Gradient Elution Of Methanol Research Articles

Rapid screening and confirming carcinogenic banned azo colorants in textiles by high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry

A method of high performance liquid chromatography-linear ion trap/orbitrap highresolution mass spectrometry (HPLC-LTP/Orbitrap MS) was ued to screen and confirm-banned azo colorants in textiles rapidly. The analytes were reduced to carcinogenic aromatic amines with sodium dithionite in citrate buffer solution. The reduced solution was extracted bydiatomite, and loadd onto an Acquity UPLC BEH C18 column (50 mm x 2.1 MM. 1.7 microm) with a gradient elution of methanol and 0.1% (v/v) methane acid aqueous solution, and finally detected by linear ion trap/orbitrap high-resolution mass spectrometry in positive ESI mode. In mass spectrometry method, the MS spectrum of high-resolution and the collision induced dissociation (CID) spectrum of data-dependent scan mode were used for screening analysis and conformation, respectively. The calibration curves showed a good linearity in the range of 0.05 -2.00 mg/b, and the correlation coefficients (r) were higher than 0.99. By detecting spiked samples, the limits of quantification were 0.08 mg/kg for all the residues and the recoveries were in the range of 65.5% - 111.5% with the relative standard deviations (RSDs) between 0.87% and 2.49%. The results indicate that the method is simple, rapid, sensitive and suitable for the qualitative and quantitative analysis of carcinogenic aromatic amines in textiles.

Simultaneous quantification of six major flavonoids from Fructus sophorae by LC-ESI-MS/MS and statistical analysis

A new, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method has been developed for the determination of six major flavonoids including sophoricoside, genistin, genistein, rutin, quercetin, kaempferol in Fructus sophorae. Principal component analysis and hierarchical clustering analysis were used to classify and differentiate these samples. Chromatographic separation was performed on a C18 column with linear gradient elution of methanol and 0.05% acetic acid (v/v) at a flow rate of 0.8 ml/min. The detection was accomplished in the negative mode using multiple-reaction monitoring. The total run time was 8.0 min. Full validation of the assay was carried out including linearity, precision, accuracy, recovery, limit of detection and limit of quantification. The validated method was successfully applied to the simultaneous determination of these active components in Fructus sophorae. The results demonstrated that the quantitative difference in content of six active compounds was useful for chemotaxonomy of many samples from different sources and the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by high-performance liquid chromatography–tandem mass spectrometric method coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of Fructus sophorae.

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods

A novel method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed-phase C(18) column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.

An in vitro Study on the DNA Damaging Effects of Phytochemicals Partially Isolated from an Extract of Glinus lotoides

An extract of Glinus lotoides, a medicinal plant used in Africa and Asia for various therapeutic purposes, was recently shown to cause DNA damage in vitro. To further explore the potential genotoxicity of this plant, fractionation of the crude extract was performed using reverse phase solid-phase extraction and a stepwise gradient elution of methanol in water. Four fractions were collected and subsequently analysed for their DNA damaging effects in mouse lymphoma cells using an alkaline version of the comet assay. To identify potential genotoxic and non-genotoxic principles, each fraction was then subjected to liquid chromatography coupled to mass spectrometry, LC-MS/MS. 1D and 2D nuclear magnetic resonance analyses were used to confirm the identity of some saponins. Although fractions containing a mixture of flavonoids and oleanane-type saponins or oleanane-type saponins alone produced no DNA damage, those containing hopane-type saponins exhibited a pronounced DNA damaging effect without affecting the viability of the cells. To conclude, even if this study presents evidence that hopane-type of saponins are endowed with a DNA damaging ability, further studies are needed before individual saponins can be cited as a culprit for the previously reported genotoxicity of the crude extract of G. lotoides.

Simultaneous Determination of Four Main Isosteroidal Alkaloids of Bulbus Fritillariae Cirrhosae in Rat Plasma by LC–MS–MS

