method was established and validated for the first time for simultaneous determination of 10 phenolic acids and flavonoids (including neochlorogenic acid , protocatechuic aldehyde, p‐hydroxybenzoic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, p‐coumaric acid, ferulic acid, rutin and kaempferol‐3‐O‐rutinoside) in LLB. The separation was performed on an Acquity UPLC C₁₈ chromatographic column (100 × 2.1 mm internal diameter, 1.7 μm particle size) with 0.1% formic acid in water (A)–acetonitrile (B) as the mobile phase under gradient elution. Multiple reaction monitoring mode was adopted to simultaneously monitor the target components. The developed method was fully validated in terms of linearity (r² ≥ 0.9860), precision (RSD ≤ 6.58%), repeatability (RSD ≤ 6.60%), stability (RSD ≤ 6.17%), recovery (95.56–108.06%, RSD ≤ 4.64%) and limit of detection (0.021–0.664 ng/mL) and limit of quantitation (0.069–2.210 ng/mL), and then successfully applied to evaluate the quality of 64 batches of LLB collected from 41 producing areas in four different provinces of China. The results showed that the LLB, especially collected from Inner Mongolia regions, were rich in the phenolic acids and flavonoids. Rutin, kaempferol‐3‐O‐rutinoside and chlorogenic acid are the predominant compounds contained in LLB. The above findings will provide helpful information for the effective utilization of LLB.     

Liquid phase microextraction‐ion exchange‐high performance thin layer chromatography for the preconcentration,separation, and determination of plasticizers in aqueous samples

Liquid phase microextraction combined with ion‐exchange‐high performance thin layer chromatography has been developed for analysis of four plasticizers in aqueous samples. After their preconcentration by liquid phase microextraction, fast separation on thin layers of inorganic ion‐exchanger stannic silicate has been developed using a mixture of toluene + ethyl acetate (10:1, v/v) as mobile phase. Consequently, densitometric quantitative determination of the plasticizers has been made at λ = 280 nm in reflection–absorption mode by Camag TLC scanner‐3. The effects of type and volume of extraction solvent, stirring rate, extraction time, and ionic strength in the microextraction method have been also evaluated and optimized. The results show that the proposed method provides enhanced accuracy, linear range, LOD, and LOQ, and is very effective for analyzing the target compounds in water samples. Under the optimized conditions, preconcentration factor of 149–279 and extraction efficiency of 31–59% have been obtained. Repeatability (5.67–7.26%) and intermediate precision (6.21–8.17%) were in acceptable range. The relative recovery obtained for each analyte in different water samples was higher than 82.3% at three fortification levels with RSD <7.9%.     

Simultaneous determination of wogonin,oroxylin a,schisandrin, paeoniflorin and emodin in rat serum by HPLC–MS/MS and application to pharmacokinetic studies

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti‐inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high‐performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). The separation was performed on a Symmetry C₁₈ column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple‐reaction monitoring with electrospray ionization source in positive–negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu‐gan‐kan‐kang‐yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.     

Development and validation of a UPLC–MS method for the determination of galantamine in guinea pig plasma and its application to a pre‐clinical bioavailability study of novel galantamine formulations

To evaluate the bioavailability and pharmacokinetic profiles of two novel galantamine formulations as medical countermeasure products, an ultra‐performance liquid chromatography–single quadrupole mass spectrometry (UPLC–MS) method was developed and validated for quantifying galantamine in guinea pig plasma using solid‐phase extraction with a mixed mode strong cation exchange reversed‐phase cartridge. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column maintained at 40°C. The mobile phases were solution A, acetonitrile–water, 5:95 (v/v) and solution B, acetonitrile–water 90:10 (v/v), both containing 2 mM ammonium formate and 0.2% formic acid. The mobile phase was delivered utilizing a 3 min gradient program start with 95%A–5%B at a flow rate of 0.6 mL/min. The analyte and internal standard, galantamine‐d3, were detected by selected ion monitoring mode on a Waters 3100 single quadrupole mass spectrometer with positive electrospray ionization. The method was validated according to the US Food and Drug Administration bioanalytical guidance. The method was selective and was linear over the analytical range of 2–2000 ng/mL. Accuracy and precision were acceptable with intra‐ and inter‐day accuracies between 96.8 and 101% and precisions (RSD) <4.88%. The method was successfully implemented to measure galantamine plasma levels in a series of pre‐clinical bioavailability studies for the evaluation of novel galantamine formulations.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

