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A novel quality evaluation method of Corydalis yanhusuo was established by researching the high-performance liquid chromatography behavior of alkaloids under different buffer solutions and exploring the correlation between alkaloids in C. yanhusuo. The retention times of tetrahydropalmatine and corydaline were significantly influenced by pH, while the peak shape was affected by buffer types and ionic strength. The resolution of compounds in fingerprint was satisfactory under acetonitrile-0.2% phosphoric acid buffer (adjusted pH to 5.0 with triethylamine). Twelve common peaks were found by comparing 20 batches of C. yanhusuo fingerprints, and three tertiary alkaloids and four quaternary alkaloids were identified. The fingerprints were analyzed by similarity analysis, hierarchical cluster analysis, principal component analysis, and partial least squares discriminant analysis. All samples were divided into three groups, and the contents of dehydrocorydaline and coptisine from Zhejiang province were relatively higher than other origins. There were six components performing more contributions to the quality of C. yanhusuo. The correlations between alkaloids were conducted by Pearson correlation analysis and mathematical model analysis. The content correlation between palmatine and berberine was y = 0.28x2 + 0.03x + 0.03, and the dehydrocorydaline and coptisine was y = -7.54/(1 + (x/0.14)0.5 ) + 2.61. The established mathematical model of alkaloids provided a guiding significance for the quality control of C. yanhusuo.

A method for quantitative determination of the main active ingredient in traditional Chinese medicine was developed and validated, using liquid-liquid chromatography combined with high performance ...

A rapid, sensitive, environmental friendly dual preconcentration method by combining micro matrix solid-phase dispersion extraction with field-enhanced sample injection and micelle to cyclodextrin stacking has been developed for the determination of furocoumarins. Molecular sieve, KIT-6, was used as an adsorbent in micro matrix solid-phase dispersion process. The important parameters affecting off-line and on-line CE preconcentration efficiency were optimized. Under the optimal experimental conditions, all analytes showed good linearity (R2 > 0.999). The LODs of notopterol, isoimperatorin and imperatorin were 0.1 μg/mL, 1.2 mg/kg and 1.0 mg/kg respectively. Compared with the normal CE method, the enrichment times were up to 300. Moreover, Angelicae Dahuricae Radix was used as the mode of complex solid sample matrix to demonstrate the prospect of application of this methodology. The results showed the proposed strategy is promising for determining trace furocoumarins in complex matrix samples, which might be applied as a powerful and economic tool in monitoring illegal cosmetic adding. This article is protected by copyright. All rights reserved.

In traditional Chinese therapy, the decoction of herbal products is commonly used as a therapeutic drug, which takes water as the extraction medium and contains many hydrophilic compounds. The main...

As a liquid-liquid partition chromatography, counter-current chromatography has advantages in large sample loading capacity without irreversible adsorption, which has been widely applied in separation and purification fields. The main factors, including partition coefficient, two-phase solvent systems, apparatus, and operating parameters greatly affect the separation process of counter-current chromatography. To promote the applications of counter-current chromatography, it is essential to develop theoretical research to master the principles of counter-current chromatographic separations so as to achieve predictions before laborious trials. In this article, recent progress about separation prediction methods are reviewed from a point of the steady and unsteady state of the mass transfer process of counter-current chromatography and its mass transfer characteristics, and then it is divided into three aspects: prediction of partition coefficient, modeling the thermodynamic process of counter-current chromatography, and modeling the dynamic process of counter-current chromatography.

A simple, efficient, and sensitive strategy by coupling matrix solid-phase dispersion with ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry was proposed to extract and determine three types of components (including seven analytes) in Chinese patent medicines Chenxiangqu. The highly ordered mesoporous material Fe-SBA-15 synthesized under weakly acidic conditions was selected as a dispersant in matrix solid phase dispersion extraction for the first time. Several parameters including the mass ratio of sample to dispersant, the type of dispersant, the grinding time, and the elution condition were investigated in this work. Under the optimized conditions, 20 compounds were identified by quadrupole time-of-flight mass spectrometry and seven analytes were quantified. The results demonstrated that the developed method has good linearity (r > 0.9995), and the limits of detection of the analytes were as low as 0.55 ng/mL. The recoveries of all seven analytes ranged from 97.6 to 104.6% (relative standard deviation < 3.4%). Finally, the improved method was successfully applied to determination of five batches of Chenxiangqu samples, which provided a robust method in quality control of Chinese patent medicines Chenxiangqu. The developed strategy also shows its great potential in analysis of complex matrix samples.

