Simultaneous determination of eight bioactive alkaloids in Corydalis saxicola by high-performance liquid chromatography coupled with diode array detection

Corydalis saxicola Bunting (Papaveraceae), a traditional folk medicine, has been used to treat hepatic diseases for a long time. Owing to its signicant clinical effectiveness against hepatitis, cirrhosis and hepatoma, C. saxicola and its preparation are widely applied. In this study, eight alkaloids, namely isocorydine, scoulerine, dehydrocheilanthifoline, dehydrodiscretamine, dehydroisoapocavidine, dehydrocavidine, palmatine and berberine, which have been previously proven to possess potential antitumour activity, were selected as the chemical markers of C. saxicola. To evaluate the quality of C. saxicola, a simple, accurate and reliable HPLC-DAD method was developed for the simultaneous determination of the above eight compounds. Separation was achieved on a Gemini C(18) column (5 microm, 250 x 4.6 mm i.d., Phenomenex Inc., CA, USA) with a gradient solvent system of 20 mM aqueous ammonium acetate-acetonitrile, at a flow-rate of 1.0 mL/min and detected at 270 and 280 nm. All eight calibration curves showed good linearity (R(2) > 0.9992). The method was reproducible with intra- and inter-day variations of less than 5%. The recovery was in the range of 96.09-102.80%. This assay was successfully utilised to quantify the eight alkaloids in C. saxicola from different locations. The results demonstrated that this method is simple, reliable and suitable for the quality control of this medicinal herb.     

Fast high-performance liquid chromatographic analysis of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.     

Simultaneous quantitation of five flavonoid glycosides in Herba Epimedii by high-performance liquid chromatography-tandem mass spectrometry

A rapid and accurate reversed-phase liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the quantitative determination of five flavonoid glycosides, icariin, epimedin A, epimedin B, epimedin C and hyperin in Herba Epimedii. Chromatographic separations were performed using a C(18) narrow-bore HPLC column; a mixture of an aqueous solution of ammonium formate (pH 4.0) and acetonitrile was used as the mobile phase, with compounds detected in the positive ion mode with multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with an electrospray ionisation interface. This method for the determination of the reported flavonoid glycosides was accurate and reproducible, with a lower limit of quantication of 0.5 microg/mL. The standard calibration curves for the above-mentioned compounds were linear (r(2) > 0.998) over the concentration range 0.5-10.0 microg/mL. The relative standard deviations for intra- and inter-day precision over the concentration range for the flavonoid glycosides were lower than 7.8% with accuracy between 90.1 and 111.0%. The established method was successfully applied to the quality assessment of samples of Herba Epimedii collected from Korea and China.     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

Two benzophenone O-arabinosides and a chromone from Hypericum annulatum

Two benzophenone O-arabinosides, annulatophenonoside (1) and acetylannulatophenonoside (2) were isolated from the methanol extract of the herb of Hypericum annulatum. The structures of the benzophenones were established as 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone (1) and 2-O-alpha-L-3"-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone (2) based on spectral and chemical evidence. A chromone, 5,7-dihydroxy-3-methylchromone (3) was isolated from the chloroform extract. Although it has been previously synthesized it is encountered in a plant source for the first time. Co-occurrence of the two new benzophenone O-arabinosides along with the biogenetically related 1,5,7-trihydroxy-3-methoxyxanthone was not found.     

A high-performance liquid chromatography with electrochemical detection for the determination of total hypericin in extracts of St. John's Wort

An HPLC method for the quantitation of hypericin using a new and sensitive amperometric detection is presented. Hypericin was eluted isocratically using a mobile phase consisting of ammonium acetate, methanol and acetonitrile. The oxidation was carried out with a glassy carbon electrode at a potential of + 1.1 V vs. an Ag-AgCl-KCl reference electrode. Under the conditions described, hypericin was separated at a retention time (Rt) of 12 min. Linearity was obtained over the range 0.035-1.30 microg/mL (r = 0.9994). The limit of detection was determined to be 0.010 ng on-column for hypericin. The method was applied to the determination of total hypericin (hypericin, pseudohypericin, protohypericin and protopseudohypericin) in extracts of St. John's wort using hypericin as an external standard. The protoforms were converted into hypericin and pseudohypericin by subjecting the sample to artificial light prior to chromatographic analysis. For the evaluation of total hypericin, the peak areas of pseudohypericin (Rt 3.7 min) and hypericin (Rt 12.0 min) were combined. The relative standard deviation in analysing samples containing Hypericum ranged from 2.5 to 5.4%.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Simultaneous determination of three naphthoquinones in the leaves of Impatiens balsamina L. by reversed-phase high-performance liquid chromatography