Rapid separation of cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside from crude mulberry extract using high‐performance countercurrent chromatography and establishment of a volumetric scale‐up process

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside, using high‐performance countercurrent chromatography, and the establishment of a volumetric scale‐up process from semi‐preparative to preparative‐scale. To optimize the separation parameters, biphasic solvent systems composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi‐preparative‐scale high‐performance countercurrent chromatography. The optimized semi‐preparative‐scale high‐performance countercurrent chromatography parameters (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200–1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative‐scale (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0–10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside. The separation of mulberry anthocyanins using semi‐preparative high‐performance countercurrent chromatography and its volumetric scale‐up to preparative‐scale was addressed for the first time in this report.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Determination of pethidine in human plasma by LC–MS/MS

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of pethidine in human plasma was developed and validated over the concentration range of 4–2000 ng/mL. After addition of ketamine as internal standard, liquid–liquid extraction was used to produce a protein‐free extract. Chromatographic separation was achieved on a 100 × 2.1 mm, 5 µm particle, Allure^TM PFP propyl column, with 45:40:15 (v/v/v) acetonitrile–methanol–water containing 0.2% formic acid as mobile phase. The MS data acquisition was accomplished by multiple reactions monitoring mode with positive electrospray ionization interface. The lower limit of quantification was 4 ng/mL; for inter‐day and intra‐day tests, the precision (RSD) for the entire validation was less than 7%, and the accuracy was within 95.9–106.5%. The method is sensitive and simple, and was successfully applied to analysis of samples of clinical intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of 4‐hydroxyphenyl lactic acid, 4‐hydroxyphenyl acetic acid,and 3,4‐hydroxyphenyl propionic acid in human urine by ultra‐high performance liquid chromatography with fluorescence detection

A simple and reliable method was established for simultaneous determination of 4‐hydroxyphenyl acetic acid, 4‐hydroxyphenyl lactic acid, and 3,4‐hydroxyphenyl propionic acid in human urine by high‐performance liquid chromatography with fluorescence detection. Solid‐phase extraction was used to eliminate the interferences in urine. The separation of three analytes was achieved using a C₁₈ column and a mobile phase formed by a 95:5 v/v mixture of 50 mmol/L ammonium acetate buffer at pH 6.8 that contained 5 mmol/L tetrabutyl ammonium bromide and acetonitrile. Under the optimized conditions, the detection limits of 4‐hydroxyphenyl acetic acid, 4‐hydroxyphenyl lactic acid, and 3,4‐hydroxyphenyl propionic acid were 4.8 × 10⁻³, 8.80 × 10⁻³, and 9.00 × 10⁻³ mg/L, respectively, and the recoveries were in the range of 85.0–120.0% with relative standard deviations of 1.5–3.1%. This method was used to analyze urine samples from breast cancer patients, healthy people and post‐surgery breast cancer patients. Significant differences in urinary levels of 4‐hydroxyphenyl acetic acid and 4‐hydroxyphenyl lactic acid could be found between the breast cancer patients group and other two groups. No effect of age and sex was observed on the urinary levels of 4‐hydroxyphenyl acetic acid and 4‐hydroxyphenyl lactic acid. This method might be helpful for cancer biomarkers discovery in urine.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Liquid chromatographic resolution of racemic rasagiline and its analogues on a chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A liquid chromatographic chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was applied to the resolution of 15 analytes, including racemic rasagiline, a chiral drug for the treatment of Parkinson's disease, and its analogues. The composition of mobile phase was optimized to be ethanol/acetonitrile/acetic acid/triethylamine (80:20:0.2:0.3, v/v/v/v) by evaluating the chromatographic results for the resolution of five selected analytes under various mobile phase conditions. Under the optimized mobile phase conditions, racemic rasagiline was resolved quite well with a separation factor of 1.48 and resolution of 2.71 and its 14 analogues were also resolved reasonably well with separation factors of 1.06–1.54 and resolutions of 0.54–2.11. Among 15 analytes, racemic rasagiline was resolved best except for just one analyte. The analyte structure–enantioselectivity relationship indicated that racemic rasagiline has the most appropriate structural characteristics for resolution on the chiral stationary phase.     