Background Honokiol and magnolol were considered as markers for the analysis of Cortex Magnoliae Officinalis, its related Chinese Patent Medicines and their metabolites. However, the determination of these two analytes in a water-soluble sample is difficult and therefore requires a more efficient method. Objective To develop a sensitive method for the determination of honokiol and magnolol in a water-soluble sample for better quality control of Cortex Magnoliae Officinalis and its related Chinese Patent Medicines. Method In this work, a combination of dispersive micro-solid-phase extraction (DMSPE) and high-performance liquid chromatography (HPLC) has been developed for simultaneous preconcentration and determination of honokiol and magnolol in complex bio-samples. Several experimental factors affecting the extraction efficiency were optimized by single factor test. Results Under the optimized extraction conditions, the proposed method exhibited good linearity of not less than 0.9998, satisfactory precision with relative standard deviation of less than 1.3%, and acceptable mean recoveries of 97.3% and 101.5% for honokiol and magnolol, respectively. Furthermore, the method exhibits extremely high sensitivity with detection limits of 0.0097 and 0.0231 ng/mL, which is even more sensitive than those methods developed by MS. Conclusions The method established in this study is fast, economic, accurate, easy to operate, and importantly well suited to the extraction and analysis of honokiol and magnolol in a real complex sample matrix.

Despite the recent increase in the development of bioactive molecules in the drug industry, the enormous chemical space and lack of productivity are still important issues. Additional alternative approaches to screen and locate bioactive molecules are urgently needed. Label-free bio-affinity mass spectrometry (BA-MS) provides opportunities for the discovery and development of innovative drugs. This review provides a comprehensive portrayal of BA-MS techniques and of their applications in screening and locating bioactive molecules. After introducing the basic principles, alongside some application notes, the current state-of-the-art of BA-MS-assisted drug discovery is discussed, including native MS, size-exclusion chromatography-MS, ultrafiltration-MS, solid-phase micro-extraction-MS, and cell membrane chromatography-MS. Finally, several challenges and limitations of the current methods are summarized, with a view to potential future directions for BA-MS-assisted drug discovery. © 2019 Wiley Periodicals, Inc. Mass Spec Rev.

Four stereoisomeric components were produced during the synthesis of the antidepressant drug (1S, 4S)-sertraline hydrochloride due to the two chiral carbon centers in its chemical structure, including (1S, 4S), (1R, 4R), (1S, 4R), and (1R, 4S)-isomer. Stereoselective separation of the target isomer (1S, 4S)-sertraline from the medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl-β-cyclodextrin as the stereoselective selector was investigated. A biphasic solvent system composed of n-hexane/0.20 mol/L phosphate buffer solution with pH 7.6 containing 0.10 mol/L of hydroxypropyl-β-cyclodextrin (1:1, v/v) was selected for separation of cis-sertraline and trans-sertraline using reverse phase elution mode and (1S, 4S)-sertraline was separated with (1R, 4R)-sertraline using recycling elution mode. A fabricated in-house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions. Stationary phase retention and peak resolution were investigated for separation of cis-sertraline and trans-sertraline by the analytical apparatus.

Introduction Germacrone, furanodiene and β-elemene are the representative bioactive compounds in Curcuma species. The conventional extraction methods of these three sesquiterpenes are usually time-consuming and require a large volume of hazardous organic solvents. Thus, a fast and reliable method for extracting these sesquiterpenes from Curcuma plant is required urgently. Objective To establish a novel and simple extraction method for quantitative analysis of small amounts of sesquiterpenes in C. wenyujin plant. Methodology A method using microwave-assisted ionic liquid-micelle extraction combined with dispersive micro-solid-phase extraction (DMSPE) has been proposed for the extraction of three sesquiterpenoids in Curcuma plant. Fluorinated carbon nanotubes (FCNTs) were used as an adsorbent in DMSPE for the first time. Parameters concerning the microwave-assisted extraction (MAE) conditions and the DMSPE were investigated and evaluated to achieve optimum extraction efficiency of target analytes. Results The final conditions of ionic liquid-micelle based MAE were selected to be 0.25 M of 1-decyl-3-vinylimidazolium bromide as the extraction solvent, microwave irradiation for 10 min at 60°C. And the optimal DMSPE conditions were found to be 2 μg/mL of FCNTs as the adsorbent, extraction time of 2 min and 100 μL of acetonitrile as the elution solvent. The developed method exhibited good linearities (R > 0.9990), high repeatability and recoveries. The proposed method has been successfully applied in determination of sesquiterpenes in C. wenyujin samples. Conclusion The work shows a potential in analysing small amounts of sesquiterpenes in complex samples and represents the first attempt of using FCNTs as an adsorbent for the microextraction mode.

Background: Aflatoxins (AFs) have attracted increasing amounts of attention in recent years for their high toxicity. Previously, AF-polluted Chinese Patent Medicines (CPMs) were ignored, but their quality, safety, and efficacy might be influenced by the toxic compounds. Objective: The objective is to develop a simple, low-cost, and efficient method for quantitative analysis of aflatoxins B1 (AFB1) and B2 (AFB2) in CPMs to ensure their safe use. Methods: A simple, modified, magnetic nanoparticle-based solid-phase extraction (SPE) combined with HPLC-fluorescence detection for the determination of trace amounts of AFB1 and AFB2 was established. The main parameters affecting the efficiency of modified magnetic nanoparticle-based SPE, such as pH of sample solution, adsorbent amount, adsorption time, and desorption condition, were investigated. Results: Under the optimum conditions, AFB1 and AFB2 were linear in the ranges of 0.3–10 and 0.04–3 ng/mL with the correlation coefficient (R) of 0.9998 and 0.9999, respectively. Their intraday precisions were 1.16 and 2.30% and the interday precisions were 1.28 and 1.87% for AFB1 and AFB2, respectively. The developed SPE was applied for AFB1 and AFB2 extraction in three commercially available Fupuganmao granule samples, and the results were compared with the official immunoaffinity column chromatography method. Conclusions: The method provided a preferable candidate for the determination approach of AF measurement in CPMs. Highlights: Amine-functionalized magnetic nanoparticles were successfully applied to SPE for adsorbing AFB1 and AFB2 in CPMs for the first time.

