Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

Homoisoflavonoids profiling of Ophiopogon japonicus by off‐line coupling high‐speed countercurrent chromatography with high‐performance liquid chromatography–diode array detector‒quadrupole time‐of‐flight tandem mass spectrometry

Roots of Ophiopogon japonicus have been used as a functional food ingredient and traditional Chinese medicine for a long time in China. Homoisoflavonoids are one of the major kinds of bioactive compounds in O. japonicus; however, literature data about its homoisoflavonoids profile are scarce because of the complex ingredients with low abundance. Here, homoisoflavonoid fraction was prepared by petroleum ether extraction. Then, a high‐speed countercurrent chromatography off‐line coupling with high‐performance liquid chromatography–diode array detector?quadrupole time‐of‐flight tandem mass spectrometry was developed for systematic identification of homoisoflavonoids. After that, 39 homoisoflavonoids, including 29 homoisoflavanone and 10 homoisoflavone, were unambiguously or tentatively identified, while 12 of them were reported in O. japonicus for the first time. Finally, eight available homoisoflavonoids were sensitively, precisely, and accurately determined by standard calibration curves, with limit of detection and limit of quantification in the range of 0.05–0.30 μg/mL and 0.12–0.66 μg/mL, relative standard deviation less than 7.3% for intra‐ and interday variations, and recovery at 94.5–105.2%. Collectively, our developed method is efficient, reliable, and valuable to profile chemical components of complex natural products.     

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.     

Separation of acteoside and linarin from Buddlejae Flos by high‐speed countercurrent chromatography and their anti‐inflammatory activities

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high‐speed countercurrent chromatography with a two‐phase solvent system composed of ethyl acetate: n‐butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one‐step high‐speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization‐mass spectrometry and nuclear magnetic resonance. After separation, the anti‐inflammatory effects of the two compounds were evaluated using lipopolysaccharide‐induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1β, which demonstrated that acteoside and linarin possessed anti‐inflammatory activity.     

Non‐polar lipids characterization of Quinoa (Chenopodium quinoa) seed by comprehensive two‐dimensional gas chromatography with flame ionization/mass spectrometry detection and non‐aqueous reversed‐phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection

A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non‐aqueous reversed‐phase high‐performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two‐dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high‐resolution time‐of‐flight mass spectrometry.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Preparative isolation and purification of hainanmurpanin,meranzin, and phebalosin from leaves of Murraya exotica L. using supercritical fluid extraction combined with consecutive high‐speed countercurrent chromatography

The objective of this study was to develop a consecutive preparation method for the isolation and purification of hainanmurpanin, meranzin, and phebalosin from leaves of Murraya exotica L. The process involved supercritical fluid extraction with CO₂, solvent extraction, and two‐step high‐speed countercurrent chromatography. Pressure, temperature, and the volume of entrainer were optimized as 27 MPa, 52°C, and 60 mL by response surface methodology in supercritical fluid extraction with CO₂, and the yield of the crude extracts was 7.91 g from 100 g of leaves. Subsequently, 80% methanol/water was used to extract and condense the three compounds from the crude extracts, and 4.23 g of methanol/water extracts was obtained. Then, a two‐step high‐speed countercurrent chromatography procedure was developed for the isolation of the three target compounds from methanol/water extracts, including conventional high‐speed countercurrent chromatography for further enrichment and consecutive high‐speed countercurrent chromatography for purification. The yield of concentrates from high‐speed countercurrent chromatography was 2.50 g from 4.23 g of methanol/water extracts. Finally, the consecutive high‐speed countercurrent chromatography produced 103.2 mg of hainanmurpanin, 244.7 mg of meranzin, and 255.4 mg of phebalosin with purities up to 97.66, 99.36, and 98.64%, respectively, from 900 mg of high‐speed countercurrent chromatography concentrates in one run of three consecutive sample loadings without exchanging a solvent system.     

