Simultaneous quantification and rat pharmacokinetics of formononetin‐7‐O‐β‐d‐glucoside and its metabolite formononetin by high‐performance liquid chromatography–tandem mass spectrometry

Formononetin‐7‐O‐β‐d ‐glucoside has been proved to have significant anti‐inflammatory effect. To evaluate its rat pharmacokinetics, a rapid, sensitive, and specific liquid chromatography–tandem mass spectrometry method has been developed and validated for the quantification of formononetin‐7‐O‐β‐d ‐glucoside and its main metabolite formononetin in rat plasma. Samples were pretreated using a simple protein precipitation and the chromatographic separation was performed on a C₁₈ column by a gradient elution using a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Both analytes were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. The assay showed wide linear dynamic ranges of both 0.10–100 ng/mL, with acceptable intra‐ and inter‐batch accuracy and precision. The lower limits of quantification were both 0.10 ng/mL using 50 μL of rat plasma for two analytes. The method has been successfully used to investigate the oral pharmacokinetic profiles of both analytes in rats. After oral administration of formononetin‐7‐O‐β‐d ‐glucoside at the dose of 50 mg/kg, it was rapidly absorbed in vivo and metabolized to its metabolite formononetin. The plasma concentration‐time profiles both showed double‐peak phenomena, which would be attributed to the strong enterohepatic circulation of formononetin‐7‐O‐β‐d ‐glucoside.     

Development of a sensitive ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of nine active compounds in rat plasma and its application to a pharmacokinetic study after administration of Viscum coloratum extracts

A simple, specific, and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous quantification of nine compounds including a new compound, rhamnazin‐3‐Ο‐β‐d ‐(6″‐β‐hydroxy‐β‐methyglutaryl)‐β‐d ‐glucoside‐4′‐Ο‐β‐d ‐glucoside, in rat plasma using baicalin as an internal standard. The plasma samples were pretreated and extracted by protein precipitation with 0.2% formic acid in acetonitrile. The analytes were separated on a Thermo Syncronis C₁₈ column by gradient elution with a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at a flow rate of 0.25 mL/min. The detection of the analytes was performed on an electrospray ionization interface operating in positive‐ion and multiple reaction monitoring acquisition modes. The calibration curves of these analytes showed good linearity (r > 0.99) within the test ranges. The lower limit of quantification ranged from 0.4 to 20.1 ng/mL for the analytes. The intra‐ and interday precision and accuracy were all within ±15%, and the recoveries were higher than 80.0%. The validated method was successfully applied to a pharmacokinetic study of the nine flavonoids after administration of the Viscum coloratum extracts by intravenous injection.     

A validated high‐performance thin‐layer chromatography method for the identification and simultaneous quantification of six markers from Platanus orientalis and their cytotoxic profiles against skin cancer cell lines

Betulinic acid ( 1 ), betulinic acid‐3‐acetate ( 2 ), 3‐acetylbetulinaldehyde ( 3 ), oleanolic acid‐3‐acetate ( 4 ), 3‐β‐hydroxy‐28,19‐β‐olenolide ( 5 ), and β‐sitosterol ( 6 ) were isolated from Platanus orientalis and a high‐performance thin‐layer chromatography method was developed for their simultaneous quantification. The markers were first derivatized on the chromatogram with ceric ammonium sulfate and then high‐performance thin‐layer chromatography densitometry was carried out. Chromatographic separation of these markers was carried out on silica gel 60 plates using a ternary solvent system n‐hexane/toluene/acetone (6:3.5:1 v/v/v) as a mobile phase. For marker 1 , a deuterium (D₂) lamp and wavelength of 420 nm was used. A tungsten (W) lamp was used for markers 2 and 3 at 550 nm and for 4 – 6 at 500 nm. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.9919). The precision evaluated by an intra‐ and interday study showed RSDs < 2.51% and accuracy validation recovery between 95.54 and 99.33% with RSDs < 1.55%. The successful application of the validated method showed 1 as the most abundant component (4.63%) and 5 (0.017%) the least. The markers displayed a significant cytotoxic effect against human keratinocyte, mouse melanoma, and human skin epithelial carcinoma cancer cells by using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay.     