A sensitive dispersive micro solid-phase extraction coupled with HPLC has been developed for preconcentration and determination of three flavonoids (quercetin, kaempferol, and isorhamnetin) in complex matrix samples. Parameters that affect extraction efficiency have been optimized. The optimal extraction conditions are using 2 μg/mL of crab shell as the sorbent, extraction for 2 min at pH 7, and then eluting with 100 μL of methanol. As a result, the method shows good linearity (R > 0.9994), low LODs (even 0.08 ng/ml) and satisfactory recovery in real honey and rat urine samples. As an eco-friendly biomaterial, crab shell powder is used as sorbent in pretreatment of flavonoids, and its adsorption mechanism has been investigated for the first time. Compared with the other reported methods, the proposed strategy is time-saving, eco-friendly, and highly sensitive using HPLC (even achieving MS grade sensitivity).

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic-acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high-performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0-5 min, 5-19% A; 5-10 min, 19% A; 10-50 min, 19-42% A; 50-54 min, 42-100% A; 54-60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.

A rapid and sensitive UPLC-MS/MS method was established for the simultaneous quantification of 6'''-feruloylspinosin, spinosin, jujuboside A, and jujuboside B in rat plasma after the oral administration of traditional and dispensing granules (DG) decoction of Ziziphi Spinosae Semen (ZSS). The four components were separated using 0.1% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.3 mL/min equipped with a C18 column (2.1 × 50 mm, 1.7 μm particle size, Acquity BEH C18 ). The mass spectrometer was operated under multiple reaction monitoring mode. An aliquot of 100 μL rat plasma was deproteinized by 300 μL methanol. The supernatant was injected into the UPLC-MS/MS system for analysis. The calibration curves displayed good linearity. The intra-day and inter-day precisions (RSD) were less than 7.3%. The accuracies ranged from -1.3 to 6.1%. The extraction recoveries ranged from 95.8 to 101.9%, and the matrix effects were satisfactory. For DG, half-life values (t1/2 ) of 6'''-feruloylspinosin and Cmax of jujuboside A were elevated remarkably. MRT0-t of jujuboside B was significantly increased. No significant variation was observed for the pharmacokinetic parameters of spinosin. The results could provide a scientific basis for the clinical application of traditional and DG decoction of ZSS.

A simple, convenient, and sensitive method that involves combining matrix solid-phase dispersion extraction with field-amplified sample stacking in capillary electrophoresis has been developed for the determination of organic acids in a complex solid matrix. Mesoporous molecular sieve, MCM-48, was synthesized by a hydrothermal method and selected as the adsorbent in matrix solid-phase dispersion. After fast extraction, the enriched analytes were back-extracted into a basic aqueous solution for field-amplified sample stacking in capillary electrophoresis. Parameters that affect extraction efficiency and sample stacking were optimized. Under the optimal conditions, approximately 42-, 49-, and 56-fold sensitivity enhancements were achieved for danshensu, protocatechuic acid, and cinnamic acid, respectively, when compared to normal injection. A satisfactory correlation coefficient (r > 0.99) was obtained. Both intra- and interday precision were lower than 2.53%. And the limits of detection of the three organic acids ranged between 0.01 and 0.029 μg/mL. Finally, the newly proposed method was successfully applied to the determination of organic acids in Fufang Danshen tablets, which indicates its great potential in analyzing organic acids in a complex matrix.

Three key intermediates in the preparation of the nonsteroidal anti-inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin-layer chromatography. Fifty milligrams of the synthetic mixture after the three-step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0 mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.

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.

A simple, efficient and environmental friendly method was proposed for determining five sesquiterpenoids of Curcuma wenyujin by MSPD extraction coupled with MEEKC separation. Molecular sieve was applied as a solid support for extraction of sesquiterpenoids for the first time. Various parameters affecting extraction and separation efficiency were investigated. The optimized conditions involved dispersing sample (200 mg) with 200 mg of TS-1 for 150 s and using 1000 μL of methanol to elute five target analytes. Finally, they were well separated by using a running buffer containing 1.3% SDS, 5.0% 1-butanol, 0.5% ethyl acetate and 10% acetonitrile in 10 mM borate buffer at pH 9.0. Consequently, the developed method was fully validated and successfully applied to determine the five sesquiterpenoids including curdine, curcumenol, germacrone, furanodiene and β-elemene in Curcuma wenyujin origin's Chinese herbal medicines. Furthermore, hierarchical cluster analysis was performed based on the contents of target compounds for distinguishing steamed and non-steamed drugs. The present study provided a promising method for fast investigation and discrimination of chemical difference in steam & non-steamed Chinese medicines from Curcuma wenyujin origin.

