Determination of the immunosuppressant mycophenolic acid in human serum by solid-phase microextraction coupled to liquid chromatography

A solid phase microextraction (SPME)-HPLC-UV method for the determination of the immunosuppressant mycophenolic acid (MPA) in human serum samples was developed for the first time. The procedure, that employed a carbowax/templated resin (Carbowax/TPR-100) as fiber coating, required a very simple sample pretreatment, an isocratic elution, and provides an highly selective extraction. The linear range was 0.2-100 microg x ml(-1). Recovery was practically unchanged (63+/- 4%) passing from 0.2 to 100 microg x ml(-1) level. Within-day and between-days coefficient of variation ranged from 5.9 to 6.5% and from 8.8 to 9.2%, respectively. A detection limit of 0.05 microg x ml(-1) was estimated in spiked serum. The method was successfully applied to the determination of MPA in serum of a patient under mycophenolate mophetil ester (MMF) therapy, as demonstrated by the relevant concentration-time profiles.     

Simultaneous determination of free mycophenolic acid and its glucuronide in serum of patients under mycophenolate mophetil therapy by ion-pair reversed-phase liquid chromatography with diode array UV detection

An high performance liquid chromatography (HPLC)-UV method for the simultaneous determination of the free forms of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) in human serum samples was developed for the first time. Chromatographic separation was performed on octadecylsilane based stationary phase in combination with a mobile phase of methanol/buffered tetrabutylammonium (TBA) salt mixture. Sample pretreatment consisted of an ultrafiltration step followed by clean-up/enrichment on a C(18) solid-phase extraction (SPE) cartridge. Average recoveries of (99.7 +/- 0.2)% and (64.1 +/- 6.9)% for free MPA and MPAG, respectively, were estimated in the concentration range from 0.5 to 10 microg/ml. The within-day and between-days coefficients of variation were 0.4 and 0.8% for free MPA (0.1 microg/ml spiking level) and 0.8 and 1.6% for free MPAG (5 microg/ml spiking level), respectively. The linear ranges for free MPA and MPAG were 0.06-1 and 0.2-10microg/ml, respectively. Detection limits of 4 and 17 ng/ml for free MPA and MPAG were estimated in spiked serum. The same HPLC method was also capable of a simultaneous determination of the total concentration of MPA and MPAG when coupled to a proper sample pretreatment step. The potential of the method is demonstrated by excretion kinetics measurement in serum of patients receiving MMF therapy.     

Determination of vancomycin in serum by liquid chromatography-high resolution full scan mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the analysis of vancomycin (VCM) in human serum. The method was based on full scan data with extracted ions for the accurate masses of VCM and the atenolol internal standard obtained by Fourier transform MS. VCM was extracted from serum using strong cation exchange (SCX) solid phase extraction (SPE). The method was found to be linear in the range 0.05-10 microg/ml, which was adequate for quantification of VCM in serum samples, with a limit of quantification (LOQ) of 0.005 microg/ml and a limit of detection (LOD) of 0.001 microg/ml. Intra-day precision (n=5) was +/-3.5%, +/-2.5%, +/-0.7% at 0.05, 0.5 and 5 microg/ml, respectively. Inter-day precision (n=5) was +/-7.6%, +/-6.4%, +/-3.9% at 0.05, 0.5 and 5 microg/ml, respectively. The process efficiency for VCM was in the range 89.2-98.1% with the recovery for the atenolol internal standard (IS) being 97.3%. The method was used to determine VCM levels in patients during peri-operative infusion of the drug, which was found to result in drug levels within the required therapeutic window.     

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of cefixime in human plasma: application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine cefixime ((6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino)acetamido]-8-oxo-3-vinyl-5-thia-1-azabicyclo-[4,2,0]-oct-2-ene-2-carboxylic acid) in human plasma. After a simple protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C(8) column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water-formic acid (40:60:0.5, v/v/v). The analyte and internal standard cefetamet were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.05-8.0 microg/ml. The lower limit of quantification was 0.05 microg/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.7%. The accuracy determined at three concentrations (0.05, 0.80 and 7.2 microg/ml for cefixime) was within +/-2.0% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefixime capsule in 24 healthy volunteers.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.     

