Sensitive Reversed-phase High-performance Liquid Chromatography Research Articles

Method development and validation for simulataneous estimation of albendazole and praziquantel in bulk and in a synthetic mixture

A simple, rapid, sensitive reversed-phase high-performance liquid chromatography method was developed and validated for simultaneous measurement of albendazole and praziquantel with an internal standard, simvastatin, at single wavelength of 225nm. Chromatographic separation was performed on an Enable C18 column (250mm×4.6mm, 5μm: Spinco Biotech Pvt Ltd) and a mobile phase consisting of acetonitrile:water (60:40, v/v) with 10% orthophosphoric acid to adjust the pH to 3.2, at a flow rate of 1.0ml/min. The calibration curve was linear (r2≥0.999) over the concentration range 0.05–8.0μg/ml. The concentrations of simvastatin was 1.0μg/ml. The limit of quantification was 0.05μg/ml for both albendazole and praziquantel. No interference was found by the excipients in the synthetic mixture. The proposed methods were validated as per International Conference on Harmonisation guidelines for linearity, accuracy, precision and robustness for estimation of albendazole and praziquantel in bulk and in a synthetic mixture, and the results were found to be satisfactory.

Simultaneous determination of metolazone and losartan potassium in their binary mixtures using high‐performance liquid chromatography with fluorimetric detection: application to combined tablets and spiked human plasma

A new, specific and sensitive reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of metolazone (MET) and losartan potassium (LOS). Good chromatographic separation was achieved within 6.0 min on a 150 × 4.6 mm i.d., 5 µm Waters, Ireland and ProDIGY 5 ODS 3 100 A column. A mobile phase containing a mixture of methanol and 0.02 M phosphate buffer (65:35, v/v) at pH 3.0 was used. The analysis was performed at a flow rate of 1 mL/min with fluorescence detection at 410 nm after excitation at 230 nm. Aspirin (ASP) was used as an internal standard. The proposed method was rectilinear over 2.0-40.0 (MET) and 40.0-800.0 ng/mL (LOS), with limits of detection of 0.22 and 4.52 ng/mL and limits of quantification of 0.68 and 13.70 ng/mL for MET and LOS, respectively. The method was successfully applied for the simultaneous analysis of the studied drugs in their laboratory-prepared mixtures, single tablets and co-formulated tablets. Moreover, the method was applied to an in vitro drug release (dissolution) test. The method was further extended to the determination of LOS in spiked human plasma. Statistical evaluation and comparison of data obtained using the proposed and comparison methods revealed no significant difference between the two methods in addition to good accuracy and precision for the proposed method.

The pharmacokinetics and oral bioavailability studies of columbianetin in rats after oral and intravenous administration

Ethnopharmacological relevanceThe roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan (RAP) has been used as Traditional Chinese medicine to treat rheumatic disease in China since ancient times, but its action mechanisms was not well understood. Columbianetin is one of the main active constituents isolated from RAP, which has been shown to have various biological activities, but the absorption characteristics and oral bioavailability dose proportionality of columbianetin in vivo were not studied. Materials and methodsMale Sprague Dawley rats (210–230g) received either an intravenous (i.v. 5, 10 and 20mgkg−1) or oral (5, 10 and 20mgkg−1) dose of columbianetin. The levels of columbianetin in plasma were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The simple liquid–liquid extraction with ethyl acetate was used for sample preparation. Osthole was selected as internal standard (IS). ResultsThe chromatographic separation was accomplished on a C18 column at a flow rate of 1mLmin−1, where water–methanol was used as mobile phase. The calibration curve of the method was linear in the concentration range of 0.05–2000μgmL−1. The intra and inter-day accuracy for columbianetin in rat plasma samples were within 8% and the variation was less than 8.3%. This method was suitable for the determination and pharmacokinetic study of columbianetin in rat plasma after both intravenous and oral administration. The results indicated that maximum plasma concentrations(Cmax) for the columbianetin (17–42μgmL−1) were achieved at 0.3–0.5h post-oral dosing and the apparent volume of distribution (V/F) ranged from 0.38 to 0.44L. Absolute bioavailability of columbianetin was assessed to be 81.13±45.85, 81.09±33.63 and 54.30±23.19%, respectively. Terminal elimination half-life (T1/2) of the columbianetin after oral dosing was 60–90min and were 2.5–3.3 fold longer than those observed for the i.v. dosing. ConclusionsThe pharmacokinetic properties of columbianetin in rat after oral administration were characterized as rapid oral absorption, quick clearance and good absolute bioavailability. The bioavailability of columbianetin ranged from 54 to 81% for 5, 10 and 20mgkg−1 oral doses. The bioavailability of columbianetin is independent of the doses studied. Columbianetin showed dose proportionality over the dose range 5–20mgkg−1. The results clearly demonstrated that columbianetin was one of the material bases of RAP. Furthermore, an HPLC method was demonstrated in this study for the research of traditional Chinese medicine.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Rapid and Sensitive Reverse-phase High-performance Liquid Chromatography Method for Estimation of Ketorolac in Pharmaceuticals Using Weighted Regression