Microwave‐assisted extraction coupled with countercurrent chromatography for the rapid preparation of flavonoids from Scutellaria barbata D. Don

As a famous Chinese herb having good inhibitory effects on numerous human cancers both in vitro and in vivo, Scutellaria barbata D. Don attracts extensive attention worldwide. In this work, four flavonoids named scutellarin, baicalin, luteolin, and apigenin were simply and rapidly prepared from S. barbata by microwave‐assisted extraction coupled to countercurrent chromatography. Extraction conditions including irradiation time, extraction temperature, liquid/solid ratio, and microwave power were optimized using an orthogonal array design method. The extract of S. barbata was separated and purified with a two‐phase solvent system composed of hexane/ethyl acetate/methanol/acetic acid/water (1:5:1.5:1:4, v/v/v/v/v) and 4.5 mg of scutellarin, 4.6 mg of baicalin, 1.1 mg of luteolin, 2.1 mg of apigenin were obtained from 2.0 g original sample in a single run. The purities of scutellarin, baicalin, luteolin, and apigenin determined by HPLC were 93.6, 97.3, 97.6, and 98.4%, respectively. The targeted compounds were identified by LC with MS and ¹H NMR spectroscopy. The total time including extraction, separation, and purification was <300 min. Compared to traditional methods, microwave‐assisted extraction coupled to countercurrent chromatography method is more simple and rapid for the extraction, separation, and purification of flavonoid compounds from natural products.     

Rapid characterization and determination of multiple components in Bu‐Shen‐Yi‐Qi‐Fang by high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry

In this study, a qualitative and quantitative analysis using high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry was performed for the quality control of Bu‐Shen‐Yi‐Qi‐Fang, a traditional Chinese formula used for asthma. Thirty‐four compounds, including flavonoids, isoflavonoids, triterpenoid saponins, and iridoid glycosides were identified or tentatively characterized by comparing their retention times and mass spectra with those of authentic standards or literature data. Sixteen components were considered as the main bioactive constituents of Bu‐Shen‐Yi‐Qi‐Fang and they were chosen as the chemical markers in quantitative analysis, including catalpol, leonuride, calycosin‐7‐O‐β‐d ‐glucoside, hyperoside, acteoside, formononetin‐7‐O‐β‐d ‐glucoside, epimedin A, calycosin, icariin, epimedin B, epimedin C, formononetin, astragaloside IV, astragaloside II, baohuoside‐I, and astragaloside I. The total run time was 20 min. It was found that the calibration curves for all analytes showed good linearity (R² > 0.99) within the test ranges. The relative standard deviations for intra‐ and inter‐day precisions were below 3.9 and 11.7%, respectively. The accuracy was evaluated by the recovery test within the range of 89.20–110.71% with the relative standard deviation < 4.8%. The sample was stable for at least 48 h at 4°C. The results showed that the new approach was effective for the quality control of Bu‐Shen‐Yi‐Qi‐Fang.     

Separation and purification of four flavonol diglucosides from the flower of Meconopsis integrifolia by high‐speed counter‐current chromatography

Flavonoids are the main components of Meconopsis integrifolia (Maxim.) Franch, which is a traditional Tibetan medicine. However, traditional chromatography separation requires a large quantity of raw M. integrifolia and is very time consuming. Herein, we applied high‐speed counter‐current chromatography in the separation and purification of flavonoids from the ethanol extracts of M. integrifolia flower. Ethyl acetate/n‐butanol/water (2:3:5, v/v/v) was selected as the optimum solvent system to purify the four components, namely quercetin‐3‐O‐β‐d‐ glucopyrannosy‐(1→6)‐β‐d‐ glucopyranoside (compound 1 , 60 mg), quercetin 3‐O‐[2’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 2 , 40 mg), quercetin 3‐O‐[3’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 3 , 11 mg), and quercetin 3‐O‐[6’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 4 , 16 mg). Among the four compounds, 3 and 4 were new acetylated flavonol diglucosides. After the high‐speed counter‐current chromatography separation, the purities of the four flavonol diglucosides were 98, 95, 90, and 92%, respectively. The structures of these compounds were identified by mass spectrometry and NMR spectroscopy.     

Determination of L‐trantinterol in rat plasma by using chiral liquid chromatography‐tandem mass spectrometry

A simple, sensitive, and rapid method for determination of L‐trantinterol in rat plasma was developed for the first time by using LC coupled to MS/MS based on chiral stationary phase. A baseline separation of the enantiomers of trantinterol was achieved on a Chirobiotic V column, using a mixture of acetonitrile–methanol–ammonia–acetic acid (80:20:0.01:0.02, v/v/v/v) as the mobile phase. The detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple reaction monitoring mode via ESI. The calibration curve was linear in concentration range from 0.270 to 108 ng/mL in plasma with the lower limit of quantification of 0.270 ng/mL. The intra‐ and interday precision (relative standard deviation) values were within 10.9% and the accuracy (relative error) was from 2.6 to 9.2% at all quality control levels. The method has been successfully applied to a study of L‐trantinterol pharmacokinetics in rats.     

Separation of three anthraquinone glycosides including two isomers by preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf

Anthraquinone glycosides, such as chrysophanol 1‐O‐β‐d‐ glucoside, chrysophanol 8‐O‐β‐d‐ glucoside, and physion 8‐O‐β‐d‐ glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above‐mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1‐O‐β‐d‐ glucoside and chrysophanol 8‐O‐β‐d‐ glucoside. This study demonstrated an efficient strategy based on preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography for the separation of the above‐mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.     

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Simultaneous determination of ten flavonoids of crude and wine‐processed Radix Scutellariae aqueous extracts in rat plasma by UPLC‐ESI‐MS/MS and its application to a comparative pharmacokinetic study

Radix Scutellariae (RS) is a herbal medicine with various pharmacological activities to treat inflammation, respiratory and gastrointestinal infections, etc. In this study, a rapid, sensitive and selective UPLC‐ESI‐MS/MS method was developed for simultaneous determination of 10 flavonoids – scutellarin, scutellarein, chrysin, wogonin, baicalein, apigenin, wogonoside, oroxylin A‐7‐O‐glucuronide, oroxylin A and baicalin – from RS aqueous extracts in rat plasma with propyl paraben as internal standard (IS). Chromatographic separation was achieved on a C₁₈ column using gradient elution with the mobile phase consisting of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detection was performed in multiple reaction monitoring mode using electrospray ionization in negative mode. The validated method showed good linearity over a wide concentration range (r >0.9935). The intra‐ and interday assay variabilities were <9.5% and <12.4% for all analytes, respectively. The extraction recovery ranged from 71.2 to 89.7% for each analyte and IS. This method was successfully applied to pharmacokinetic comparision after oral administration of crude and wine‐processed RS aqueous extracts. There were significant differences in some pharmacokinetic parameters of most analytes between crude and wine‐processed RS. This suggested that wine‐processing exerted effects absorption of most flavonoids. Copyright © 2014 John Wiley & Sons, Ltd.     

Constituents of the Whole Herb of Clinoponium laxiflorum

The isolation and identification of twenty‐two components (including one new compound) from the whole herb of Clinoponium laxiflorum (Hay) Matsum (Labiatae) are described. Their structures were determined on the basis of spectral and chemical transformation. One new compound is methyl rosmarinate. The other twenty‐one compounds include three steroids (α‐spinasterol, α‐spinasteryl‐3‐O‐β‐D‐glucopyranoside, and β‐sitosteryl‐3‐O‐β‐glucopyranoside), three triterpenes (oleanolic acid, ursolic acid, and betulinic acid), nine flavonoids (didymin, apigenin‐7‐O‐β‐glucopyranoside, luteolin‐7‐O‐β‐glucopyranoside, isosakuranetin, narigenin, apigenin, luteolin, narirutin, and hesperidin), three lignolic acids (rosmarinic acid, 3‐(3,4‐dihydroxyphenyl)lactic acid, and caffeic acid), and three phenols (4‐hydroxybenzaldehyde, 3,4‐dihydroxybenzaldehyde, and 3,4‐dihydroxybenzoic acid).     

Isolation,cytotoxic evaluation,and simultaneous quantification of eight bioactive secondary metabolites from Cicer microphyllum by high‐performance thin‐layer chromatography

Chemical investigation of Cicer microphyllum resulted in the isolation and characterization of eight natural products viz. Stigmasterol, Oleanolic acid‐3‐acetate, Oleanolic acid, Biochanin A, Genistein, Pratensein, Chrysoeriol, and Luteolin. Herein, we report a novel, accurate, and cost‐effective high‐performance thin‐layer chromatography method for the simultaneous quantification of the isolated natural products on silica‐gel 60F₂₅₄ plates using the solvent system n‐hexane/ethyl acetate/formic acid (9.0:6.5:0.8, v/v/v). Natural products were quantified after postchromatographic derivatization with ceric ammonium sulfate. The method was validated as per the International Conference on Harmonization guidelines. All calibration curves showed a good linear relationship (r > 0.9943) within the test range. Precision was assessed by intra‐ and interday tests with relative standard deviations <1.82%, accuracy validation recovery 98.38–99.57% with relative standard deviations <1.00%. On quantification, Pratensein was a major constituent (0.921%). The screening for cytotoxic activity using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay resulted into identification of Luteolin as potent molecule with IC₅₀ 3.5 and 25.6 μg/mL against murine melanoma and human epidermoid carcinoma cell lines, respectively.     

Pharmacokinetics of luteolin and tetra‐acetyl‐luteolin assayed by HPLC in rats after oral administration

Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra‐acetyl‐luteolin (TALT) in rat plasma. HPLC analyses were performed on an Agilent TC‐C₁₈ column protected by a guard Agilent Zorbax Eclipse Plus. The mobile phase for LT was a binary mixture of acetonitrile–water (40:60, v/v) containing 0.5% phosphoric acid at a flow rate of 1.0 mL/min, and that for TALT was a binary mixture of methanol–water (70 : 30, v/v) containing 0.5% glacial acetic acid at the same flow rate. The UV detection wavelength for both analytes was set at 350 nm. The calibration curve was linear over the range of 40–1800 ng/mL, the lower limit of quantitation was 40 ng/mL and the lower limit of detection was 20 ng/mL for both LT and TALT. The intra‐ and inter‐day precision (RSD) values for all samples were within 7.9%. The concentration–time curves of LT and TALT after oral administration (30 mg/kg) were both fitted to a two‐compartment model. The pharmacokinetic characteristics of TALT were better than that of LT in the maximum plasma concentration (C_max) and the area under the concentration–time curve (AUC). Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical Components of Seriphidium Santolium Poljak

A new biflavonoid glucoside, apigenin‐7‐O‐β‐D‐glucopyranoside‐(3′‐O‐7″)‐quercetin‐3″‐methyl ether ( 1 ) together with twenty known compounds, apigenin ( 2 ), luteolin ( 3 ), chrysoeriol ( 4 ), tricin ( 5 ), hispidulin ( 6 ), pectolinarigenin ( 7 ), eupatilin ( 8 ), 5,7‐dihydroxy‐6,3′,4′,5′‐tetramethoxyflavone ( 9 ), 5,7,4′‐trihydroxy‐6,3′,5′‐trimethoxyflavone ( 10 ), 3,6‐O‐dimethylquercetagetin‐7‐O‐β‐D‐glucoside ( 11 ), 6‐hydroxy‐5,7‐dimethoxy‐coumarin ( 12 ), taraxerol ( 13 ), taraxeryl acetate ( 14 ), a mixture of β‐sitosterol ( 15 ) and stigmasterol ( 16 ), a mixture of the n‐alkyl trans‐p‐coumarates ( 17 ), a mixture of the n‐alkyl trans‐ferulates ( 18 ), 2‐hydroxy‐4,6‐dimethoxyacetophenone ( 19 ), 4‐hydroxy‐2,6‐dimethoxyphenol‐1‐O‐β‐D‐glucopyranoside ( 20 ), and 2‐hydroxycinnamoyl‐β‐D‐glucopyranoside ( 21 ), were isolated from the whole plant of Seriphidium santolium Poljak. The structures of these compounds were determined by means of spectral and chemical studies.     

Specific Deuteration in Patuletin and Related Flavonoids via Keto–Enol Tautomerism: Solvent‐ and Temperature‐Dependent 1H‐NMR Studies

An H/D exchange process in patuletin ( 1 ) and its derivatives in D‐donor solvents (e.g., CF₃COOD), which occurs regioselectively at C(8) was observed for the first time during NMR studies. The effect of substituents and temperature on the deuteration of various flavonoids (see Fig. 1) which include apigenin, chrysin, galangin, kaempferol, luteolin, morin, myricetin, patuletin, patulitrin, and quercetin, as well as derivatives of patuletin was examined extensively under NMR conditions. The rate constant of deuteration at C(8) of patuletin ( 1 ) and two flavones, luteolin ( 3 ) and apigenin ( 12 ), was also determined in CF₃COOD. The D‐atom was introduced into the flavonoids via a keto–enol tautomerism (Scheme 1). During these studies, monodeuterated patuletin was also obtained as a new compound. The examined flavonoids have been reported to possess significant pharmacological activities, and their deuterated derivatives would be of importance for the identification and quantification of these compounds in biological matrices.     

Development of a novel dual CD‐MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.—Loranthaceae—extracts

The present work deals with the development and validation of a novel dual CD‐MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.—Loranthaceae—extracts. The BGE consisted of 20 mM pH 8.3 borate buffer, 50 mM SDS, a dual CD system based on the combination of 5 mM β‐CD and 2% w/v S‐β‐CD, and 10% v/v methanol. The proposed method has been successfully applied to the comparative analysis of extracts from aerial parts and different hosts, geographical areas, and extraction procedures in order to establish the flavonoid fingerprint of L. cuneifolia . The method was validated according to international guidelines. LOD and LOQ, intra and interday precision, and linearity were determined for catechin, epicatechin, procyanidin B2, rutin, quercetin‐3‐O‐glucoside, quercetin‐3‐O‐xyloside, quercetin‐3‐O‐rhamnoside, quercetin‐3‐O‐arabinofuranoside, quercetin‐3‐O‐arabinopyranoside, and quercetin. The CD‐MEKC methodology emerges as a suitable alternative to the traditional HPLC for quality control, fingerprinting, and standardization of L. cuneifolia extracts from different sources.     

Isolation,purification, and identification of the main phenolic compounds from leaves of celery (Apium graveolens L. var. dulce Mill./Pers.)

We developed a simple and meaningful preparative method for the separation and purification of the main phenolic compounds from the leaves of celery (Apium graveolens L. var. dulce Mill./Pers.) and we established an accurate and specific analytical method for the identification of the main phenolic compounds from celery leaves. The crude extract from celery leaves was prefractioned by polyamide resin to enrich the phenolic compounds. They were then purified further by preparative high‐performance liquid chromatography, and seven main phenolic compounds were obtained: including chlorogenic acid, luteolin 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐β‐d‐ glucopyranoside, apiin, chrysoeriol 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside, and apigenin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside. Their purities were measured by using high‐performance liquid chromatography, and their chemical structures were confirmed using UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy. Our studies indicate that preparative high‐performance liquid chromatography combined with polyamide resin is a simple and meaningful preparative method for the separation and purification of phenolic compounds from the leaves of celery or other plants, and the use of UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy is an accurate and specific analytical method for the identification of phenolic compounds.     

Ionic liquid vortex‐simplified matrix solid‐phase dispersion for the simultaneous determination of terpenoids,crocins, quinic acid derivatives and flavonoids in Gardeniae fructus by UHPLC

A novel ionic‐liquid‐based vortex‐simplified matrix solid‐phase dispersion method using 2,6‐dimethyl‐β‐cyclodextrin was established by ultra high performance liquid chromatography coupled with a photodiode array detector. 2,6‐Dimethyl‐β‐cyclodextrin was first used as a promising adsorbent in this proposed method for simultaneous determination of eight compounds in Gardeniae fructus. These compounds are terpenoids (geniposidic acid, genipin‐1‐β‐D‐gentiobioside, geniposide, 8‐o‐acetyl shanzhiside methyl ester), crocins (crocin‐I, crocin‐II), quinic acid derivatives (chlorogenic acid), and flavonoids (isoquercitrin), respectively. Several parameters were investigated in the adsorption and desorption processes to obtain the optimal conditions, including 2,6‐dimethyl‐β‐cyclodextrin as sorbent, 0.5 mL 100 mM 1‐dodecyl‐3‐methylimidazolium hydrogen sulfate as the extraction solvent, 2:1 of sample/sorbent ratio, grinding for 2 min and vortexing for 60 s. The recoveries of the eight compounds ranged from 96.6 to 100% (<3.50%). The limits of detection and quantification were in the range of 0.02–0.30 and 0.06–1.25  μg/mL, respectively. Meanwhile, a good linearity was attained with r values (>0.9997). The established method showed higher extraction efficiency and less reagent consumption than traditional matrix solid phase dispersion and ultrasonic‐assisted extraction. Hence, it could be applied for sample preparation and analysis of natural products.     

Development and validation of a high performance liquid chromatography quantification method of levo‐tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study

Levo ‐tetrahydropalmatine (l‐ THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l‐ THP and its desmethyl metabolites l‐ corydalmine (l‐ CD) and l‐ corypalmine (l‐ CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid–liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed‐phase Symmetry® C₁₈ column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile–methanol–10 mm ammonium phosphate (pH 3) (10:30:60, v /v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1–10,000 ng/mL. The intra‐ and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l‐ THP in rats. Taken together, the developed method can be applied for bioanalysis of l‐ THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.     

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2‐carboxyethyl)phosphine as a reducing agent by LC–MS/MS

A simple, robust, and rapid LC–MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2‐carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide‐bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel‐d₄ as an internal standard (IS). The mixtures were separated on a C₁₈ RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI‐MS. The detector was operated in selected reaction‐monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05–20 and 0.5–200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra‐ and interday, was less than 8.26% with an accuracy of 87.6–106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.