Simple liquid chromatography-tandem mass spectrometry method for determination of novel anti-methicillin-resistant Staphylococcus aureus fluoroquinolone WCK 771 in human serum

A simple, rapid, specific, precise, accurate and sensitive method for determination of WCK 771 in human serum has been developed. The method uses high performance liquid chromatography with tandem mass spectrometric detection. Sample preparation involves protein precipitation method by addition of acetonitrile. Gatifloxacin was used as internal standard. The response was found to be linear from 0.312 to 40 microg/ml of serum with correlation coefficient greater than 0.99. Limit of detection and lower limit of quantification for WCK 771 was found to be 0.078 microg/ml and 0.312 microg/ml, respectively. The intra-day precision and accuracy from analysis of quality control (QC) samples at four concentrations was in the range of 2.36-2.58% and from 96.71 to 103.2%, respectively. The inter-day precision and accuracy from analysis of quality control samples at four concentrations was in the range of 3.14-6.82% and from 96.84 to 105.76%, respectively. WCK 771 was found to be stable for 24 h at auto-injector environment. WCK 771 was also found to be stable for 2h in serum at 25+/-3 degrees C and for 3 months at -20 degrees C. Mean absolute recovery at four different concentrations was 86.92% with standard deviation of 1.79. Throughput of the method is approximately one sample every 4 min. The method was also reproduced with monkey serum. The method was employed for estimation of drug serum levels during pre-clinical and clinical trials.     

Simultaneous determination and pharmacokinetic studies of dihydromyricetin and myricetin in rat plasma by HPLC-DAD after oral administration of Ampelopsis grossedentata decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of dihydromyricetin (1) and myricetin (2) in rat plasma after orally administrating the decoction of Ampelopsis grossedentata. Plasma samples were acidified with 0.375% phosphoric acid and extracted with ethyl acetate. Analysis of the extract was performed on reversed-phase C(18) column with a gradient eluent composed of acetonitrile and 0.04% phosphoric acid. The flow rate was kept at 1 ml/min and the detection wavelength was set at 290 and 370 nm for 1 and 2, respectively. The calibration curves were linear in the range of 0.247-4.114 microg/ml and 0.150-2.501 microg/ml for 1 and 2, respectively. The intra-day and inter-day precisions were better than 4.9 and 6.2%, respectively. The limits of detection (LOD) for 1 and 2 in plasma were 21.600 and 52.530 ng/ml, and the limits of quantification (LOQ) were 0.247 and 0.150 microg/ml, respectively. The mean recoveries for 1 and 2 were 92.0 and 93.3%, respectively. The accuracy and precision were well within the acceptable range and R.S.D. of measured rat samples was less than 7.5%. This validated method has been successfully applied in the pharmacokinetics study of dihydromyricetin and myricetin in vivo after orally administrating the decoction of A. grossedentata to rats.     

Determination of paeoniflorin in rat hippocampus by high-performance liquid chromatography after intravenous administration of Paeoniae Radix extract

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of paeoniflorin in rat hippocampus was developed in this study. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Zorbax SB-C(18) column, a mobile phase of methanol-water (32:68, v/v), and detection by ultraviolet (UV) absorption at 233 nm. The lower limits of quantitation (LLQ) were 1 microg/ml for paeoniflorin. The calibration curve for paeoniflorin was linear (r = 0.9999) over the concentration range of 1-50 microg/ml. The coefficients of variation of intra- and inter-day assays were 7.00, 0.58, 1.46% and 5.48, 1.79, 1.70% at concentrations of 1, 10, 50 microg/ml, respectively. The recoveries of paeoniflorin from rat hippocampus were 98.28 +/- 2.14, 98.96 +/- 1.48, and 95.34 +/- 0.92 at concentrations of 1, 10 and 50 microg/ml, respectively. Stability studies showed that paeoniflorin was stable at temperatures of 2-8 degrees C in methanol for at least 20 days. The method was applied to determine the time course of paeoniflorin in rat hippocampus, following the administration of a 60 mg/kg i.v. dose of paeoniflorin in Paeoniae Radix extract to a male Wistar rat.     

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate

A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1-150 microg/ml in serum and 25-1500 microg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41+/-19.99 ml/min/1.73 m(2) in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and beta-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either 3H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (14C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1-10 microg ml(-1)) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1- 6 h. The results were expressed as pmol min(-1) (mg cell protein)(-1), n = 6-8 for each value. Basal 3H-deoxyglucose and 14C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means +/- S.E.M.) 32.14 +/- 1.34 and 13.48 +/- 1.86 pmol min(-1) (mg cell protein)(-1), respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in 3H-deoxyglucose and 14C-Me-AIB uptakes. Typically, 3H-deoxyglucose and 14C-Me-AIB uptakes in the presence of insulin were 58.57 +/- 4.49 and 29.52 +/- 3.41 pmol min(-1) (mg cell protein(-1)), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 microg ml(-1)) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in 3H-deoxy-D-glucose and 14C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 microg ml(-1). Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of 3H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Determination of chromium in urine samples by complexation-supercritical fluid extraction and liquid or gas chromatography