A reliable, rapid and sensitive isocratic reverse phase high-performance liquid chromatography method has been developed and validated for assay of ketorolac tromethamine in tablets and ophthalmic dosage forms using diclofenac sodium as an internal standard. An isocratic separation of ketorolac tromethamine was achieved on Oyster BDS (150×4.6 mm i.d., 5 μm particle size) column using mobile phase of methanol:acetonitrile:sodium dihydrogen phosphate (20 mM; pH 5.5) (50:10:40, %v/v) at a flow rate of 1.0 ml/min. The eluents were monitored at 322 nm for ketorolac and at 282 nm for diclofenac sodium with a photodiode array detector. The retention times of ketorolac and diclofenac sodium were found to be 1.9 min and 4.6 min, respectively. Response was a linear function of drug concentration in the range of 0.01-15 μg/ml (R2=0.994; linear regression model using weighing factor 1/x2) with a limit of detection and quantification of 0.002 μg/ml and 0.007 μg/ml, respectively. The % recovery and % relative standard deviation values indicated the method was accurate and precise.

Pharmacokinetics and Tissue Distribution Study of Praeruptorin D from Radix Peucedani in Rats by High-Performance Liquid Chromatography (HPLC)

Praeruptorin D (PD), a major pyranocoumarin isolated from Radix Peucedani, exhibited antitumor and anti-inflammatory activities. The aim of this study was to investigate the pharmacokinetics and tissue distribution of PD in rats following intravenous (i.v.) administration. The levels of PD in plasma and tissues were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The biosamples were treated by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) and osthole was used as the internal standard (IS). The chromatographic separation was accomplished on a reversed-phase C18 column using methanol-water (75:25, v/v) as mobile phase at a flow rate of 0.8 mL/min and ultraviolet detection wave length was set at 323 nm. The results demonstrate that this method has excellent specificity, linearity, precision, accuracy and recovery. The pharmacokinetic study found that PD fitted well into a two-compartment model with a fast distribution phase and a relative slow elimination phase. Tissue distribution showed that the highest concentration was observed in the lung, followed by heart, liver and kidney. Furthermore, PD can also be detected in the brain, which indicated that PD could cross the blood-brain barrier after i.v. administration.

Determination and Stability Assessment of Clarithromycin in Human Plasma using RP-LC with Electrochemical Detection

A selective, sensitive and stability-indicating reversed-phase high-performance liquid chromatography method was developed and validated for the determination of clarithromycin antibiotic in human plasma. Liquid chromatography was performed on a 5-µm (100 × 4.6 mm) C8 column at 40°C. The mobile phase consisted of acetonitrile with 0.045M H(3)PO(4) (37:63, v/v) adjusted to pH 6.7 and pumped at a flow rate of 1.2 mL/min. Detections were monitored on an electrochemical detector operated at a potential of 0.85 V with glassy carbon electrode against Ag/AgCl reference electrode. Each analysis required 13 min and quantification over the range of 0.05-5.0 µg/mL of plasma was linear, as indicated by a correlation coefficient (R(2)), 0.9999. The method was validated according to international guidelines. Data with respect to accuracy, within-run and between run, were close to 100% with 4% precision. Absolute recovery was 95%. The limit of quantification was 0.05 µg/mL. Neither endogenous substances nor commonly used drugs were found to interfere with the retention times of analytes. Stock solutions and calibration standards of the drug and quality control preparations were demonstrated to be stable at room temperature and -20°C for long and short periods of time. Eventually, the proposed method was successfully applied to quantify clarithromycin in spiked human plasma and real samples from healthy volunteers, indicating the utility and throughput of this method for clinical and bioavailability studies.

