An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug combination

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile-0.05 M acetic acid adjusted to pH 5.2 with 1.0M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol-acetonitrile-0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30-750 ng/0.5 ml plasma and MQ over a concentration of 75-1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0-1.4% for AS, 0.4-3.4% for DHA and 0.7-1.5% for MQ. The day-to-day coefficients of variation were 1.3-7.6%, 1.8-7.8% and 2.0-3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with recutive electrochemical detection for quantitatvie determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: and β isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a μBondapak CN column. The method was capable of separating the two isomeric forms of DHA (, β). The retention times of -DHA, β-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80–640 ng/ml were 86–93%. The coefficients of variation were below 10% for all three drugs (ART, -DHA, ARN). The minimum detectable concentrations for ART and -DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Determination of metformin in human plasma by high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of antihyperglycemic agent metformin in human plasma using a novel sample extraction procedure. Liquid-liquid extraction of metformin and ranitidine (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (50:50, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a silica column (250 mmx4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (25:75, v/v), pH 6. The limit of quantification (LOQ) was 15.6 ng/ml and the calibration curves were linear up to 2000 ng/ml. The mean absolute recoveries for metformin and internal standard using the present extraction procedure were 98 and 95%, respectively. The intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 8.3%.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.     

Simultaneous determination of piroxicam, meloxicam and tenoxicam in human plasma by liquid chromatography with tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of piroxicam, meloxicam and tenoxicam in human plasma was developed. Piroxicam, meloxicam, tenoxicam and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analyzed on a Sunfire column with the mobile phase of methanol:ammonium formate (15 mM, pH 3.0) (60:40, v/v). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring (MRM) mode. The standard curve was linear (r=1.000) over the concentration range of 0.50-200 ng/ml. The coefficient of variation (CV) and relative error (RE) for intra- and inter-assay statistics at three QC levels were 1.0-5.4% and -5.9 to 2.8%, respectively. The recoveries of piroxicam, meloxicam and tenoxicam ranged from 78.3 to 87.1%, with that of isoxicam being 59.7%. The lower limit of quantification for piroxicam, meloxicam and tenoxicam was 0.50 ng/ml using a 100 microl plasma sample. This method was successfully applied to a pharmacokinetic study of piroxicam after application of transdermal piroxicam patches to humans.     

Simultaneous high-performance liquid chromatographic determination of Cedrus deodara active constituents and their pharmacokinetic profile in mice

A specific and sensitive high-performance liquid chromatographic (HPLC) method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three bioactive constituents of Cedrus deodara namely wikstromol, matairesinol and dibenzylbutyrolactol in mouse plasma. In solid-phase extraction (SPE) these constituents were successfully separated using a C18 column by isocratic elution using acetonitrile:water containing hexanesulphonic acid, 32:68 (v/v). The flow rate was set at 1ml/min and detector wavelength at 225nm. Good linearity (r2>0.999) was observed over the studied range of 0.015-5.0microg/ml for wikstromol and 0.030-5.0microg/ml for matairesinol and dibenzylbutyrolactol. The CV values of intra-day precision for wikstromol, matairesinol and dibenzylbutyrolactol were in between 1.8-6.9, 1.7-4.9 and 1.6-4.2% and values of inter-day precision were in between 10.4-12.2, 9.7-11 and 10-11.2%, respectively. The extraction recoveries at low to high concentration were greater than 98, 83 and 87% for each analyte, respectively. The LOQ for wikstromol was 0.015microg/ml and for both matairesinol and dibenzylbutyrolactol it was 0.030microg/ml. The developed method was used to determine the pharmacokinetics of the three analytes in mice after intraperitoneal administration of CD-3.     

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid-liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-(S)- and (-)-(R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm x 4.6 mm, 10 microm particle size) protected with a CN guard column, using hexane-ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-(S)- and (-)-(R)-mirtazapine were in the 88-111% range with a linear response over the 6.25-625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence

A sensitive capillary electrophoretic method for the determination of carvedilol enantiomers in 100 μl of human plasma has been developed and validated. Carvedilol and the internal standard carazolol are isolated from plasma samples by liquid–liquid extraction using diethylether. A sensitive and selective detection is provided by helium–cadmium laser-induced fluorescence. The total analysis time is 17.5 min, about 30 min are needed for the sample preparation. The linearity of the assay ranges from 1.56 to 50 ng/ml per carvedilol enantiomer. The limits of quantification (LOQ) for the carvedilol enantiomers in 100 μl of human plasma are 1.56 ng/ml. The inter-day accuracy for R-carvedilol is between 95.8 and 103% (104% at LOQ) and for S-carvedilol between 97.1 and 103% (107% at LOQ); the inter-day precision values are between 3.81 and 8.64% (10.9% at LOQ) and between 5.47 and 7.86% (7.91% at LOQ) for R- and S-carvedilol, respectively. The small sample volume needed is especially advantageous for the application in clinical studies in pediatric patients. As an application of the assay concentration/time profiles of the carvedilol enantiomers in a 5-year-old patient receiving a test dose of 0.09 mg/kg carvedilol are reported.     

HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 degrees C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5-500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were 0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(-)- and R-(+)-bufuralol from plasma was in the range 97-102% at 15-400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6-102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Simultaneous determination of thienorphine and its active metabolite thienorphine glucuronide in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies

A simple, sensitive and reliable method was developed to determine simultaneously the concentrations of thienorphine and its metabolite thienorphine glucuronide conjugate in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolite was identified by MS: thienorphine glucuronide conjugate. Sample preparation involved protein precipitation with methanol. Analytes were separated on Finnigan BetaBasic-18 column (150 mm x 2.1mm i.d., 5 microm) using methanol: water: formic acid (56:44:0.1, v/v/v) as mobile phase at a flow rate of 0.2 ml/min. The method had a linear calibration curve over the concentration range of 0.1-50 ng/ml for thienorphine and 2-1000 ng/ml for thienorphine glucuronide conjugate, respectively. LOQ of thienorphine and thienorphine glucuronide conjugate was 0.1 and 2 ng/ml, respectively. The intra- and inter-batch precisions were less than 12% and their recoveries were greater than 80%. Pharmacokinetic data of thienorphine and its metabolite thienorphine glucuronide conjugate obtained with this method following a single oral dose of 3mg/kg thienorphine to rats were also reported for the first time.     

Determination of N,N-dimethyltryptamine and beta-carboline alkaloids in human plasma following oral administration of Ayahuasca

Ayahuasca is a South American psychotropic beverage prepared from plants native to the Amazon River Basin. It combines the hallucinogenic agent and 5-HT(2A/2C) agonist N,N-dimethyltryptamine (DMT) with beta-carboline alkaloids showing monoamine oxidase-inhibiting properties. In the present paper, an analytical methodology for the plasma quantification of the four main alkaloids present in ayahuasca plus two major metabolites is described. DMT was extracted by liquid-liquid extraction with n-pentane and quantified by gas chromatography with nitrogen-phosphorus detection. Recovery was 74%, and precision and accuracy were better than 9.9%. The limit of quantification (LOQ) was 1.6 ng/ml. Harmine, harmaline, and tetrahydroharmine (THH), the three main beta-carbolines present in ayahuasca, and harmol and harmalol (O-demethylation metabolites of harmine and harmaline, respectively) were measured in plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Sample preparation was accomplished by solid-phase extraction, which facilitated the automation of the process. All five beta-carbolines were measured using a single detector by switching wavelengths. Separation of harmol and harmalol required only slight changes in the chromatographic conditions. Method validation demonstrated good recoveries, above 87%, and accuracy and precision better than 13.4%. The LOQ was 0.5 ng/ml for harmine, 0.3 ng/ml for harmaline, 1.0 ng/ml for THH, and 0.3 ng/ml for harmol and harmalol. Good linearity was observed in the concentration ranges evaluated for DMT (2.5-50 ng/ml) and the beta-carbolines (0.3-100 ng/ml). The gas chromatography and HPLC methods described allowed adequate characterization of the pharmacokinetics of the four main alkaloids present in ayahuasca, and also of two major beta-carboline metabolites not previously described in the literature.     

Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection

A fast, simple, and a reliable high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method for the assessment of lipoic acid (LA) and dihydrolipoic acid (DHLA) in plasma was developed using naproxen sodium as an internal standard (IS) and validated according to standard guidelines. Extraction of both analytes and IS from plasma (250 μl) was carried out with a single step liquid-liquid extraction applying dichloromethane. The separated organic layer was dried under stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both compounds and IS at 30°C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 9 min using acetonitrile: 0.05 M phosphate buffer (pH 2.4 adjusted with phosphoric acid) (52:48, v/v) as a mobile phase pumped at flow rate of 1.5 ml min(-1) using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification for lipoic acid were 500 pg/ml and 3 ng/ml, and for dihydrolipoic acid were 3 ng/ml and 10 ng/ml, respectively. The absolute recoveries of lipoic acid and dihydrolipoic acid determined on three nominal concentrations were in the range of 93.40-97.06, and 93.00-97.10, respectively. Similarly coefficient of variations (% CV) for both intra-day and inter-day were between 0.829 and 3.097% for lipoic acid and between 1.620 and 5.681% for dihydrolipoic acid, respectively. This validated method was applied for the analysis of lipoic acid/dihydrolipoic acid in the plasma of human volunteers and will be used for the quantification of these compounds in patients with oxidative stress induced pathologies.     

A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.     

Assay for levormeloxifene, a selective estrogen receptor modulator, in human and monkey plasma employing high-performance liquid chromatography and solid-phase extraction

Assays for levormeloxifene, a new selective estrogen receptor modulator, and its 7-desmethyl metabolite in human and cynomolgus monkey plasma are described. Plasma was extracted on mixed-mode bonded sorbent material (C₈/SCX) and the extracts were analysed by high-performance liquid chromatography with fluorescence detection. Recoveries of levormeloxifene and the metabolite exceeded 70%. Within and total assay precision calculated as a coefficient of variation (C.V.) were <8% for both compounds at all concentration levels, except at the limit of quantitation (LOQ) where the C.V. was 15%. Within and total-assay accuracy calculated as a percentage of the nominal value were between 90 and 114% for both analytes. The LOQ was for levormeloxifene and 7-desmethyllevormeloxifene, respectively, 1.5 and 2.5 ng/ml (man) and 5.2 and 6.9 ng/ml (monkey). In the monkey plasma assay, human plasma could substitute monkey plasma as blank plasma.