A method was developed for the determination of chromium as the Cr(acac)(3) complex in urine using SFE and chromatography. Quantitative extractions were achieved when the experiments were carried out under 3000 p.s.i. of pressure, at a temperature of 120 degrees C, with 2.0 ml of methanol, 30 min of static extraction and 5 min of dynamic extraction. The chromium was quantified by GC-FID detection. The calibration graph of Cr(acac)(3) solutions was linear between 0.50 and 43.0 microg ml(-1) of chromium (DL 0.18 microg ml(-1), R=0.9994). The same extracts were quantified by HPLC-340 nm detection. The calibration curve of the Cr(acac)(3) solutions was linear over a range of 0.013 to 60.0 microg ml(-1) (DL 0.02 microg ml(-1), R=0.9999). This method was applied to urine samples from 60 diabetic patients and 21 healthy volunteers. Chromium concentration ranges were 2.5-29.5 microg l(-1) for the diabetics and 5.9-12.3 microg l(-1) for the normal subjects.     

Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample

A flow injection analysis (FIA) system combined with Rayleigh light scattering (RLS) detection is developed for the sensitive and rapid determination of protein concentration in human serum sample. This method is based on the weak intensity of RLS of Eriochrome Black T (EBT, 2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitronaphthalene-4-sulfonic acid sodium salt), which can be enhanced by the addition of protein in weakly acidic solution. The effects of pH and interfering species on the determination of protein were examined. Calibrations for protein, based on RLS intensity, were linear in the concentration ranges of 7-36 microg/ml for human serum album (HSA) and 8-44 microg/ml for bovine serum album (BSA). The detection limits of the method were found to be 0.882 and 2.507 microg/ml for HSA and BSA, respectively. A relative standard deviation of 0.76% (n=5) was obtained with 20 microg/ml HSA standard solution. The FIA-RLS method was more stable than the general RLS method, and the average RSD value of FIA-RLS was less than that of the general RLS. The sample rate was determined to be 90 samples per hour.     

Improvement and validation of an HPLC method for examining the effects of the MDR1 gene polymorphism on sparfloxacin pharmacokinetics

A rapid, simple, accurate, and precise reverse-phase high-performance liquid chromatography (HPLC) method for measuring sparfloxacin in human serum was improved, validated, and applied to determine the influence of polymorphisms in MDR1 (exons 12, 21, and 26) gene on sparfloxacin pharmacokinetics. Sparfloxacin and an internal standard, ciprofloxacin, were extracted from human serum by protein precipitation with dilution and analyzed on a Luna C(18) 5-microm column in a mobile phase of acetonitrile-0.035 M perchloric acid (28:72, v/v, adjusted to pH 2.0 with 0.015 M triethylamine) and UV detection at 300 nm. This analysis was performed at three different laboratories using the same quality control (QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum. The method showed linear responses over a concentration range of 0.05-2 microg/ml, with correlation coefficients of greater than 0.999 at the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. The mean absolute recovery for human serum was 98.8+/-5.7%. Sparfloxacin in human serum was stable during storage and the assay procedure. The lower limit of quantification using 0.2 ml of serum was 0.05 microg/ml, which was sensitive enough for pharmacokinetic studies. This method was used to study the pharmacokinetics of sparfloxacin in human volunteers, following a single oral administration of sparfloxacin (100 mg) two tablets at three different laboratories. MDR1 polymorphisms did not significantly (P < 0.01) affect the pharmacokinetic parameters (AUC and C(max)) of sparfloxacin.     