N-Carbamoylputrescine, a citrulline-derived polyamine, is not a significant citrulline metabolite in rats

Citrulline, a key amino acid of the urea cycle, has been shown to play a regulatory role in protein and energy metabolism in mammals. We questioned whether N-carbamoyl-putrescine (NCP), the decarboxylated derivative of citrulline, could play a role in the biological properties of this amino acid. To evidence the presence of NCP in mammalian tissues, we developed a sensitive reverse-phase high-performance liquid chromatography (HPLC) with fluorimetric detection method with precolumn dansyl derivatization and solid-phase extraction for the determination of NCP together with polyamines in biological samples. Dansyl NCP was identified with a 5.85-min retention time. Linearity was obtained in a concentration range of 0.125 to 12.5μM. Intraday and day-to-day relative coefficients of variation ranged from 8.9% to 12.3% and from 14% to 14.3%, respectively. Recovery rates in serum ranged from 75% to 83%. Thereafter, we used this method to search for the presence of NCP in serum, muscle, liver, jejunum, and ileum in rats after both short-term intraperitoneal injection and long-term oral citrulline supplementation. We failed to detect NCP in these animals. These data suggest that NCP is not a significant citrulline metabolite in rats.

Pharmacokinetic profile of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside in mice after oral administration of Polygonum multiflorum extract

Context: Stilbene glycoside (2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside) is a main bioactive component of Polygonum multiflorum, a traditional Chinese medicine (TCM) commonly used in clinic for anti-aging treatment. Its medicinal activities, such as anti-oxidation, anti-inflammation and endothelial protection, have been extensively studied, but its pharmacokinetic property is still unclear.Objective: A pharmacokinetic study was undertaken to quantitatively determine P. multiflorum stilbene glycoside (PM-SG) in mouse plasma after oral administration of 100 mg/kg P. multiflorum extract.Materials and methods: A sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with liquid-liquid phase extraction method was employed for this study. Pharmacokinetic parameters of PM-SG were determined in mice applying both compartmental and non-compartmental analyses.Results and discussion: The calibration curve for PM-SG in the plasma was linear (r2 > 0.99) over the range of 0.66 to 56.40 μg/ml, and the concentration-time curve was plotted with the maximum concentration (Cmax) and time to reach maximum concentration (Tmax) of 29.62 μg/ml and 60 min, respectively. The intra- and inter-day variations were less than 3% for relative standard deviation (RSD) and relative error (RE), with a good recovery of more than 97% (RSD <3%). All pharmacokinetic parameters estimated by compartmental and non-compartmental models reached a same conclusion that PM-SG was rapidly absorbed and widely distributed throughout the body with a great efficiency of utility, followed by quick elimination and clearance.Conclusions: This was the first report on determination of the pharmacokinetic profile of PM-SG in mice after oral administration. The result may provide a meaningful basis for evaluating the clinical applications of such a bioactive compound from herbal medicines.

DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS

□ A simple, rapid and sensitive isocratic reversed-phase (RP) high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of chlorhexidine (CHX) and p-chloroaniline (CAL) in various pharmaceutical formulations. Compound separation was achieved in less than 10 min with an XBridge C18 column that was maintained at 40°C and a mobile phase consisting of 32:68 (v/v) of acetonitrile and a pH 3.0 phosphate buffer solution (a 0.05 M monobasic sodium phosphate solution containing 0.2% of triethylamine). Analyses were performed at a flow rate of 2 mL min−1 and at a detection wavelength of 239 nm. The method was shown to be selective, linear, accurate, and precise in intra-day and inter-day analyses. The robustness of the method was shown by slightly changing the flow rate, column oven temperature, and proportion of acetonitrile in the mobile phase. The method was found, however, to be very sensitive to the pH of the mobile phase buffer. The method was successfully validated following the guidelines of the International Conference on Harmonization (ICH). This validation proved that the method was suitable for the determination of CHX and CAL in toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants.

