Plasma pharmacochemistry combined with pharmacokinetics and pattern recognition analysis to screen potentially bioactive components from Daming capsule using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Daming capsule is a traditional Chinese medicine for hyperlipidemia treatment. However, the vague understanding of the bioactive components of Daming capsule hampers its modernization and internationalization. This work first developed a high‐throughput, high‐resolution, and high‐sensitivity ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method for identifying the absorbed compounds and monitoring the pharmacokinetics of Daming capsule. A high‐throughput strategy integrating plasma pharmacochemistry, pharmacokinetics, and pattern recognition analysis was also established for screening the bioactive components of Daming capsule in vivo. The established strategy based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was successfully applied to screen the bioactive components of Daming capsule. Up to 53 absorbed compounds were identified. Six anthraquinones with fast and high absorption, namely, emodin‐O‐glucoside, aurantio‐obtusin, aloe‐emodin, rhein, emodin, and chrysophanol, were screened as potentially bioactive components of Daming capsule. The plasma pharmacochemistry and pharmacokinetics of Daming capsule were reported for the first time. Notably, the high‐throughput and reliable strategy facilitated the screening and identification of bioactive components of traditional Chinese medicine, thereby providing novel insights into the research and development of new drugs.     

Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFI^TM platform based on the in‐house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty‐five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry‐oriented method for the discovery of the potential new compounds.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

Identification of short‐chain poly‐3‐hydroxybutyrates in Saiga horn extracts using LC–MS/MS

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high‐performance liquid chromatography coupled with high‐resolution mass spectrometry, we have been able to identify a series of short‐chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high‐performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short‐chain poly‐3‐hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.     

Screening and analysis of potentially active components in Shenxiong glucose injection using UHPLC coupled with photodiode array detection and MS/MS

Shenxiong glucose injection, a pharmaceutical preparation containing a water extract of the roots of Salvia miltiorrhizae and ligustrazine hydrochloride, is widely used in clinical to treat cardiovascular diseases in China. The chemical components of the water extract have been reported and the cardioprotective effects of the injection have been evaluated. However, the chemical constituents of the injection and their correlations with its pharmacological effects have not been established. In this study, 13 chemical constituents of the injection have been identified or characterized by ultra‐high performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry. Besides, the potentially active compounds of this preparation that directly act on cardiac cells have been screened by cell extraction and ultra high performance liquid chromatography targeted multiple reaction monitoring. As a result, eight potentially active compounds, danshensu ( 1 ), ligustrazine hydrochloride ( 4 ), salvianolic acid I/H ( 7 ), lithospermic acid ( 8 ), salvianolic acid D ( 9 ), rosmarinic acid ( 10 ), salvianolic acid B ( 12 ), and salvianolic acid C ( 13 ), were obtained and structurally characterized from the 11 target compounds used for screening. The liquid chromatography with quadrupole time‐of‐flight mass spectrometry and liquid chromatography with multiple reaction monitoring tandem mass spectrometry combination method has demonstrated its potency for the screening, detection, and structural identification of bioactive compounds in a complex matrix.     

Determination of azithromycin residue in pork using a molecularly imprinted monolithic microcolumn coupled to liquid chromatography with tandem mass spectrometry

Using spiramycin as a dummy template, a molecularly imprinted polymer monolithic micro‐column with high selection to azithromycin was prepared in a micropipette tip. The imprinting factor of the monolithic micro‐column prepared was approximately 2.67 and the morphological structure of the polymers was characterized by scanning electron microscopy. A simple, sensitive, and reproducible method based on the imprinted monolithic micro‐column coupled to liquid chromatography with tandem mass spectrometry was developed for determining the residues of azithromycin in pork. Pork samples were extracted with acetonitrile, cleaned up under the optimal monolithic micro‐column conditions, and analyzed using liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode. The assay exhibited a linear dynamic range of 0.50–50 μg/L with the correlation coefficient (r²) above 0.99. In the three spiking levels of 0.50, 1.0, and 10 μg/kg, the average recoveries of azithromycin from pork samples were between 85.8 and 96.5% with a relative standard deviation below 10%. The limit of detection and limit of quantitation were 0.03 and 0.1 μg/kg, respectively.     

Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high‐throughput UHPLC–MS with a multivariate analysis approach

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in‐depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese medicine.     

Rapid screening,identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC–ESI‐TOF‐MS combined with semipreparative HPLC

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.     