Isolation of a furan fatty acid from Hevea brasiliensis latex employing the combined use of pH‐zone‐refining and conventional countercurrent chromatography

Furan fatty acids are valuable and bioactive minor fatty acids that usually contribute <0.1% to the fatty acid content of food samples. Their biological role still remains unclear as authentic furan fatty acid standards are not readily available and thorough experimental studies verifying the relevance of furan fatty acids are thus virtually impossible. An efficient protocol for the isolation of the furan fatty acid 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from hydrolyzed and centrifuged latex of Hevea brasiliensis was developed using countercurrent chromatography. A first run using pH‐zone‐refining countercurrent chromatography provided 48.4 mg of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from 210 mg latex extract in a purity of 95%. The purity was increased to 99% by means of one second run in conventional countercurrent chromatography mode. The Structure and purity of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid were determined by gas chromatography coupled to mass spectrometry and ¹H and ¹³C NMR spectroscopy.     

Effective on‐line high‐speed shear dispersing emulsifier technique coupled with high‐performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits

An efficient combination strategy based on high‐speed shear dispersing emulsifier technique and high‐performance countercurrent chromatography was developed for on‐line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high‐speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high‐performance countercurrent chromatography solvent system composed of n‐hexane?dichloromethane?acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high‐performance counter‐current chromatography process adopts elution–extrusion mode and the upper phase of the solvent system as stationary phase (reverse‐phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction‐separation operation within 120 min. The targeted compounds were analyzed and identified by high‐performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on‐line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

Extraction and purification of five terpenoids from olibanum by ultrahigh pressure technique and high‐speed countercurrent chromatography

Five terpenoids, including two new ones, 3,7‐dioxo‐tirucalla‐8,24‐dien‐21‐oic acid ( 2 ) and 3α‐acetoxyl‐7‐oxo‐tirucalla‐8,24‐dien‐21‐oic acid ( 3 ), and three known ones, boscartol A ( 1 ), 11‐keto‐β‐boswellic acid ( 4 ), and acetyl‐11‐keto‐boswellic acid ( 5 ), have been extracted by the ultrapressure extraction and purified by pH‐zone‐refining countercurrent chromatography and high‐speed countercurrent chromatography from olibanum. For ultrapressure extraction, the optimal condition including 200 MPa of extraction pressure, ethyl acetate of extraction solvent, 1:20 (g/mL) of solid/liquid ratio, and 2 min of extraction time were obtained. For the separation, from 1.5 g of the terpenoid extract, 220.1 mg of 4 , 255.5 mg of 5 , and 212.3 mg of the mixture of 1 , 2 , and 3 were obtained by pH‐zone‐refining countercurrent chromatography under the solvent system of chloroform/ethyl acetate/methanol/water (3:1:3:2, v/v/v/v) with aqueous ammonia and trifluoroacetic acid as retention and eluter agents. The enriched mixture (210 mg) was further separated by conventional high‐speed countercurrent chromatography with petroleum ether/ethyl acetate/methanol/water (1:0.8:1.1:0.6, v/v/v/v), yielding 30.1 mg of 1 , 35.5 mg of 2 , 12.3 mg of 3 . The structures of these five terpenoids were elucidated by extensive spectroscopic methods.     

Silver ion coordination countercurrent chromatography: Separation of β‐elemene from the volatile oil of Curcumae Rhizoma

In this work, the antitumor constituent β‐elemene was selectively separated from the volatile oil of the Curcumae Rhizoma by countercurrent chromatography with silver nitrate as selective reagent based on the formation of coordination complexes. A biphasic solvent system composed of n‐hexane/methanol/water (2:1.5:0.5, v/v/v) was selected, in which 0.15 mol/L of silver nitrate was added to the aqueous phase. The aqueous phase was used as the stationary phase for separation of β‐elemene by countercurrent chromatography after it was partially purified from the volatile oil by silica gel column chromatography. An enriched β‐elemene fraction was obtained by silica gel column chromatography to improve the percentage of β‐elemene from 16.5 to 46.1%. Subsequently, β‐elemene was further purified from 445 mg of the partially purified sample of volatile oil by countercurrent chromatography with silver nitrate as a selective reagent, yielding 145 mg of β‐elemene with greater than 99% purity, as determined by gas chromatography mass spectrometry. The recovery of β‐elemene from the crude volatile oil through two steps was around 63.6%.     

Ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography for screening and isolating potential α‐glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α‐glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α‐glucosidase‐ and xanthine oxidase binding activities and one lactone with α‐glucosidase‐binding activity. Subsequently, with the help of high‐speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low‐toxicity two‐phase solvent system composed of ethyl acetate/n‐butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography is not only a powerful tool for screening and isolating α‐glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.     

Developments in high‐speed countercurrent chromatography and its applications in the separation of terpenoids and saponins

High‐speed countercurrent chromatography is a liquid–liquid separation chromatographic technique, which has the unique feature of eliminating irreversible adsorption using liquid support medium, and is widely used in research and development of traditional Chinese medicine, biochemistry, food, environment analysis, and so on. In this review, some new developments of countercurrent chromatography, for instance cross‐axis countercurrent chromatography, dual countercurrent chromatography, foam countercurrent chromatography, and pH‐zone‐refining countercurrent chromatography are presented. Furthermore, the research and progress in high‐speed countercurrent chromatography techniques and its application in the separation and purification of terpenoids and saponins are reviewed.     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

Fast determination of octinoxate and oxybenzone uv filters in swimming pool waters by gas chromatography/mass spectrometry after solid‐phase microextraction

A fast gas chromatography/mass spectrometry method was developed and validated for the analysis of the potential endocrine disrupters octinoxate and oxybenzone in swimming pool water samples based on the solvent‐free solid‐phase microextraction technique. The low‐pressure gas chromatography/mass spectrometry method used for the fast identification of UV filter substances was compared to a conventional method in terms of sensitivity and speed. The fast method proposed resulted in 2 min runs, leading to an eightfold decrease in the total analysis time and a sevenfold improvement in detection limits. The main parameters affecting the solid‐phase microextraction process were also studied in detail and the optimized conditions were as follows: fiber coating, polyacrylate; extraction mode, direct immersion; extraction temperature, 25°C; sample volume, 5 mL; extraction time 45 min; pH 6.5. Under the optimized conditions, a linear response was obtained in the concentration range of 0.5–25 μg/L with correlation coefficients in the range 0.990–0.999. The limits of detection were 0.17–0.29 μg/L, and the recoveries were 80–83%. Combined method uncertainty was assessed and found to be less than 7% for both analytes for concentrations equal to or higher than 5 μg/L. Pool water samples were analyzed to demonstrate the applicability of the proposed method. Neither octinoxate nor oxybenzone were detected in the swimming pool water samples at concentrations above the respective limits of detection.     

Simultaneous determination of four antiepileptic drugs in human plasma samples using an ultra‐high‐performance liquid chromatography tandem mass spectrometry method and its application in therapeutic drug monitoring

Therapeutic drug monitoring of antiepileptic drugs is widely practiced to achieve optimal efficacy and avoid adverse side effects. We describe an ultra‐high‐performance liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method developed for the monitoring of four frequently prescribed antiepileptic drugs – lamotrigine, levetiracetam, oxcarbazepine and topiramate. The main pharmacologically active metabolite of oxcarbazepine (mono‐hydroxy‐derivative metabolite, MHD) was also quantified. After addition of internal standards and a simple stage of protein precipitation, plasmatic samples were analyzed on a C₁₈ column. All antiepileptic drugs were separated and quantified in 6 min, without interference. A good linearity was observed all over the calibration range (r² > 0.99), up to 20 μg/mL (40 μg/mL for MHD). The limit of quantification was 0.20 μg/mL (0.40 μg/mL for MHD) with precision and accuracy ranging from 1.0 to 2.1% and from 96.7 to 110.8%, respectively. Intra‐ and inter‐day precision and accuracy values were within 15%. No significant matrix effect was observed for all analytes. Clinical application was successfully evaluated in 259 samples from patients treated for epilepsy or bipolar disorders. In conclusion, a rapid, specific and sensitive UHPLC/MS/MS method was developed and validated for simultaneous quantification of antiepileptic drugs, suitable for therapeutic drug monitoring in neurology and psychiatry.     