Simplified RP-HPLC method for multi-residue analysis of abamectin, emamectin benzoate and ivermectin in rice

A rapid, reliable and sensitive reverse-phase high-performance liquid chromatography method with fluorescence detection (RP-FLD-HPLC) was developed and validated for simultaneous analysis of the abamectin (ABA), emamectin (EMA) benzoate and ivermectin (IVM) residues in rice. After extraction with acetonitrile/water (2 : 1) with sonication, the avermectin (AVMs) residues were directly derivatised by N-methylimidazole (N-NMIM) and trifluoroacetic anhydride (TFAA) and then analysed on RP-FLD-HPLC. A good linear relationship (r 2 > 0.99) was obtained for three AVMs ranging from 0.01 to 5 µg ml−1, i.e. 0.01–5.0 µg g−1 in rice matrix. The limit of detection (LOD) and the limit of quantification (LOQ) were between 0.001 and 0.002 µg g−1 and between 0.004 and 0.006 µg g−1, respectively. Recoveries were from 81.9% to 105.4% and precision less than 12.4%. The proposed method was successfully applied to routine analysis of the AVMs residues in rice.

Determination of retinol, retinyl palmitate, and retinoic acid in consumer cosmetic products

Retinol and retinyl palmitate are frequently used in cosmetic products. A simple, rapid, and sensitive reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed for the quantitation of retinol, retinyl palmitate, and retinoic acid in cosmetic preparations. The analytes were extracted from a cosmetic/Celite mixture using a solvent system composed of equal amounts of hexane, isopropanol, and ethyl acetate, and the extract was injected directly into an HPLC chromatograph with a C18 column and UV detector set at 330 nm. Chromatographic separation was achieved by gradient elution with a mobile phase, starting with aqueous ammonium acetate buffer/methanol that was gradually changed to methanol/dichloromethane. The average recoveries of retinol, retinyl palmitate, and retinoic acid from spiked cosmetic products were 95% or higher. In a survey of twenty-nine consumer cosmetic skin care products labeled to contain retinoids, most products were found to contain either retinol or retinyl palmitate at concentrations up to 2.2% (w/w), while a few products contained both ingredients. A number of products also contained cis isomers of retinol that could be quantitatively distinguished from the all-trans compound. The method can be used to quantitate several retinoids and their isomers in cosmetic products. The method will be useful for obtaining information needed to estimate levels of exposure to retinoids from cosmetic products.

Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography–diode array–fluorescence (HPLC–DAD–FLD) in human urine samples

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N(4)-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N(4)-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N(4)-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex Gemini C(18) (150mm x 4.6mm I.D., particle size 5microm) analytical column with LiChroCART LiChrospher C(18) (4mm x 4mm, particle size 5microm) guard column. Analyzed drugs were determined within 34min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut Plexa columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP-1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C(18) column and acetonitrile-0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane-isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08-50 microg/mL for levonorgestrel and 0.12-50 microg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 microg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter-day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma.

Determination of Cefaclor in Human Plasma by Reversed-Phase High-Performance Liquid Chromatography with UV Detection and Its Application to the Bioequivalence Studies

A selective and sensitive reversed-phase high-performance liquid chromatography method was developed and validated for quantitation of cefaclor in human plasma using cefradine as an internal standard. Calibration curve was linear over range of 0.1–20 mg · L−1. The intra- and inter-run relative standard deviations of the assay were less than 7%. The mean absolute recoveries determined at the concentrations of 0.3, 3.0, 8.0, and 15.0 mg · L−1 were 69.9%, 69.9%, 77.1% and 72.0%, respectively. The analytical method established was proved to be specific, precise, sensitive, and suitable for applying in the pharmacokinetic and bioequivalence studies of cefaclor in human.

A simple and reliable preparative high-performance liquid chromatographic technique for isolation of a bioactive flavone diglycoside from and extract ofCuminum cyminumseeds

Summary In this paper we describe a sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) method with photodiode-array detection for isolation and quantification of the bioactive hydrophilic constituent 7-(1-O-β-d-galacturonide-4′-(1-O-β-d-glucopyranosyl)-3′,4′,5,7-tetrahydroxyflavone, 1, from the seeds of Cuminum cyminum. Compound 1 was separated isocratically on a C18 preparative column, in high purity, after removal of solvents. The purity and identity of the compound were established by use of LC-mass spectrometry and by spectroscopic techniques (1H and 13C NMR). The purity of 1 was also confirmed by HPTLC.