Screening and investigation of triplex DNA binders from Stephania tetrandra S. Moore by a combination of peak area‐fading ultra high‐performance liquid chromatography with orbitrap mass spectrometry and optical spectroscopies

The identification and screening of triplex DNA binders are important because these compounds, in many cases, are potential anticancer agents as well as promising drug candidates. Therefore, the ability to screen for these compounds in a high‐throughput mode could dramatically improve the drug screening process. A method involving a combination of 96‐well plate format and peak area‐fading ultra high‐performance liquid chromatography coupled with Orbitrap mass spectrometry was employed for screening bioactive compounds binding to the triplex DNA from the extracts of Stephania tetrandra S. Moore. Two compounds were screened out and identified as fangchinoline and tetrandrine based on the comparison of retention time and tandem mass spectrometry data with those of standards. The binding mechanisms of fangchinoline and tetrandrine at the molecular level were explored using tandem mass spectrometry, fluorescence spectroscopy, ultraviolet‐visible spectroscopy, and circular dichroism. Collision‐induced dissociation experiments showed that the complexes with fangchinoline and tetrandrine were dissociated by ligand elimination. According to these measurements, an intercalating binding is the most appropriate binding mode of these two alkaloids to the triplex DNA. The current work provides not only deep insight into alkaloid‐triplex DNA complexes but also useful guidelines for the design of efficient anticancer agents.     

Simultaneous analysis by Quadrupole‐Orbitrap mass spectrometry and UHPLC‐MS/MS for the determination of sedative‐hypnotics and sleep inducers in adulterated products

Adulterated products are continuously detected in society and cause problems. In this study, we developed and validated a method for determining synthetic sedative‐hypnotics and sleep inducers, including barbital, benzodiazepam, zolpidem, and first‐generation antihistamines, in adulterated products using Quadrupole‐Orbitrap mass spectrometry and ultrahigh performance liquid chromatography with tandem mass spectrometry. In Quadrupole‐Orbitrap mass spectrometry analysis, target compounds were confirmed using a combination of retention time, mass tolerance, mass accuracy, and fragment ions. For quantification, several validation parameters were employed using ultrahigh performance liquid chromatography with tandem mass spectrometry. The limit of detection and limit of quantitation was 0.05–53 and 0.17–177 ng/mL, respectively. The correlation coefficient for linearity was more than 0.995. The intra‐ and interassay accuracies were 86–110 and 84–111%, respectively. Their precision values were evaluated as within 4.0 (intraday) and 10.7% (interday). Mean recoveries of target compounds in adulterated products ranged from 85 to 116%. The relative standard deviation of stability was less than 10.7% at 4°C for 48 h. The 144 adulterated products obtained over 3 years (2014–2016) from online and in‐person vendors were tested using established methods. After rapidly screening with Quadrupole‐Orbitrap mass spectrometry, the detected samples were quantified using ultrahigh performance liquid chromatography with tandem mass spectrometry. Two of them were adulterated with phenobarbital.     

One‐step strategy for the synthesis of a derivatized cyclodextrin‐based monolithic column

Derivatized β‐cyclodextrin (β‐CD) functionalized monolithic columns were prepared by a “one‐step” strategy using click chemistry. First, the intended derivatized β‐CD monomers were synthesized by a click reaction between propargyl methacrylate and mono‐6‐azido‐β‐CD and then sulfonation or methylation was carried out. Finally, monolithic columns were prepared through a one‐step in situ copolymerization of the derivatized β‐CD monomer and ethylene glycol dimethacrylate. The sulfated β‐CD‐based monolith was successfully applied to the hydrophilic interaction liquid chromatography separation of nucleosides and small peptides, while the methylated β‐CD‐functionalized monolith was useful for the separation of nonpolar compounds and drug enantiomers in capillary reversed‐phase liquid chromatography. The structures of the monomers were characterized by Fourier transform infrared spectroscopy and mass spectrometry. The physicochemical properties and column performance of monoliths were evaluated by scanning electron microscopy and micro high performance liquid chromatography. This strategy has considerable prospects for the preparation of other derivatized CD‐functionalized methacrylate monoliths.     

Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Extraction and isolation of acetylcholinesterase inhibitors from Citrus limon peel using an in vitro method

A simple and efficient ultrafiltration–liquid chromatography–mass spectrometry–based method was developed for the rapid screening and identification of ligands from Citrus limon peel, which are suitable acetylcholinesterase inhibitors. Subsequently, the anti‐Alzheimer's activity of these compounds was assessed using a PC12 cell model. Six major compounds, viz. neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin, were identified as potent acetylcholinesterase inhibitors. A continuous and efficient online method, which involved the use of a microwave‐assisted extraction device, solvent concentration tank, and centrifugal partition chromatography column, was developed for the scale‐up of these compounds, and the obtained compounds presented high purity. Next, their bioactivity was evaluated using a PC12 cell model. This novel approach, which was based on ultrafiltration–liquid chromatography–mass spectrometry, microwave‐assisted extraction online coupled with solvent concentration tank, and centrifugal partition chromatography along with in vitro evaluation, could represent a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices, and could be a useful platform for the large‐scale production of bioactive and nutraceutical ingredients.     

Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry,and complex chromatography

Nonsteroidal anti‐inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti‐inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase‐2. The cyclooxygenase‐2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase‐2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi‐preparative liquid chromatography was developed for the efficient scaled‐up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase‐2 inhibitors from complex samples. This could be used as an efficient method for the large‐scale production of functional ingredients.     