Ginseng reduces the micronuclei yield in lymphocytes after irradiation

To assess the effect of Chinese ginseng in modifying the radiation-induced micronuclei (MN) yield in human G(o) peripheral blood lymphocytes (PBL), we conducted the cytokinesis-blocked (CB) MN assay in blood samples obtained from healthy volunteers (n=4). Before (137)Cs ex vivo irradiation, mononuclear cell cultures from each sample were incubated 24 h with different concentrations (0-2000 microg ml(-1)) of crude water extract of ginseng dry root. We found that (1) at 0 Gy and without the presence of ginseng, MN yield (mean+/-S.E.M.) was 11.7+/-2.7 per 1000 binucleated (BN) cells. Different concentrations of ginseng crude water extract did not affect the MN yields and the proliferative activity of PBL; (2) after 1 and 2 Gy exposure, radiation alone sharply increased the MN yields, respectively, to 119.6+/-17.4 and 340.5+/-20.9 per 1000 BN cells. However, treatment with ginseng for 24 h before radiation exposure, resulted in a significant linear decline of MN yields as ginseng concentration increases. Compared to radiation alone, the extent to which ginseng water extract reduced the MN yields induced by 1 Gy exposure was 46.0% at 1500 microg ml(-1) and 61.5% at 2000 microg ml(-1), and with 2 Gy exposure, it was 38.6% at 1500 microg ml(-1) and 46.5% at 2000 microg ml(-1); (3) MN data suggested a tendency for overdispersion relative to the Poisson model; and (4) over the different levels of ginseng concentrations, the trend in micronucleated BN index was as similar as that of the MN yields. These results indicated that (1) ginseng crude water extract exerts no apparent cytogentic effect on human PBL at concentrations up to 2000 microg ml(-1) as evaluated by the CBMN assay; and (2) the protection of ginseng water extract against (137)Cs-induced MN in human PBL is concentration-dependence. Therefore, our findings indicated that ginseng may have therapeutic value as a possible radioprotector for normal tissue during radiotherapy of cancer patients.     

Measurement of total mirtazapine and normirtazapine in plasma/serum by liquid chromatography with fluorescence detection

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean +/- S.D.) was 79 +/- 2, and 64 +/- 3%, respectively. The LOD was estimated as 0.5 microg/l, LLOQ was 1 microg/l, with a linear response over the concentration range 4-1000 microg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at -20 degrees C. Intra- and inter-day accuracy were in the range 91-107 and 93-103%, respectively. In clinical samples (n = 14, median mirtazapine dose 45 mg per day, range 15-45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8-40) and 21 (8-32) microg/l, respectively.     

Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study

A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm x 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm x 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1-20 microg/ml. With UV detection, the limit of quantitation was 0.1 microg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 microl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 +/- 3.9, 93.5 +/- 3.2, and 93.7 +/- 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 microl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.     

Studies on the antioxidant activity of the essential oil and methanol extract of Marrubium globosum subsp. globosum (lamiaceae) by three different chemical assays

This study is designed to examine the chemical composition and in vitro antioxidant activity of the essential oil and sub-fractions of the methanol extract of Marrubium globosum subsp. globosum. The GC and GC-MS analysis of the essential oil were resulted in the determination of 84 components representing 88.2% of the oil. The major constituents of the oil were spathulenol (15.8%), beta-caryophyllene (9.0%), caryophyllene oxide (7.9%), germacrene D (6.5%), and bicyclogermacrene (3.1%). Antioxidant activities of the samples were determined by three different test systems namely DPPH, beta-carotene/linoleic acid and reducing power assay. In DPPH system, the weakest radical scavenging activity was exhibited by the essential oil (1203.38+/-7.18 microg ml(-1)). Antioxidant activity of the polar sub-fraction of methanol extract was superior to the all samples tested with an EC(50) value of 157.26+/-1.12 microg ml(-1). In the second case, the inhibition capacity (%) of the polar sub-fraction of methanol extract (97.39%+/-0.84) was found the strongest one, which is almost equal to the inhibition capacity of positive control BHT (97.44%+/-0.74). In the case of reducing power assay, a similar activity pattern was observed as given in the first two systems. Polar sub-fraction was the strongest radical reducer when compared with the non-polar one, with an EC(50) value of 625.63+/-1.02 microg ml(-1). The amount of the total phenolics was highest in polar sub-fraction (25.60+/-0.74 microg/mg). A positive correlation was observed between the antioxidant activity potential and total phenolic level of the extracts. On the other hand, total flavonoid content was found equal for the both sub-fractions.     

Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract

Quantification of ellagic acid, the principal bioactive component of pomegranate leaf extract, in rats plasma following oral administration of pomegranate leaf extract was achieved by using a high-performance liquid chromatographic method. The calibration curve for ellagic acid was linear (r2=0.9998) ver the concentration range 0.026-1.3 microg/ml. The intra- and inter-day assays of ellagic acid from rat plasma were less than 6.52% at concentration range from 26 to 1300 ng/ml and good overall recoveries (94.5-102.4%) were found on same concentrations. The concentration-time profile was fitted with an open two-compartment system with lag time and its max concentration of ellagic acid in plasma was 213 ng/ml only 0.55 h after oral administration extract 0.8 g/kg. The pharmacokinetic profile indicates that ellagic acid has poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach.