Simultaneous quantification of marker components in Ojeok-san by HPLC–DAD

A systematic high-performance liquid chromatography–diode array detection (HPLC–DAD) method was developed for the rapid, accurate, and simultaneous quantification of eight marker compounds, paeoniflorin, 6-gingerol, decursin, glycyrrhizin, cinnamic acid, hesperidin, poncirin and magnolol, in Ojeok-san, a traditional Korean herbal medicine. These compounds were separated in less than 50 min using a Dionex C₁₈ column with a gradient elution system of water and methanol at a flow rate of 1 ml/min. All calibration curve of standard components showed excellent linearity (R ² > 0.9922). Limits of detection (LOD) and limits of quantification (LOQ) ranged from 0.01 to 0.11 μg/ml and 0.03 to 0.34 μg/ml, respectively. The relative standard deviations (RSDs) of data of the intra- and inter-day experiments were less than 2.15 and 2.60%, respectively. The accuracy of recovery test ranged from 94.27 to 107.68% with RSD values 0.15–3.61%. The results of validation showed that the HPLC method was stable and very accurate for the quantification of eight marker components in Ojeok-san.     

Simultaneous analysis of bioactive metabolites from Ziziphus jujuba by HPLC–DAD–ELSD–MS/MS

A high-performance liquid chromatography (HPLC) with diode array detector (DAD), evaporative light scattering detector (ELSD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of nine representative metabolites of the alkaloid, flavonoid and triterpenoid classes from Ziziphus jujuba var. spinosa. The optimal chromatographic conditions were obtained on an ODS column and the mobile phase was composed of water and methanol with 0.1% formic acid using a gradient elution system. Using the developed methods, all of the validation parameters were successfully obtained. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of representative metabolites in commercial samples of Z. jujuba and Z. mauritiana from different markets in Korea, China and Myanmar. The analytical results showed that the contents of the nine analytes vary significantly with sources and species, thus demonstrating its potential for the detection of this plant.     

Simultaneous quantification of 19 diterpenoids in Isodon amethystoides by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method was developed to characterize and quantify 19 diterpenoid compounds in Isodon amethystoides simultaneously. By employing a Diamonsil C₁₈ column, 19 constituents were separated within 15 min using a gradient elution consisted of methanol containing 0.1% formic acid and 0.1% aqueous formic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode in a single run and quantified by a multiple-reaction monitor (MRM). All standard calibration curves showed good linearity (r² > 0.99) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.06–3.25% and 1.56–3.84%. The recovery studies for the quantified compounds were between 95.82 and 108.3% with RSD values less than 1.86%. The results indicated that the method is simple, rapid, specific and reliable. This method was successfully applied for identification and quantification of 19 diterpenoids in 11 batches of I. amethystoides. The results showed that the contents of diterpenoids in I. amethystoides from different sources were widely varied.     

Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC–DAD–ESI-MS

A high-performance liquid chromatography–diode array detection–mass spectrometry method with electrospray ionization mode (HPLC–DAD–ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC–DAD–ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70 min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile–water (94:6, v/v) with flow rate at 1 mL/min, column temperature at 30 °C and detection wavelength at 280 nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6 μg/g), followed by quercetin-3-O-rutinoside (281.3 μg/g), dicaffeoylquinic acid isomers (250.1 μg/g), chlorogenic acid (237.0 μg/g), quercetin-di-(rhamnohexoside) (117.5 μg/g), quercetin-di-(rhamno)-hexoside (116.8 μg/g), kaempferol-3-O-rutinoside (97.7 μg/g), isorhamnetin-3-O-rutinoside (72.1 μg/g), p-coumaric acid (64.0 μg/g), caffeic acid (23.7 μg/g) and vanillic acid (22.8 μg/g).     

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC–DAD and HPLC–ESI-MS

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC–DAD–ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC–ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids.     

Simultaneous quantification of antibiotic dyes in aquatic products and feeds by liquid chromatography–tandem mass spectrometry

A confirmatory and quantitative method based on liquid chromatography–tandem mass spectrometry (LC/MS/MS) has been developed for the determination of low-level residues of three antibiotic dyes and two metabolites in fish muscle and feed. The target compounds include methylene blue (MB), crystal violet (CV), leucocrystal violet (LCV), malachite green (MG), and leucomalachite green (LMG). The procedures involve solvent extraction by 50% McIlvaine’s buffer with acetonitrile, followed by solid phase extraction (SPE) with an MCX cartridge. High performance liquid chromatography (HPLC) and positive electrospray ionization (ESI) MS with multiple reaction monitoring of two transition reactions was applied for each compound. The detected ion ratios of MB, CV, LCV, MG, and LMG were 11.8, 34.9, 88.4, 25.6, and 42.0, respectively. The average fortification recoveries of the MB, CV, LCV, MG, and LMG of the level of 0.8 μg/kg tested in fish muscle and feed samples were 99.68, 98.93, 100.49, 100.01, and 100.00%, respectively. The precision of analysis of analytes in fish muscle and feed ranged from 4% to 14% and from 7% to 14%, respectively. The decision limits (CCα) were 0.28–0.54 μg/kg, and the detection capabilities (CCβ) were 0.35–0.67 μg/kg (n = 99).     

Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography–mass spectrometry

Si-Wu-Tang (SWT), comprising Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular Traditional Chinese Medicine (TCM) formulae for woman's health. Data mining from the available Chinese and English literatures indicated that the major bioactive components of SWT consist of paeoniflorin, paeonol, gallic acid, ferulic acid, Z-ligustilide, ligustrazine, butylphthalide, senkyunolide A and catalpol. Since content determination of the marker compounds is generally considered as an initial step for quality control of TCM product, a high performance liquid chromatography–mass spectrometric method employing both positive and negative electrospray ionization was developed for the simultaneous determination of the nine identified compounds in the raw herbs and products of SWT. The LOQ of the developed assay method for the tested components was 10 ng/ml for ligustrazine, 200 ng/ml for catalpol, and 100 ng/ml for the other seven compounds. The intra-day and inter-day variations of the current assay were within 17.5%. Paeoniflorin, ferulic acid, gallic acid, Z-ligustilide and senkyunolide A were found in all SWT products investigated. Variations in the contents of the studied compounds were observed among batches of raw herbs and SWT products. The currently developed method provides a sensitive and rapid quantification approach that can be useful in the quality control of raw herbs and products of SWT.     

Simultaneous determination of five lignan constituents of Wuzhi capsule in rat plasma by LC–MS/MS: Application to pharmacokinetic study

A rapid sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC–MS/MS method. Liquid–liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100 mm × 2.1 mm i.d., 3.5 μm) by isocratic elution using a mobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at a flow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r² > 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule.     

Gastrointestinal gene delivery by cyclodextrins – In vitro quantification of extracellular barriers

Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers.     

Simple and reproducible HPLC–DAD–ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical study on this species roots. Alkaloids in aqueous extracts, the usual form of consumption of this matrix, were studied using HPLC–DAD–ESI-MS/MS, which allowed the identification of 19-S-vindolinine, vindolinine, ajmalicine and an ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer. Quantification of the identified compounds revealed that serpentine and its isomer were predominant (64.7%) over the other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The used procedure revealed to be simple, sensitive and reproducible.     

Simultaneous quantification combined with multivariate statistical analysis of multiple chemical markers of Wu Ji Bai Feng Pill by UHPLC–MS/MS

Wu Ji Bai Feng Pill (WJBFP) is a traditional Chinese medicine (TCM) complex formula, which has been widely used in the treatment of various gynecological disorders. However, the quality control of multiple components in WJBFP is challengeable by using the methods applicable to analysis of several phytochemicals in single herbs or simple herbal preparations. The purpose of this study is to establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC–MS/MS) method for the quantitative determination of 20 bioactive compounds in WJBFP. The modified chromatographic conditions were achieved on an Agilent Poroshell 120 EC-C₁₈ column with a gradient elution consisted of 0.1% formic acid in acetonitrile and 0.1% aqueous formic acid (v/v). All analytes were determined using a triple quadrupole mass spectrometry in positive or negative ionization modes with multiple reaction monitoring (MRM) mode. An UHPLC–MS/MS method was optimized and validated for linearity, limits of detection and quantification, precision, repeatability, stability and recovery. The proposed method was applied for the analysis of 20 compounds in 19 batches of commercial WJBFP products. principal component analysis and hierarchical cluster analysis were applied to evaluate intrinsic quality and to identify chemical markers most responsible for quality evaluation. In conclusion, the established method offered speedy and sensitive determination for 20 compounds and is helpful for chemical standardization of commercial WJBFP products.     

Quantitative analysis of six polyynes and one polyene in Oplopanax horridus and Oplopanax elatus by pressurized liquid extraction and on-line SPE–HPLC

A pressurized liquid extraction and on-line SPE–HPLC method was developed for simultaneous determination of six polyynes, including falcarindiol, oplopandiol, (11S,16S,9Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate, oplopandiol acetate, oplopantriol A, oplopantriol B, and one polyene, (S,E)-nerolidol, in Oplopanax horridus and Oplpanax elatus. The analysis was conducted on a Grace Prevail C₁₈ column (3 μm, 7 mm × 33 mm) with gradient elution of acetonitrile and water after the sample loaded and washed with 42%ACN in 0.3 min on a phenomenex Strata-X on-line Extraction Cartridge SPE column (2.5 μm, 2.0 × 20 mm). All calibration curves of seven analytes showed good linearity within the test ranges. The validated method was successfully applied to quantify six polyynes and one polyene in two species of Oplopanax, O. horridus and O. elatus.     

Simultaneous quantification of enalapril and enalaprilat in human plasma by high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to simultaneously determine enalapril and enalaprilat in human plasma. With benazepril as internal standard, sample pretreatment involved in a one-step protein precipitation (PPT) with methanol of 0.2 ml plasma. Analysis was performed on an Ultimate™ XB-C₁₈ column (50 mm × 2.1 mm, i.d., 3 μm) with mobile phase consisting of methanol–water–formic acid (62:38:0.2, v/v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction-monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for enalapril and enalaprilat were both obtained in the concentration range of 0.638–255 ng/ml (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.638 ng/ml. The intra-day precision (R.S.D.) was below 7.2% and inter-day R.S.D. was less than 14%, while accuracy (relative error R.E.) was within ±8.7 and ±5.5%, determined from QC samples for enalapril and enalaprilat which corresponded to requirement of the guidance of FDA. The HPLC–MS/MS method herein described was fully validated and successfully applied to the pharmacokinetic study of enalapril maleate capsules in 20 healthy male volunteers after oral administration.     

Simultaneous quantification of losartan and active metabolite in human plasma by liquid chromatography–tandem mass spectrometry using irbesartan as internal standard

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing electronspray ionization was developed and validated for quantification of losartan and its carboxylic acid metabolite in human plasma using irbesartan as internal standard (IS). Following a simple pretreatment procedure, the analytes were separated using a gradient mobile phase on reverse phase C₁₈ column. Selected reaction monitoring was specific for losartan, losartan acid and irbesartan. The method validation demonstrated the specificity, lower limit of quantification, accuracy and precision of measurements. The assay exhibited a linear dynamic range of 2.0–400 ng/mL for losartan and 1.85–370 ng/mL for losartan acid. A run time of 3.5 min for each sample made it possible to analyze more than 200 samples per day. The validated method has been successfully used to analyze human plasma samples for application in bioavailability/bioequivalence studies.     

Quantitative determination of flavonoids and cycloartanol glycosides from aerial parts of Sutherlandia frutescens (L.) R. BR. by using LC-UV/ELSD methods and confirmation by using LC–MS method

This paper describes the first analytical method for the determination of four flavonoids (sutherlandins A–D) and four cycloartanol glycosides (sutherlandiosides A–D) from the aerial parts of Sutherlandia frutescens (L.) R. Br. A separation by HPLC was achieved by using a reversed phase (RP-18) column, PDA with ELS detection, and a water/acetonitrile gradient as the mobile phase. The wavelength used for quantification of four flavonoids with the diode array detector was 260 nm. Owing to their low UV absorption, the cycloartanol glycosides were detected by evaporative light scattering. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The limits of detection and limits of quantification of eight compounds were found to be in the range from 0.1 to 7.5 μg/mL and 0.5 to 25 μg/mL, respectively. The analysis of products showed considerable variation of 1.099–5.224 mg/average weight for the major compound, sutherlandioside B. The eight compounds in plant sample and products of S. frutescens were further confirmed by LC–ESI-TOF. This method involved the use of the [M+H]⁺ and [M+Na]⁺ ions in the positive ion mode with extractive ion monitoring (EIM).     

Determination of nucleosides and nucleobases in different species of Cordyceps by capillary electrophoresis–mass spectrometry

In present study, a capillary electrophoresis–mass spectrometry (CE–MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2′-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 μL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 °C, respectively. The optimized CE–MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138 ± 4823 μg/g for cultured C. sinensis; 3722 ± 1446 μg/g for C. militaris) than in natural ones (2167 ± 412 μg/g). However, the hypoxanthine (131 ± 47 μg/g) and inosine (335 ± 90 μg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5 ± 842.6 μg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.     

Development and validation of an HPLC–UV method for the quantification of carbamazepine in rabbit plasma

An isocratic simple rapid assay has been developed and validated for the determination of carbamazepine (CBZ) in both solution form and rabbit plasma using propylparaben as an internal standard. The assay was performed using a μ-Bondapak C₁₈ (150 mm × 4.6 mm i.d) with a mobile phase consisting of methanol and water (50:50), the flow rate was 1 ml/min and UV detection at 285 nm. The method was found to be specific for CBZ, no interfering peaks were observed with an overall analytical run time of 15 min. Accuracy reported as % recovery were found to be 98.37–100.45% and 97.53–103.58% for inter-day and intra-day accuracies, respectively. Inter-day precision (reproducibility) was found to be 0.53–2.75% RSD, while intra-day precision (repeatability) was found to be 1.06–3.7% RSD for the samples studied. The calibration curve was found to be linear with the equation y = 0.2847x + 0.0138, with a correlation coefficient of 0.9999 (R²) over a concentration range of 0.5–40 μg/ml. The limit of quantitation was the lowest concentration. The method is simple and rapid and does not require any preliminary treatment of the sample. The method was fully validated.     

Simultaneous determination and pharmacokinetic analysis of seven alkaloids and two flavonoids from rat plasma by HPLC–DAD after oral administration of Wuzhuyu decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic analysis of seven alkaloids dehydroevodiamine (DHED), 10-hydroxyrutaecarpine (HDR), evodiamine (EDM), rutaecarpine (RCP), 1-methyl-2-n-nonyl-4(1H)quinolone (MNQ), evocarpine (ECP), and dihydroevocarpine (DHE), and two flavonoids isorhamnetin-7-O-rutinoside (RIM) and diosmetin-7-O-β-d-glucopyranoside (GRD) in rat plasma after oral administration of Wuzhuyu decoction. The flow rate was kept at 1.0 ml/min and the detection wavelength was set at 300 nm. The calibration curves were linear in the range of 0.5013–30.076 μg/ml for DHED, 0.2161–21.608 μg/ml for RIM, 0.161–12.876 μg/ml for HDR, 0.2146–21.457 μg/ml for GRD, 2.0464–40.928 μg/ml for EDM, 1.0398–31.194 μg/ml for RCP, 0.5970–35.818 μg/ml for MNQ, 0.8371–20.928 μg/ml for ECP, and 0.5167–31.003 μg/ml for DHE. The precision (relative standard deviation (RSD), %) for all was less than 10% and the accuracy (relative error (RE), %) was within ± 10%. The results demonstrated that the assay had remarkable reproducibility with acceptable accuracy and precision. The lower limit of quantifications for the compounds in plasma ranged from 0.12 to 0.23 μg/ml and the lower limit of detections ranged from 0.024 to 0.076 μg/ml. This validated method has been successfully applied in the pharmacokinetics study of seven alkaloids and two flavonoids after orally administrating the Wuzhuyu decoction to rats.