3-Phenyllactic acid is an antimicrobial compound with broad-spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3-phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3-phenyllactic acid with a synthesized chiral ligand. A two-phase solvent system was composed of n-butanol/hexane/water (0.4:0.6:1, v/v/v) to which N-n-dodecyl-l-hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid-liquid extraction. Baseline enantioseparation of racemic 3-phenyllactic acid by analytical high-speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3-phenyllactic acid reached 99.0%, as determined by chiral high-performance liquid chromatography.

To promote the detection sensitivity of neutral analytes, a sensitive two-step stacking strategy by coupling field-enhanced sample injection and micelle to cyclodextrin stacking in reverse migrating micelles MEKC has been established by selecting jujuboside A and B as models. The stacking mechanism, affecting factors as well as analytical performances of the proposed method were investigated. Compared with typical injection, the sensitivity enhancement factors of this method were up to 140- and 152-fold for jujuboside A and B, respectively. The enhancements were 8–10 times better than only single stacking strategy by MCDS. The LODs were 0.2–0.3 μg mL−1, interday and intraday%RSD were both lower than 4.3%, and a good linearity (r>0.999) was obtained. Feasibility in real samples analysis was evaluated by using Semen Ziziphi Spinosae dispensing granule and rat's urine. The results suggested that the strategy was simple, reliable, sensitive and promising for the analysis of neutral analytes in complex sample matrix.

We proposed a biochemometrics strategy for tracing diuretic components of herbs based on quantitative determination and pharmacological evaluation. First, a sensitive and robust liquid chromatography coupled with tandem mass spectrometry approach was established for simultaneous quantification of six major triterpenoids in crude and salt-processed Alisma orientale. The separation of triterpenoids was achieved on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid. Six major triterpenoids were detected by multiple reaction monitoring in the negative ion mode. Glycyrrhetinic acid was used as the internal standard. The approach showed good linearity. Intra- and inter-day precisions were all within 2.9%. The recovery rates of each triterpenoid ranged from 97.9% to 103.2%. The approach was then successfully employed for quantitative analysis of six triterpenoids in ten batches of crude and salt-processed A. orientale. Second, the diuretic effects of crude and salt-processed A. orientale were evaluated in mice. Third, principal component analysis and canonical correlation analysis were used to uncover the relationship between the contents of six major triterpenoids and the diuretic effect of different crude and salt-processed samples. Alisol B, alisol F, and alisol A have a close positive correlation with the diuretic effect.

Acetyltropic acid is an important synthetic intermediate for preparation of tropane alkaloid derivatives, which can be used as anticholinergic drugs, deliriants, and stimulants. In the present work, acetyltropic acid was successfully enantioseparated by countercurrent chromatography using sulfobutyl ether-β-cyclodextrin as chiral selector. A biphasic solvent system composed of n-butyl acetate/n-hexane/0.1 mol/L citrate buffer at pH = 2.2 containing 0.1 mol/L of sulfobutyl ether-β-cyclodextrin (7:3:10, v/v) was selected, which produced a suitable distribution ratio DS = 1.14, DR = 2.31 and a high enantioseparation factor α = 2.03. Baseline separation was achieved for preparative enantioseparation of 50 mg of racemic acetyltropic acid. A method for chiral analysis of acetyltropic acid by conventional reverse phase liquid chromatography with hydroxylpropyl-β-cyclodextrin as mobile phase additive was established, and formation constants of inclusion complex were determined. It was found that different substituted β-cyclodextrin should be selected for enantioseparation of acetyltropic acid by countercurrent chromatography and reverse phase liquid chromatography.

A novel on-line synergistic proconcentration strategy coupling field-amplified sample stacking and micelle to cyclodextrin stacking for cationic analytes in capillary zone electrophoresis has been proposed and applied for the separation and determination of two alkaloids, matrine, and oxymatrine in complicated matrix samples. The approach was performed by the long injection of sample in a low-conductivity sodium dodecyl benzene sulfonate solution followed by the injection of hydroxypropyl-β-cyclodextrin solution in higher conductivity. The stacking mechanism of this method has been expounded and parameters affecting stacking effect have been optimized in our study. Under the optimum experimental conditions, 169- and 218-fold sensitivity improvements were achieved for matrine and oxymatrine when compared with normal injection. Analytical indicators including linearity, limits of detection, and reproducibility (intra- and inter-day relative standard deviations) were evaluated. Moreover, sample matrix effect was studied using compound flavescent sophora and salicylic acid powder and spiked urine samples. The developed method is an attempt for the combination of micelle to cyclodextrin stacking with other stacking methods. It could be a good alternative choice for the determination of alkaloids in a complex sample matrix.