Simultaneous determination of ginsenoside Rb1, ginsenoside Rg1, paeoniflorin,albiflorin and oxypaeoniflorin in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study of wen‐Yang‐Huo‐Xue soft capsule

A simple and sensitive HPLC–MS/MS method was developed and fully validated for simultaneous determination of ginsenoside Rb1, ginsenoside Rg1, paeoniflorin, albiflorin and oxypaeoniflorin in rat plasma. Plasma samples were pretreated with protein precipitation using acetonitrile. The chromatographic separation was carried out on a C₁₈ column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). All analytes and digoxin (internal stand, IS) were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. All calibration curves exhibited good linearity (r > 0.9960) over a wide concentration range for all components. The intra‐day and inter‐day precisions (RSD) at three different levels were all <12.0% and the accuracies (RE) ranging from −6.1 to 6.2%. The extraction recoveries of the five compounds ranged from 89.2 to 97.1%. The validated method was successfully applied in a comparative pharmacokinetic study of Wen‐Yang‐Huo‐Xue soft capsule (WYHXSC) in rats. Compared with single pure component, the exposure of the investigated components, except for oxypaeoniflorin, increased after oral administration of WYHXSC in rats, which suggested a synergistic effects between the herbs in the WYHXSC preparations.     

UHPLC–MS/MS quantification combined with chemometrics for the comparative analysis of different batches of raw and wine‐processed Dipsacus asper

A rapid and sensitive ultra‐high performance liquid chromatography with tandem mass spectrometry approach was established for the simultaneous determination of 4‐caffeoylquinic acid, loganic acid, chlorogenic acid, loganin, 3,5‐dicaffeoylquinic acid, dipsacoside B, asperosaponin VI, and sweroside in raw and wine‐processed Dipsacus asper . Chloramphenicol and glycyrrhetinic acid were employed as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Intra‐ and interassay variability for all analytes were 2.8–4.9 and 1.7–4.8%, respectively. The standard addition method determined recovery rates for each analytes (96.8–104.6%). In addition, the developed approach was applied to 20 batches of raw and wine‐processed samples of Dipsacus asper . Principle component analysis and partial least squares‐discriminate analysis revealed a clear separation between the raw group and wine‐processed group. After wine‐processing, the contents of loganic acid, chlorogenic acid, dipsacoside B, and asperosaponin VI were upregulated, while the contents of 3,5‐dicaffeoylquinic acid, 4‐caffeoylquinic acid, loganin, and sweroside were downregulated. Our results demonstrated that ultra‐high performance liquid chromatography with tandem mass spectrometry quantification combined with chemometrics is a viable method for quality evaluation of the raw Dipsacus asper and its wine‐processed products.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

Simultaneous determination of 11 fluorescent whitening agents in food‐contact paper and board by ion‐pairing high‐performance liquid chromatography with fluorescence detection

4,4′‐Diaminostilbene‐2,2′‐disulfonic acid based fluorescent whitening agents (DSD‐FWAs) are prohibited in food‐contact paper and board in many countries. In this work, a reliable high‐performance liquid chromatography method was developed for the simultaneous determination of 11 common DSD‐FWAs in paper material. Sample preparation and extraction as well as chromatographic separation of multicomponent DSD‐FWAs were successfully optimized. DSD‐FWAs in prepared samples were ultrasonically extracted with acetonitrile/water/triethylamine (40:60:1, v/v/v), separated on the C₁₈ column with the mobile phase containing tetrabutylammonium bromide, and then detected by a fluorescence detector. The limits of detection were 0.12–0.24 mg/kg, and the calibration curves showed the linear correlation (R² ≥ 0.9994) within the range of 8.0–100 ng/mL, which was equivalent to the range of 0.80–10 mg/kg in the sample. The average recoveries and the RSDs were 81–106% and 2–9% at two fortification levels (1.0 and 5.0 mg/kg) in paper bowls, respectively. The successful determination of 11 DSD‐FWAs in food‐contact paper and board obtained from local markets indicated that the newly developed method was rapid, accurate, and highly selective.