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital-pretreated rats and detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3'(R)-hydroxy-nodakenetin-3'-ol and 3'(S)-hydroxy-nodakenetin-3'-ol, respectively, on the basis of their (1)H-NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP-HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C(18) column, a mobile phase with MeOH-H(2)O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat-inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50-400, 20-120 and 150-24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI.

Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography

Taxifolin has been widely used in the treatment of cerebral infarction and sequelae, cerebral thrombus, coronary heart disease and angina pectoris. A reliable sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection for the pharmacokinetic study of taxifolin in rabbit plasma after enzymatic hydrolysis was developed and validated for the first time. Taxifolin, with biochanin A as the internal standard, was extracted from plasma samples by liquid/liquid extraction after hydrolysis with beta-glucuronidase and sulfatase. Chromatographic separation was conducted on a Luna C18 column (4.6 mm x 150 mm, 5 microm particle size) and pre-column (2.0 mm, the same sorbent). Two-step linear gradient elution with acetonitrile and 0.03% water solution of trifluoroacetic acid as mobile phase at a flow rate of 1.0 ml/min was used. The UV detector is set at 290 nm. The elution time for taxifolin and biochanin A was approximately 7.9 and 18.3 min, respectively. The calibration curve of taxifolin was linear (r > 0.9997) over the range of 0.03-5.0 microg/ml in rabbit plasma. The limit of detection (LOD) and limit of quantification (LOQ) for taxifolin were 0.03 and 0.11 microg/ml, respectively. The present method was successfully applied for the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration of lipid solution to rabbits. The absolute bioavailability of taxifolin after oral administration of lipid solution was 36%.

Determination of osthol and its metabolites in a phase I reaction system and the Caco-2 cell model by HPLC-UV and LC–MS/MS

A straightforward and sensitive reversed-phase high-performance liquid chromatography (HPLC) assay was developed and validated for the analysis of osthol and its phase I metabolites (internal standard: umbelliferone). The method was validated for the determination of osthol with respect to selectivity, precision, linearity, limit of detection, recovery, and stability. The linear response range was 0.47–60μM, and the average recoveries ranged from 98 to 101%. The inter-day and intra-day relative standard deviations were both less than 5%. Using this method, we showed that more than 80% of osthol was metabolized in 20min in a phase I metabolic reaction system. Transport experiments in the Caco-2 cell culture model indicated that osthol was easily absorbed with high absorptive permeability (>10×10−6cm/s). The permeability did not display concentration-dependence or vectorial-dependence and is mildly temperature sensitive (activation energy less than 10kcal/mol), indicating passive mechanism of transport. When analyzed by LC–MS/MS, five metabolites were detected in a phase I reaction system and in the receiver side of a modified Caco-2 cell model, which was supplemented with the phase I reaction system. The major metabolites appeared to be desmethyl-osthol and multiple isomers of dehydro-osthol. In conclusion, a likely cause of poor osthol bioavailability is rapid phase I metabolism via the cytochrome P450 pathways.

Pharmacokinetic study of three active flavonoid glycosides in rat after intravenous administration of Trollius ledebourii extract by liquid chromatography

A selective and sensitive reversed-phase high-performance liquid chromatography method was developed and validated for the simultaneous determination of orientin-2''-O-beta-L-galactopyranosyl (OGA), orientin and vitexin in rat plasma. Blood samples were collected via the fossa orbitalis vein at time intervals after intravenous administration and the concentrations of the three ingredients in plasma were analyzed by HPLC after the plasma protein had been precipitated directly with methanol. OGA, orientin and vitexin were successfully separated using a C18 column with gradient elution composed of acetonitrile and 0.1% acetic acid and were detected at the detection wavelength of 348 nm. Calibration curves of OGA, orientin and vitexin were generated over the range 0.315-161, 0.326-167 and 0.215-110 microg/mL, respectively. The intra- and inter-day precisions (relative standard deviation) for the analysis of the three analytes were between 1.68 and 8.43% with accuracies (relative error) below 8.55%. The mean extraction recoveries were between 70.35 and 86.42%. The developed method was suitable for simultaneous determination of these three active flavonoid glycosides in rat plasma and was successfully applied to investigate the pharmacokinetics of glycosides from Trollius ledebourii in rats.