Corydalis turtschaninovii (CT) is a traditional Chinese medicine which is known to have analgesic effects, and is under investigation for the management of chronic pain. Our study aims to establish a UPLC–MS/MS method for pharmacokinetic determination of nine bioactive alkaloids of raw and processed CT in rat plasma. Nitidine chloride was selected as internal standard. After protein precipitation with methanol, the plasma samples were separated on a reversed phased column with a mobile phase of acetonitrile and water (including 0.1% formic acid). The MRM parameters were optimized as follows: m/z 354.0 → 188.1 for protopine, m/z 321.0 → 293.1 for coptisine, m/z 371.1 → 189.1 for allocryptopine, m/z 357.1 → 193.3 for tetrahydropalmatine, m/z 324.1 → 176.1 for tetrahydrocoptisine, m/z 340.1 → 176.2 for tetrahydroberberine, m/z 368.1 → 289.1 for corynoline, m/z 370.5 → 192.1 for corydaline, m/z 367.2 → 351.2 for dehydrocorydaline, and m/z 406.0 → 300.3 for the IS. The linearity, accuracy, precision, stability, recovery and matrix effect of the method were well validated. This method was successfully employed for a pharmacokinetic study of raw and vinegar-processed CT in rats. The absorption of the nine alkaloids was accomplished in an hour. The double peak phenomenon of the nine alkaloids may be ascribed to enterohepatic recirculation. Compared with the raw group, AUC0→t and Cmax of the nine alkaloids were significantly elevated in the vinegar-processed group. Our findings suggest that vinegar-processing could increase the bioavailability of the nine alkaloids of CT in rats. The pharmacokinetic information obtained will provide basis for application of processed CT in future clinical therapy.

Alisma plantago-aquatica is known to regulate water and fluid balance in cells, and is under testing for the therapy for patients suffering from chronic nephritis. Herein, a UHPLC-MS/MS method was established and validated for the determination of six bioactive triterpenoids of raw and salt-processed Alisma plantago-aquatica in rat plasma. The acquired plasma was subjected to protein precipitation with acetonitrile. Glycyrrhetinic acid was employed as internal standard. The pretreated samples were separated on a reversed phased column with a mobile phase of acetonitrile and water (including 0.1% formic acid). The MRM mode for the six triterpenoids were at m/z 535.4 → 489.4 for alisol A, m/z 517.3 → 471.4 for alisol B, m/z 533.3 → 487.3 for alisol F, m/z 577.4 → 531.4 for alisol A-24-acetate, m/z 559.4 → 495.4 for alisol B-23-acetate, m/z 573.3 → 509.3 for alisol C-23-acetate, and 469.3 → 425.3 for the IS. The accuracy and precision of the method were determined as −2.2%-3.6% and 0.8%–3.0%, respectively. This approach was employed to a pharmacokinetic study of the six bioactive triterpenoids after intragastric administration of raw and processed Alisma plantago-aquatica in rats. The two-phasic pharmacokinetic of alisol B, alisol C-23-acetate and alisol F were reported for the first time, which may be ascribed to enterohepatic recirculation of these triterpenoids.

Ecdysterone and saponins are the most characteristic components of Radix Achyranthes bidentate, which acts on the human body to promote collagen synthesis and stimulates cell growth. However, the relationship between these components and the differentiation of MC3T3-E1 osteoblastic cells is unknown. We developed a rapid ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry method for direct determination of one ecdysterone and four saponins in crude and salt-processed Radix Achyranthes bidentate. The method was interrogated in terms of linearity, intra- and inter-day precision, repeatability, stability and recovery. The method was linear within the concentration ranges of 0.003-336 μg/mL for β-ecdysterone, 0.0035-130 μg/mL for 25S-inokosterone, 0.004-423 μg/mL for ginsenoside Ro, 0.0036-66 μg/mL for chikusetsusaponin IV and 0.0044-111 μg/mL for chikusetsusaponin IVa. The intra- and inter-day precisions were all within 2.7%. The standard addition method determined recovery rates for each component (98.7-102.5%). The method was successfully applied to simultaneously quantify five components in ten batches of crude and salt-processed Radix Achyranthes bidentate. Subsequently, the examination of these extracts on the differentiation of MC3T3-E1 osteoblastic cells were carried out. Finally, the relationships between the contents of five components and their anti-osteoporosis effect were investigated by using canonical correlation analysis.

Selective separation of chemical components from seven kinds of volatile oil by countercurrent chromatography with three types of cyclodextrins as selective reagent was investigated in this work. Preparative separation of chemical components from volatile oil is generally quite challenging due to their extremely complexity of the composition. A biphasic solvent system n-hexane-0.10 mol L(-1) cyclodextrin (1:1, v/v) was selected for separation of components from volatile oil and three types of cyclodextrins were investigated, including β-cyclodexrin, methyl-β-cyclodexrin and hydroxypropyl-β-cyclodexrin. All kinds of volatile oils are from seven kinds of traditional Chinese herb. Results showed that some chemical components could be well separated with high purity from each kind of volatile oil using different type of cyclodextrin as selective reagent. For example, germacrone and curcumenone could be selectively separated from volatile oil of Curcumae Rhizoma with methyl-β-cyclodexrin and hydroxypropyl-β-cyclodexrin as selector respectively, and other five components were selectively separated from volatile oil of Chuanxiong Rhizoma, Myristicae Semen, Aucklandiae Radix and Angelicae Sinensis Radix by countercurrent chromatography with different cyclodexrin as selective reagent. Separation mechanism for separation of components from volatile oil by countercurrent chromatography with cyclodextrin as selective reagent was proposed. Peak resolution of the present separation method could be greatly influenced by the chemical compositions of volatile oil.

Sertraline is an antidepressant in a group of drugs called selective serotonin reuptake inhibitors. Four stereoisomeric compounds would be produced in its synthetic preparation due to two chiral carbons on its chemical structures. In the present work, stereoselective liquid-liquid extraction of isomeric sertraline with substituted cyclodextrins as stereoselective extractant was investigated. Factors affecting the distribution performance, including organic solvents, types of extractants, pH value, buffer solution of aqueous phase, concentration of extractant and temperature, were investigated. Under optimized conditions, a stereoselectivity of 1.404 was obtained for cis-sertraline and a stereoselectivity of 2.373 was obtained for trans-sertraline when hydroxypropyl-β-cyclodextrin was used as the stereoselective extractant, and a stereoselectivity of 1.685 was achieved for trans-sertraline with methyl-β-cyclodextrin as extractant. An unusual stereoselective combination was observed for trans-sertraline and cis-sertraline when sodium carbonate buffer was used. Successful stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β-cyclodextrin as stereoselective selector was achieved, using a biphasic solvent system composed of n-hexane : 0.1 mol L−1 citrate buffer solution with pH7.6 (1:1, v/v).

This study developed a robust and reliable approach using liquid chromatography- tandem mass spectrometry for the simultaneous determination of five saponins in rat tissues: β-ecdysterone, chikusetsusaponin IV, ginsenoside Ro, 25S-inokosterone and chikusetsusaponin IVa. This is the first report on a comparative tissue distribution study of crude and salt-processed Radix Achyranthes in rats. After one-step protein precipitation by acetonitrile, the tissue samples were sent to LC-MS/MS for multiple reaction monitoring. The retention times of the five saponins and internal standard were 1.77, 3.14, 3.01, 1.83, 3.26 and 4.77 min. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.3-1562.5 ng/mL. The intra- and inter-day accuracy and precision were within 15% of the nominal concentration. The recoveries of the five saponins were 92.0-99.9%. Finally, this approach was successfully applied to tissue distribution analysis of the five saponins after oral administration of crude and salt-processed Radix Achyranthes in rats. The largest concentration of the five saponins was observed in kidney after salt-processing, which indicated that processing could enhance the bioavailability.

A sensitive, simple, and environmentally friendly online three-step stacking strategy combining field-enhanced sample injection, sweeping and micelle to solvent stacking has been developed to determine trace amounts of organic anionic analytes in complex biological samples. A green ionic liquid, 1-dodecyl-3-methylimidazolium hydrogen sulfate, was first introduced as a micellar solution and used for electroosmotic flow reversal in this stacking strategy. The mechanism of stacking has been discussed, and parameters affecting three-step stacking preconcentration efficiency have been optimized. The capillary coated with an ionic liquid was easy to prepare, regenerable and repeatable (RSD

Racemic trans-paroxol, trans-N-methylparoxetine and trans-paroxetine containing two chiral centres were stereoselectively separated using countercurrent chromatography with hydroxypropyl-β-cyclodextrin as the chiral selector. A two-phase solvent system composed of n-butyl acetate and 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 9.2 (1:1, v/v) was selected, and 0.10 mol L−1 hydroxypropyl-β-cyclodextrin was added to the aqueous phase as the chiral selector. Racemic trans-N-methylparoxetine and racemic trans-paroxol (20 mg of each) were stereoselectively separated by countercurrent chromatography in an individual run, yielding 7.1–8.3 mg of enantiomers with a purity of 95–98%, where the recovery of each separated isomer reached approximately 70–83%. Racemic trans-paroxetine (20 mg) was stereoselectively separated by countercurrent chromatography using a recycling elution mode with a biphasic solvent system composed of n-hexane: n-butyl acetate: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 9.2 (9:1:10, v/v/v), and 0.10 mol L−1 hydroxypropyl-β-cyclodextrin was added to the aqueous phase as the chiral selector, yielding 5.0–5.6 mg of enantiomer with a high purity of over 98–99%.

Triterpenoids are the major secondary metabolites and active substances in Ganoderma, considered as the “marker compounds” for the chemical evaluation or standardization of Ganoderma. A response surface methodology was used to optimize the ultrasonic-assisted extraction of triterpenoids. The extraction rate was 7.338 ± 0.150 mg/g under the optimum conditions: 87% ethanol, ratio of solid to liquid (w : v) 1 : 28, and ultrasound extraction time 36 min. Based on the high sensitivity and selectivity of HPLC-LTQ-Orbitrap-MSn, 24 components of triterpenoids were tentatively identified in the negative mode. Then, the global chemical profiling consisting of HPLC and TLC fingerprints generated by ChemPattern™ software was developed for evaluation of Ganoderma species. For fingerprint analysis, 11 peaks of triterpenoids were selected as the characteristic peaks to evaluate the similarities of different samples. The correlation coefficients of similarity were greater than 0.830. The cluster analysis showed a clear separation of three groups, and 11 peaks played key roles in differentiating these samples. The developed global chemical profiling method could be applied for rapid evaluation, quality control, and authenticity identification of Ganoderma and other herbal medicines.

Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method for the determination of triterpenoids and distinction of Ganoderma and related species. Methods: Ten ganoderma triterpenoids were determined by HPLC in addition to a QAMS method. On the basis of QAMS, principal component analysis (PCA) and cluster analysis (CA) were used to distinguish Ganoderma samples. Results: Ten types of ganoderma triterpenoids had good linearity of regression coefficients (R2 ≥ 0.9992). Relative correction factors (RCFs) of lucideric acid C, lucideric acid LM1, ganoderic acid G, lucideric acid B, lucideric acid A, ganoderenic acid D, ganoderic acid D, lucideric acid D and ganoderic acid F with reference to ganoderic acid A were 0.929, 1.011, 0.608, 1.041, 0.997, 2.464, 0.891, 0.826 and 0.722, respectively. Ten types of ganoderma triterpenoids in twenty-nine batches of Ganoderma and related species were determined by QAMS. Nineteen batches of samples containing ganoderma acid A were roughly divided into three categories by PCA and CA. Conclusion: The developed QAMS method, in combination with PCA and CA, served as an accurate method for the rapid evaluation and authenticity identification of Ganoderma.

Shenqi Jiangtang Granule, a well-known traditional Chinese herbal preparation, has been widely used for the treatment of type II diabetes mellitus. In this work, an ultrafiltration liquid chromatography with quadrupole time-of-flight mass spectrometry method was proposed for the rapid identification of bioactive ingredients from Shenqi Jiangtang Granule using α-glucosidase as an example. First, the chemical profile of this preparation was clarified, including 37 saponins, 17 flavonoids, 37 lignans, and seven other compounds. After incubation with α-glucosidase in vitro, the methanol extract with an IC50 value of 0.19 mg/mL exhibited significant inhibitory activity. Then, 18 specific binding peaks were screened, and 15 peaks were identified. Among these, ten compounds were reported to have potential α-glucosidase inhibitory activity for the first time. Subsequently, the inhibitory activities of these active compounds were evaluated by ultraviolet spectrophotometry with p-nitrophenyl α-d-glucopyranoside as a substrate. As a result, gomisin J and gomisin D exhibited stronger α-glucosidase inhibitory activities than other active compounds with IC50 values of 77.69 and 133.85 μM, respectively. The results demonstrated that the integrated ultrafiltration liquid chromatography with mass spectrometry method was an effective and powerful tool for the discovery of active ingredients in Shenqi Jiangtang Granule.

A simple and effective method was developed for determining lignans in Schisandrae Chinensis Fructus by using a micro-matrix solid phase dispersion (MSPD) technique coupled with microemulsion electrokinetic chromatography (MEEKC). Molecular sieve, TS-1, was applied as a solid supporting material in micro MSPD extraction for the first time. Parameters that affect extraction efficiency, such as type of dispersant, mass ratio of the sample to the dispersant, grinding time, elution solvent and volume were optimized. The optimal extraction conditions involve dispersing 25 mg of powdered Schisandrae samples with 50 mg of TS-1 by a mortar and pestle. A grinding time of 150 s was adopted. The blend was then transferred to a solid-phase extraction cartridge and the target analytes were eluted with 500 μL of methanol. Moreover, several parameters affecting MEEKC separation were studied, including the type of oil, SDS concentration, type and concentration of cosurfactant, and concentration of organic modifier. A satisfactory linearity (R > 0.9998) was obtained, and the calculated limits of quantitation were less than 2.77 μg/mL. Finally, the micro MSPD-MEEKC method was successfully applied to the analysis of lignans in complex Schisandrae fructus samples.

Four β-adrenergic blocking agents, including 1-[(1-methylethyl)amino]-3-phenoxy-2-propanol (1), 1-[(1-methylethyl)amino]-3-(3-methylphenoxy)-2-propanol (2), 1,1′-[1,4-phenylenebis(oxy)]bis[3-[(1-methylethyl)amino]-2-propanol (3) and 1,1′-[(4-methyl-1,2-phenylene)bis(oxy)]bis[3-[(1-methylethyl)amino]-2-propanol (4), were stereoselectively separated by countercurrent chromatography using di- n -hexyl l -tartrate and boric acid as chiral selector. The compounds (3) and (4) have four optical isomers since they contained two chiral centers. A two-phase solvent system composed of chloroform-0.05 mol L −1 of acetate buffer containing 0.10 mol L −1 of boric acid (1:1, v/v) was selected, in which 0.10 mol L −1 of di- n -hexyl l -tartrate was added in the organic phase as chiral selector. 20–42 mg of each racemate was stereoselectively separated by countercurrent chromatography in a single run with high purity of 96–98%, and the recovery of each separated compound reached around 87–93%. This is the first time report on successful stereoselective separation of optical isomeric compounds containing two chiral centers by countercurrent chromatography. At the same time, a chiral stationary phase was screened for analytical stereoselective separation of compounds (3) and (4) by high performance liquid chromatography.

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−1 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%. This article is protected by copyright. All rights reserved

The influence of chlorine substituents in chiral separation of three racemic 2-(chlorophenyl)propanoic acids by countercurrent chromatography using hydroxypropyl-β-cyclodextrin as a chiral additive were mainly investigated in the present paper, including 2-(2-chlorophenyl)propanoic acids, 2-(3-chlorophenyl)propanoic acids and 2-(4-chlorophenyl)propanoic acids. The influences of chromatographic conditions on the retention behavior were studied by enantioselective liquid-liquid extraction experiments using the methodology of response surface It was found that 2-(3-chlorophenyl)propanoic acids could be successfully chiral separated by countercurrent chromatography, while no resolution was achieved for racemic 2-(2-chlorophenyl)propanoic acids and 2-(4-chlorophenyl)propanoic acids under optimized separation conditions. The formation of 1:1 stoichiometric inclusion compounds between 2-(3-chlorophenyl)propanoic acids and HP-β-CD was determined by UV spectra measurements. The inclusion constants for 2-(3-chlorophenyl)propanoic acids and HP-β-CD were determined by the Benesi-Hildebrand equation. Meanwhile, the inclusion constants of 2-(3-chlorophenyl)-propanoic acid enantiomer and HP-β-CD were obtained by the pesudophase retention equation in countercurrent chromatography. Furthermore, the inclusion interactions of the three racemates with HP-β-CD were also investigated by the molecular docking. The results obtained from UV spectra measurements and molecular docking showed that the racemate with chlorine substituents in meta-position presented the highest enantiorecognition while the racemates with chlorine substituents in ortho-position had the lowest enantiorecognition. The above results further indicated that forming a stable inclusion complex between racemate and chiral selector is a prerequisite for a successful enantioseparation and at the same time, the difference in inclusion capacity between the two enantiomers is also essential for the enantioseparation.

This study established a rapid and reliable approach using liquid chromatography-tandem mass spectrometry for the simultaneous determination of cinnamic acid, vanillic acid and protocatechuic acid in rat plasma. This is the first report on a comparative pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs in rats. After liquid-liquid extraction by ethyl acetate, the plasma samples were subjected to LC-MS/MS for multiple reaction monitoring. The standard curves showed good linear regression (r2 > 0.9991) in the range of 10.0-16000 ng/mL. The intra- and inter-day accuracy and precision were found to be within 15% of the nominal concentration. The recoveries of the three phenolics ranged from 88.7 to 105.7%. Finally, this approach was successfully applied to pharmacokinetic analysis of the three phenolics after oral administration of standard decoction and dispensing granules of C. cassia twigs in rats. Although the values of AUC0-t of vanillic acid and protocatechuic acid in standard decoction group were larger than those of the dispensing granule group, no significant difference was observed for the two groups. Of note, the elimination rates of vanillic acid were slower in the standard decoction group than the dispensing granule group.

This paper concentrates on the enantioseparation of racemic 2-(substituted phenyl)propanoic acids by high-speed countercurrent chromatography with substituted β-cyclodextrin as the chiral selector, and an investigation of the influence of the substituent on the benzene ring in enantiorecognition between the chiral selector and enantiomer of each racemate is presented. This is an extension research of our previous work on the enantioseparation of 2-phenyl propanoic acid derivatives, to investigate the relationship between the value of enantioseparation factor and the different substituent on the benzene ring. In total, ten racemic 2-(substituted phenyl)propanoic acids were investigated, of which four including 2-(4-nitrophenyl)propanoic acid, 2-(4-methylphenyl)propanoic acid, 2-(4-hydroxyphenyl)propanoic acid, and 2-(4-chlorophenyl)propanoic acid, were studied by countercurrent chromatography for the first time, and two racemates were successfully enantioseparated. The distribution ratio and enantioseparation factor for all the ten racemates were determined by enantioselective liquid-liquid extraction. The results showed that an electron-donating group on the benzene ring presents a higher enantiorecognition induced by chiral selector than that of racemates with an electron-withdrawing group on the benzene ring.

The equilibrium model in enantioseparation of mandelic acid by chiral ligand exchange countercurrent chromatography was investigated using N-n-dodecyl-l-proline as chiral ligand and cupric ion as central metal. Important parameters, including physical partition coefficient and formation constants of binary and ternary coordination complexes in the two-phase solvent system, were determined. This equilibrium model could give an excellent prediction of distribution ratio and enantioseparation factor of the analyte in the biphasic solvent system, which was further verified by experiments. All the average relative deviations were less than 12%, indicating that the established model could provide a simple computational approach for optimization of enantioseparation conditions in chiral ligand exchange countercurrent chromatography.

Analytical enantioseparation of five β-substituted-2-phenylpropionic acids by high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral mobile phase additive was established in this paper, and chromatographic retention mechanism was studied. The effects of various factors such as the organic modifier, different ODS C18 columns and concentration of HP-β-CD were investigated. The chiral mobile phase was composed of methanol or acetonitrile and 0.5% triethylamine acetate buffer at pH 3.0 added with 25 mmol L(-1) of HP-β-CD, and baseline separations could be reached for all racemates. As for chromatographic retention mechanism, it was found that there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor under constant pH value and column temperature.