Introduction – Naphthoquinones; lawsone ( 1 ), lawsone methyl ether ( 2 ) and methylene‐3,3′‐bilawsone ( 3 ) are the main active compounds of Impatiens balsamina leaves. Objective – To develop and validate an HPLC method for simultaneous quantitative determination of 1 – 3 in I. balsamina leaf extracts. Methodology – The method utilised a Supelco® C₁₈ column (5 µm, 4.6 × 150 mm) at 25°C with the mixture of 2% aqueous acetic acid : methanol (gradient elution as follows: 0–10 min, 25 : 75; 10–20 min, 32 : 68; 20–35 min, 55 : 45) as the mobile phase at a flow‐rate of 1 mL/min, and UV detection at 280 nm. The parameters of linearity, repeatability, reproducibility, accuracy specificity and sensitivity of the method were evaluated. Results – The recovery of the method was 96–101% and linearity (r² ≥ 0.9995) was obtained for all naphthoquinones. A high degree of specificity, as well as repeatability and reproducibility (RSD less than 5%), were also achieved. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Free malonaldehyde determination in tissues by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of free malonaldehyde (MA) in tissues. HPLC separation was performed using a TSK G1000 PW column (7.5-mm i.d. X 30 cm) with a mobile phase of 0.1 M Na3PO4 buffer, pH 8.0, at a flow rate of 0.6 ml/min. The eluant was monitored at 267 nm. Free MA in the tissue sample was separated and quantified in approximately 50 min. The lowest amount of MA that can be determined by this HPLC technique is approximately 1 ng per injection. This method was successfully applied to rat liver and beef, pork, and chicken muscle and was compared to the thiobarbituric acid (TBA) test. It was found to be more sensitive, accurate, and specific for the determination of free MA than the TBA method.     

Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), deltaalpha,beta-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 microg/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus.     

Simultaneous determination of catechins in human saliva by high-performance liquid chromatography

Green tea extracts have been suggested to possess a preventive effect against dental caries. A quantitative method for their anticariogenic substances, catechins, was developed to evaluate their concentrations in human saliva after mouthrinsing with green tea extract. Salivary catechins were extracted to the organic phase after forming a complex with diphenylborate and an ion-pair with tetra-n-butylammonium, and then back-extracted to the acidic aqueous phase. The extract was analyzed by high-performance liquid chromatography using diode array detection at absorption wavelengths ranging from 269 to 278 nm. In reversed-phase chromatography by a gradient elution, eight catechins originating from green tea and an internal standard were separated in 15 min without interfering peaks. All the catechins were simultaneously and selectively determined in the concentration range 0.05–25.0 μg/ml. In replicate spiking experiments with standards, the mean recovery ranged between 86 and 99%, and both intra- and inter-assay C.V.s were within 2.3%. When mouthrinsing with an aqueous solution of green tea extract (5.0 mg/ml) containing eight catechins, the quantitative results revealed that each catechin was retained at μg/ml levels in saliva for up to 60 min.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography

A specific, sensitive, single-step solid-phase extraction and reversed-phase high-performance liquid chromatographic method for the simultaneous determination of plasma 6-mercaptopurine and azathioprine concentrations is reported. Following solid-phase extraction, analytes are separated on a C₁₈ column with mobile phase consisting of 0.8% acetonitrile in 1 mM triethylamine, pH 3.2, run on a gradient system. Quantitation limits were 5 ng/ml and 2 ng/ml for azathioprine and 6-mercaptopurine, respectively. Peak heights correlated linearly to known extracted standards for 6-mercaptopurine and azathioprine (r = 0.999) over a range of 2–200 ng/ml. No chromatographic interferences were detected.     

Simultaneous determination of misonidazole and desmethylmisonidazole in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of misonidazole and desmethylmisonidazole in plasma is described. After plasma is deproteinized with methanol and the diluted supernatant is chromatographed on a C₁₈ reversed-phase column, both compounds are quantitated by means of an internal standard. The coefficients of variation of within-day and day-to-day precision are below 5.0% for misonidazole in the concentration range of 25–250 mg/l and below 6.1% for desmethylmisonidazole in the concentration range of 2.5–25.0 mg/l. Calibration curves are linear and an analytical recovery varying from 97.6 to 99.8% is obtained. The detection limits for misonidazole and desmethylmisonidazole in plasma are 1.4 mg/l and 0.7 mg/l, respectively.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Simultaneous determination of catecholamines in rat brain tissue by high-performance liquid chromatography

A novel and highly sensitive method has been developed for the determination of catecholamines [noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA)] in brain tissue. The method uses isocratic reversed-phase HPLC with amperometric end-point detection. The calibration curve was linear over the range 10–150 pg on-column. The assay limits of detection for NA, DA, 5-HT, 5-HIAA and HVA were 3.8, 3.8, 6.8, 5 and 7.5 pg on-column, respectively. The mean inter- and intra-assay relative standard deviations (RSDs) over the range of the standard curve were less than 5%. The absolute recoveries averaged 99.1%, 99.5%, 97.7%, 99.5% and 98.8% for NA, DA, 5-HT, 5-HIAA and HVA, respectively.