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for simultaneous quantitation of four isosteroidal alkaloids (peimine, peiminine, peimisine, and delavine) in rat plasma. Carbamazepine was selected as internal standard (IS). The LC–MS–MS system coupled with an electrospray ionization (ESI) source was performed in the multiple reaction monitoring (MRM) mode. The transitions of the isosteroidal alkaloids executed as following: m/z 432.4 → 414.4 for peimine, m/z 430.5 → 412.4 for peiminine, m/z 428.4 → 114.2 for peimisine, m/z 416.5 → 105.2 for delavine, and m/z 237.2 → 194.2 for carbamazepine (IS). Sample preparation was performed with liquid–liquid extraction using ethyl acetate after carbamazepine (IS) spiked. The separation was performed on a Welch C18 column (3.5 μm, 2.1 × 100 mm), and a gradient elution of methanol and 5 mmol L−1 ammonium acetate in 0.1 % formic acid aqueous solution was used. The retention time was less than 8.0 min. Linearity was obtained over the concentration range of 0.2–200 ng mL−1 for peimine and peiminine, and 1.0–200 ng mL−1 for peimisine and delavine. The method was linear for all analytes with correlation coefficients >0.995. The intra-day and inter-day accuracy and precision of the assay were acceptable. This method has been successfully applied to the pharmacokinetic study of Bulbus Fritillariae Cirrhosae extract after oral administration to rats.

Simultaneous analysis of eight phenolic compounds in Phyllanthus simplex Retz by HPLC-DAD-ESI/MS

A new, simple and sensitive high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed for the simultaneous determination of eight phenolic components in Phyllanthus simplex Retz. The chemical profile of the eight components, including gallic acid, caffeic acid, quercetin, ferulic acid, luteolin-7-O-glucoside, 8,9-epoxy brevifolin, brevifolin and globulariacitrin were acquired by using high-performance liquid chromatography-diode array detector coupled to an electrospray ionization mass spectrometer (HPLC-DAD-ESI/MS). The optimal condition of separation was achieved on a ZORBAX Extend C 18 column (5 µm, 250◊4.6 mm i.d.) with a gradient elution of methanol and 1% acetic acid at a flow rate of 1.0 ml/min. Under ultra violent (UV) detection at 275 nm, the recoveries of the analytes were in the range of 99.2 to 99.8% with a RSD of 0.41 to 1.50%. The limits of detection and quantification for the analytes were ranged from 55 to 140 ng/ml and from 155 to 320 ng/ml, respectively. The intra-day RSD% ranged from 0.32 to 0.82% and the inter-day RSD% were not higher than 5.0%. The proposed method is suitable for quantitative and qualitative determination of the eight bioactive phenolic components in P. simplex Retz.

Rapid and efficient purification of chrysophanol in Rheum Palmatum LINN by supercritical fluid extraction coupled with preparative liquid chromatography in tandem

Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25× higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210atm and 85°C for 30min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10mL/min. According to 1H NMR and LC–MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88–91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38mg/g) and the shortest run time (3h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future.

Simultaneous Determination of Cortisol, Cortisone, and 6β-Hydroxycortisol in Human Urine by UPLC with UV Detector and Application to Determine Diurnal Variations

In the present studies, a simple rapid ultra performance liquid chromatography (UPLC) method with UV detection for the simultaneous determination of cortisol, cortisone and 6β-hydroxycortisol in human urine was developed. The three analytes and the internal standard dexamethasone were separated on a Waters Acquity UPLC-Tunable UV system with an Acquity UPLC BEH C18 column (50 × 2.1 mm ID, 1.7 μm) using a gradient elution of methanol and water (containing 0.01% formic acid) with a run time of 7 min. The method was accurate and precise, over the ranges of 5–200 ng mL−1 for cortisol, and 10–1,000 ng mL−1 for both cortisone and 6β-hydroxycortisol, and showed good linearity (r 2 > 0.999). This method was applied for the measurement of cortisol, cortisone and 6β-hydroxycortisol in samples collected over different periods as a tool to assess the activity of CYP3A and 11β-hydroxysteroid dehydrogenase type 2 enzymes.

Identification and Quantitative Analysis of Nucleosides and Nucleobases in Aqueous Extracts of Fritillaria Cirrhosa D. Don. Using HPLC–DAD and HPLC-ESI-MS

Bulbus Fritillariae cirrhosae is the most widely used medicinal herb for antitussive and apophlegmatic in China and commonly prepared as a water decoction. For clarifying the water-soluble components in Fritillaria cirrhosa D. Don., a simple and sensitive method was developed using high performance liquid chromatography coupled with a photodiode array detector (HPLC-DAD) and an electrospray ionization mass spectrometer (HPLC-ESI/MS) for the quantitative determination and identification of 10 nucleosides and nucleobases. Of the 10 compounds, two (2-deoxyadenosine and hypoxanthine) were identified and determined in F. cirrhosa for the first time. The 10 compounds were separated using a Zorbax SB-Aq column with a gradient elution of methanol and water. All the calibration curves showed good linearity (r2 ≥ 0.9991). The recoveries were between 97.7% and 103.5%. The method was validated with respect to precision, repeatability and accuracy, and was successfully applied to the simultaneous determination of the 10 analytes in 24 batches of F. cirrhosa, collected from different parts of China. This report provided a firm basis for clarifying the pharmacological effect and comprehensively evaluating the quality of F. cirrhosa and related preparations.