Trypsin immobilization on an ethylenediamine-based monolithic minidisk for rapid on-line peptide mass fingerprinting studies

The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.     

Pseudotargeted screening and determination of constituents in Qishen granule based on compound biosynthetic correlation using UHPLC coupled with high‐resolution MS

Detection and determination of many known/unknown compounds in traditional Chinese medicines have always been challenging. To comprehensively identify compounds in Qishen granule, which is a widely prescribed herbal formula for treating chronic heart failure, a pseudotargeted screening method was proposed based on compound biosynthetic correlation using ultra high‐performance liquid chromatography coupled with high‐resolution mass spectrometry. Firstly, all possible compounds of Qishen granule were classified into nine types according to their core skeletons, and potential analogue molecular formulas were predicted according to core compound‐related biosynthetic correlations, such as methylation, hydroxylation, and glucosidation. Secondly, nine pseudocompound databases consisting of core compounds, deduced biosynthetic correlations, and predicted analogue molecular formulas were established. Then, compounds of interest were directly located by pseudotargeted screening of high resolution mass spectrometry data and further verified by target tandem mass spectrometry. As a result, 213 constituents were identified and 21 of them were determined as potential new compounds. This demonstrated that pseudotargeted screening based on compound biosynthetic correlations significantly facilitated the processing of extremely large information data and improved the efficiency of compound identification. This research provided essential data for exploration of effective substances in Qishen granule and enriched the methodology for comprehensive characterization of constituents in complex traditional Chinese medicines.     

LC‐MS analysis for the components captured by ECV304 cell from extract of Aconitum szechenyianum Gay.

A novel method of cell affinity screening (CAS), cell affinity capture coupled with LC‐MS analysis, was developed for screening the bioactive compounds related to cardiovascular diseases from the natural product libraries. One of the major characteristics lies in its function in affinity‐capturing and separating the bioactive components from the natural product libraries in vitro. Another characteristic is its use in analyzing and identifying the target compounds, by employing high‐performance liquid chromatography and mass spectrometry. CAS was used for screening the bioactive components from the alkaloid extract derived from Aconitum szechenyianum Gay. Of the five components found to be bound to the oxidative‐damaged endothelial cells, the two compounds identified, mesaconitine and aconitine, were recognized in the literature as being related to cardiovascular diseases. Copyright © 2008 John Wiley & Sons, Ltd.     

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

Rapid screening,separation, and detection of hydroxyl radical scavengers from total flavonoids of Ginkgo biloba leaves by chromatography combined with molecular devices

Hydroxyl radicals are the most reactive free radical of human body, a strong contributor to tissue damage. In this study, liquid chromatography coupled to electrospray ionization mass spectrometry was applied to screen and identify hydroxyl radical scavengers from the total flavonoids of Ginkgo biloba leaves, and high‐performance counter current chromatography was used to separate and isolate the active compounds. Furthermore, molecular devices were used to determine hydroxyl radical scavenging activities of the obtained hydroxyl radical scavengers and other flavonoids from G. biloba leaves. As a result, six compounds were screened as hydroxyl radical scavengers, but only three flavonoids, namely, rutin, cosmos glycosides and apigenin‐7‐O‐Glu‐4’‐O‐Rha, were isolated successfully from total flavonoids by high‐performance counter current chromatography. The purities of the three obtained compounds were over 90%, respectively, as determined by liquid chromatography. Molecular devices with 96‐well microplates evaluation indicated that the 50% scavenging concentration values of screened compounds were lower than that of other flavonoids, they performed greater hydroxyl radical scavenging activity, and the evaluation effects were consistent with the liquid chromatography with mass spectrometry screening results. Therefore, chromatography combined with molecular devices is a feasible and an efficient method for systematic screening, identification, isolation, and evaluation of bioactive components in mixture of botanical medicines.     

Simultaneous determination of phenolic compounds in Equisetum palustre L. by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid‐phase dispersion extraction

A method based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with tandem mass spectrometry is presented for the extraction and determination of phenolic compounds in Equisetum palustre. This method combines the high efficiency of matrix solid‐phase dispersion extraction and the rapidity, sensitivity, and accuracy of ultra high performance liquid chromatography with tandem mass spectrometry. The influential parameters of the matrix solid‐phase dispersion extraction were investigated and optimized. The optimized conditions were as follows: silica gel was selected as dispersing sorbent, the ratio of silica gel to sample was selected to be 2:1 (400/200 mg), and 8 mL of 80% methanol was used as elution solvent. Furthermore, a fast and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the determination of nine phenolic compounds in E. palustre. This method was carried out within <6 min, and exhibited satisfactory linearity, precision, and recovery. Compared with ultrasound‐assisted extraction, the proposed matrix solid‐phase dispersion procedure possessed higher extraction efficiency, and was more convenient and time saving with reduced requirements on sample and solvent amounts. All these results suggest that the developed method represents an excellent alternative for the extraction and determination of active components in plant matrices.