Residue determination of plant activator benzo‐1,2,3‐thiadiazole‐7‐ carboxylic acid 2‐benzoyloxyethyl ester in water,soil, and vegetable by gas chromatography with tandem mass spectrometry

This study aimed to develop a rapid, specific and sensitive method for the residue determination of benzo‐1,2,3‐thiadiazole‐7‐carboxylic acid 2‐benzoyloxyethyl ester in soil, vegetable, and water by using gas chromatography with tandem mass spectrometry. During the extraction procedure, modified quick, easy, cheap, effective, rugged, and safe method using acetonitrile was compared with classical oscillating extraction using ethyl acetate and acetone, respectively. Before injection, a solid‐phase extraction cartridge of Bond Elut Florisil was used for sample clean‐up. The method was fully validated and showed satisfactory linearity (r² > 0.99) over the range to be assayed (10–1000 ng/mL), with the limits of detection ranging from 0.092–0.229 ng/mL and the limits of quantification ranging from 0.307–0.763 ng/mL. Recovery values at the spiked concentrations of 10, 100 and 1000 ng/g varied from 85.9–109.3%, 81.0–108.2%, 74.2–113.4% for water, soil and vegetable, respectively, with the maximum relative standard deviations of 12.2%. Results indicated that the established modified quick, easy, cheap, effective, rugged, and safe method coupled to gas chromatography with tandem mass spectrometry was promising for the residue monitoring of benzo‐1,2,3‐thiadiazole‐7‐carboxylic acid 2‐benzoyloxyethyl ester in the environment and vegetable products.     

Isolation and purification of alkaloids from lotus leaves by ionic‐liquid‐modified high‐speed countercurrent chromatography

An effective high‐speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic‐liquid‐modified high‐speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C₄mim][BF₄] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N‐nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic‐liquid‐modified high‐speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.     

Simultaneous determination of ten antiepileptic drugs in human plasma by liquid chromatography and tandem mass spectrometry with positive/negative ion‐switching electrospray ionization and its application in therapeutic drug monitoring

A simple, rapid, and high‐throughput liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of ten antiepileptic drugs in human plasma has been developed and validated. The method required only 10 μL of plasma. After simple protein precipitation using acetonitrile, the analytes and internal standard diphenhydramine were separated on a Zorbax SB‐C₁₈ column (50 × 4.6 mm, 2.7 μm) using acetonitrile/water as the mobile phase at a flow rate of 0.9 mL/min. The total run time was 6 min for each sample. The validation results of specificity, matrix effects, recovery, linearity, precision, and accuracy were satisfactory. The lower limit of quantification was 0.04 μg/mL for carbamazepine, 0.02 μg/mL for lamotrigine, 0.01 μg/mL for oxcarbazepine, 0.4 μg/mL for 10‐hydroxycarbazepine, 0.1 μg/mL for carbamazepine‐10,11‐epoxide, 0.15 μg/mL for levetiracetam, 0.06 μg/mL for phenytoin, 0.3 μg/mL for valproic acid, 0.03 μg/mL for topiramate, and 0.15 μg/mL for phenobarbital. The intraday precision and interday precision were less than 7.6%, with the accuracy ranging between –8.1 and 7.9%. The method was successfully applied to therapeutic drug monitoring of 1237 patients with epilepsy after administration of standard antiepileptic drugs. The method has been proved to meet the high‐throughput requirements in therapeutic drug monitoring.