Determination of 7 nipagin ester preservatives in leather by ultra performance liquid chromatography-tandem mass spectrometry coupled with gel permeation chromatographic clean-up

A novel method has been developed for the rapid separation and determination of 7 nipagin ester preservatives in leather by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with gel permeation chromatographic (GPC) clean-up. Nipagin ester preservatives in leather were extracted by ultrasonic extraction with methanol. The extract was dried by a rotavapor and purified by GPC, then redissolved in the solvent of methanol-water (1 : 1, v/v). The chromatographic analysis was performed on an Acquity UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 microm) with a gradient elution of methanol and water as the mobile phases. The analytes were detected by electrospray ionization (ESI) tandem mass spectrometry with multiple reaction monitoring (MRM) in negative ion mode. Good linearity (r > 0.99) was observed between 0.1 and 1.0 mg/L for all the analytes. The recoveries and relative standard deviations (RSDs) were checked by spiking samples with the 7 nipagin ester preservatives at the three levels of 0.5, 1.0 and 3.0 mg/kg. The average recoveries of the 7 nipagin ester preservatives were from (79.44 +/- 5.67)% to (98.07 +/- 9.50)%. The precision values expressed as RSD ranged from 4.24% to 14.00% (n = 6). The limits of detection were 4 -12 microg/kg and the limits of quantification were 13.2 - 39.6 microg/kg for the analytes. The method is simple, rapid, sensitive and accurate, and suitable for the quantitative determination and confirmation of 7 nipagin ester preservatives in leather.

Catanionic surfactant ambient cloud point extraction and high-performance liquid chromatography for simultaneous analysis of organophosphorus pesticide residues in water and fruit juice samples

A mixed anionic-cationic surfactant cloud point extraction (CPE) has been developed using sodium dodecyl sulfate (SDS) and tetrabutylammonium bromide (TBABr) for the extraction and preconcentration of organophosphorus pesticides (OPPs) at ambient temperature before analysis by high-performance liquid chromatography. The studied OPPs were azinphos-methyl, parathion-methyl, fenitrothion, diazinon, chlorpyrifos, and prothiophos. The optimum conditions of the mixed anionic-cationic CPE were 50 mmol L(-1) SDS, 100 mmol L(-1) TBABr, and 10% (w/v) NaCl. The extracted OPPs were successfully separated within 11 min using the conditions of a Waters Symmetry C8 column, a flow rate of 0.8 mL min(-1), a gradient elution of methanol and water, and detection at 210 nm. Linearity was found over the range 0.05-5 μg mL(-1), with the correlation coefficients higher than 0.996. The enrichment factor of the target analytes was in the range 6-11, which corresponds to their limits of detection from 1 to 30 ng mL(-1). High precisions (intra-day and inter-day) were obtained with relative standard deviation <1.5% (t(R)) and 10% (peak area). Accuracies (% recovery) of the different spiked OPP concentrations were 82.7-109.1% (water samples) and 80.3-113.3% (fruit juice samples). No contamination by the OPPs was observed in any studied samples.

Simultaneous quantification of flavonoids and phenolic acids in Herba Scutellariae barbatae and its confused plants by high performance liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography–tandem mass spectrometric method (HPLC–MS/MS) has been developed for simultaneous analysis of 11 components (7 flavonoids and 4 phenolic acids) in Herba Scutellariae barbatae. Simultaneous separation of these 11 compounds was achieved on a C18 analytical column with gradient elution of methanol and 0.1‰ acetic acid (v/v) at a flow rate of 0.8mL/min. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in negative mode using multiple-reaction monitoring (MRM). Full validation of the assay was carried out including linearity, precision, accuracy, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid and specific. Then the method was successfully applied to differentiate 18 batches of Herba Scutellariae barbatae samples and 3 batches of its confused plants from different sources.

Sensitive measurement of vinorelbine in dog plasma by liquid chromatography–electrospray ionization tandem mass spectrometry utilizing transitions from double‐charged precursor ions

A sensitive high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for measuring vinorelbine was developed. A 100 µL aliquot of plasma was spiked with deuterium-labeled internal standard and subjected to solid-phase extraction using an Oasis HLB μ-elution plate. Two microliters of the extracted samples was directly injected into LC/MS/MS. Chromatographic separation was achieved on a Capcell Pak C18 UG column (2 × 75 mm) with a gradient elution of methanol (mobile phase B) against 0.05% formic acid in aqueous 10 mm ammonium formate (mobile phase A). The LC flow rate was set to 0.28 mL/min and the gradient (solvent B concentration) was processed from 40 to 90%. In mass spectrometric detection, observation of the reaction from a double-charged precursor ion [M + 2H](2+) (m/z 390) to product ion m/z 122 provided very high sensitivity. The method was validated with a lower limit of detection of 0.2 ng/mL with 0.1 mL of plasma, and the method was used to determine the plasma pharmacokinetics of vinorelbine in dogs.

Solid‐phase extraction and ultra high‐performance liquid chromatography tandem mass spectrometry analysis of the gastrointestinal absorption of emodin in different digestive segments of rats

A rapid, simple and sensitive ultra high-performance liquid chromatography (UHPLC-MS/MS) method was established for determining the absorption amount of emodin in the five digestive segments of rat. Plasma samples were pre-purified by solid-phase extraction (SPE) with Oasis MAX cartridge. Separation of emodin and 1,8-dihydroxyanthraquinone (internal standard) was performed on an Acquity BEH UHPLC C(18) column (100 mm × 2.1 mm, 1.7 μm) with gradient elution of methanol and 0.1% formic acid aqueous solution. The LC/MS system was operated under multiple reaction monitoring mode using electrospray ionization (ESI) in negative ion mode. The results showed that this established method was valid and sensitive for emodin within 0.04-16.4 μg/mL, with low limits of detection and quantification of 0.6 ng/mL and 2.4 ng/mL, respectively and upper limit of quantification of 220.0 ng/mL. The intra- and interday variations were below 4.9% of RSD. The extraction recoveries were 98.9-106.1% with RSD of 1.9-3.2%. The plasma concentration-time relationship showed that the absorption of emodin in stomach was faster than in intestine segments. The sequence of absorption amount was: ileum>jejunum>colon≈duodenum>stomach. Most of emodin was absorbed in ileum, and the absorption amount was increased with prolonged retention of drug form in intestine, especially in ileum and jejunum. The developed UHPLC-ESI-MS/MS method was appropriate for determining the in vivo absorption of emodin in other herbal medicines or preparations containing this compound.

RP-LC with Fluorescence Detection of Amino Acids in Rat Brain Synaptosomes

For the first time, a simple and validated reversed-phase liquid chromatography (RP-LC) with fluorescence detection has been developed for the simultaneous analysis of glutamate (Glu), γ-aminobutyric acid (GABA), glycine (Gly) and taurine (Tau) in Wistar and tremor rats brain synaptosomes. The samples were separated on a C18 analytical column with gradient elution of methanol and 0.1 mol L−1 potassium acetate at a flow rate of 1 mL min−1. Total run time was approximately 25 min. All calibration curves exhibited good linearity (r 2 > 0.999) within test ranges. The reproducibility was estimated by intra-and inter-day assays and RSD values were less than 2.48%. The recoveries were between 96.32 and 105.21%. The method was successfully applied to the quantification of amino acids in Wistar and tremor rats brain synaptosomes. Through this developed protocol, the levels of Glu in hippocampal and prefrontal cortical synaptosomes of tremor rats were both significantly elevated than those of adult Wistar rats whereas significantly decreased concentrations of GABA and Gly were observed in the hippocampal region of tremor rats without evident difference in the prefrontal cortex between experimental and control groups. In addition, our studies also showed a marked elevation of Tau in tremor rats hippocampal synaptosomes although there was no pronounced difference in the prefrontal cortical region of Wistar and tremor rats.

Simultaneous Identification and Quantification of Canrenone and 11‐α‐Hydroxy‐Canrenone by LC‐MS and HPLC‐UVD

A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r2 ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.

Establishment of a Drug Screening System by LC/MSn—Applications of a Liquid Chromatography/Mass Spectrometry System based on Retention Indices—

As the first step to develop a liquid chromatography/multiple-stage mass spectrometry (LC/MSn) analytical system which allows us to simultaneously screen relevant drugs and poisons in forensic specimens, an efficient and practical analytical method for correcting LC retention data has been produced utilizing a liquid chromatography/mass spectrometry (LC/MS). The method is based on retention indices (RI). LC/MS employed a C18 (ODS) semi-micro column with a methanol gradient elution program, and the RI values of target substances were calculated on the basis of the retention times of a substance and tri-n-alkylamines, which were chosen as reference markers (internal standards) and respectively assigned RI values. The present study has indicated that RI provides a very reproducible method for correction of retention data. This system has also been found to be a potentially important tool in simultaneously correcting LC retention data of target substances even when we modify the LC conditions such as the solvent-gradient and flow rate. This method will, thus, be successfully incorporated into routine work for forensic investigation.

The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts

Water, water/methanol (1/1), methanol and ethanol crude extracts from a brown alga Stypocaulon scoparium were examined for total phenolic contents (TPC) using Folin–Ciocalteu method. DPPH scavenging assay was performed to measure the radical scavenging activities (RSA) of the extracts. Results showed a significant association between the antioxidant potency and the TPC. The aqueous extract showed both, the highest antioxidant activity and highest phenolic contents. The identification and quantification of phenolic antioxidants were carried out with a rapid and simple method of reverse phase high performance liquid chromatography (RP-HPLC). This method was developed for the simultaneous analysis of 14 polyphenols, namely gallic acid, catechin, epicatechin, rutin, p-coumaric acid, myricetin, quercetin and protocatechuic, vanillic, caffeic, ferulic, chlorogenic, syringic and gentisic acids. The chromatographic separation of 14 polyphenols was achieved in less than 40 min by RP-HPLC (Varian, Pursuit XRs C18 column, 5 μm, 250 mm × 4.6 mm) using linear gradient elution of methanol and water (0.1% formic acid) with a flow rate of 1 ml/min. Gallic acid was by far the predominant polyphenol.

Simultaneous Determination of Triterpenoid Saponins from Pulsatilla koreana using High Performance Liquid Chromatography Coupled with a Charged Aerosol Detector (HPLC-CAD)

Several triterpenoid saponins from root of Pulsatilla koreana Nakai (Ranunculaceae) were studied and their biological activities were reported. It is difficult to analyze triterpenoid saponins using HPLC-UV due to the lack of chromophores. So, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we developed and validated a novel method of high performance liquid chromatography coupled with a charged aerosol detector for the simultaneous determination of four triterpenoid saponins: pulsatilloside E, pulsatilla saponin H, anemoside B4 and cussosaponin C. Analytes were separated by the Supelco Ascentis ® Express C18 column (4.6 mm × 150 mm, 2.7 µm) with gradient elution of methanol and water at a flow rate of 0.8 mL/min at 30 o C. We examined various factors that could affect the sensitivity of the detectors, including various concentrations of additives, the pH of the mobile phase, and the CAD range. Linear calibration curves were obtained within the concentration ranges of 2 - 200 µg/mL for pulsatilloside E, anemoside B4 and cussosaponin C, and 5 - 500 µg/mL for pulsatilla saponin H with correlation coefficient (R 2 ) greater than 0.995. The limit of detection (LOD) and quantification (LOQ) were 0.04 - 0.2 and 2 - 5 µg/mL, respectively. The validity of the developed HPLC-CAD method was confirmed by satisfactory values of linearity, intra- and inter-day accuracy and precision. This method could be successfully applied to quality evaluation, quality control and monitoring of Pulsatilla koreana.

Simultaneous quantification of 14 bioactive constituents in Forsythia Suspensa by liquid chromatography–electrospray ionisation–mass spectrometry

Forsythia suspensa is a commonly used traditional Chinese medicine including phenylethanoid glycosides, lignans, flavonoids, terpenes and volatile oils. Quantification of multi-components is important for the quality control of Forsythia suspensa. To establish a liquid chromatography-electrospray ionisation-mass spectrometry method for simultaneous quantification of 14 bioactive constituents of Forsythia suspensa in different places of China and different parts of this herb. The optimal chromatographic conditions were achieved on a Kromasil C(18) column (150 yen 4.6 mm, 5 microm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. Detection was performed in negative ionisation mode by monitoring the precursor-product combination in multiple reaction monitoring (MRM) mode. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. All calibration curves showed good linear regression (r > 0.9990) within test ranges. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.7-4.3 and 1.1-3.9% respectively, and overall recoveries of 96.65-101.2% for the compounds analysed. The proposed method was successfully applied for the quantitative analysis of 14 constituents in 12 Forsythia suspensa samples.