Determination of milnacipran, a serotonin and noradrenaline reuptake inhibitor, in human plasma using liquid chromatography with spectrofluorimetric detection

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid-liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).     

Liquid chromatography-tandem mass spectrometry of I3,II8-biapigenin, the major biflavone in Hypericum perforatum extracts

High-performance liquid chromatography method coupled with tandem mass spectrometry was developed for the quantitative determination of I3,II8-biapigenin. The procedure includes solid-phase extraction and separation on an XTerra MS C18. The assay was linear over a wide range; precision and accuracy were acceptable. Biapigenin was present in mouse and rat plasma after a standardized Hypericum perforatum extract. It was not detected in brain (<5ngg(-1)), suggesting poor brain-to-blood permeability. Biapigenin concentrations were measurable in mice after intraperitoneal biapigenin (10mgkg(-1)) but these amounted to about 2% of the equivalent systemic exposure, after correction for the contribution from residual blood.     

Determination of cediranib in mouse plasma and brain tissue using high-performance liquid chromatography-mass spectrometry

A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid-liquid extraction. The assay was validated for a 2.5-2500 ng/mL concentration range for plasma, and for 1-2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1-14.3% for plasma and 1.5-9.4% for brain homogenate; between-assay variabilities were 2.4-9.2% for plasma, and 4.9-10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a brain distribution study in mice at an oral dose of 5 mg/kg.     

Simultaneous determination of lansoprazole enantiomers and their metabolites in plasma by liquid chromatography with solid-phase extraction

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5M NaClO(4)-acetonitrile-methanol (6:3:1 (v/v/v)). The analysis required only 100 microl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10-1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.     

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.     

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.     

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.     

Molecularly imprinted solid-phase extraction for rapid screening of mycophenolic acid in human plasma

A molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography (MISPE-HPLC) method was developed for rapid screening of mycophenolic acid (MPA) in human plasma. MPA imprinted polymers (MPA-MIP) were synthesized and then tested for their performance both in organic and in aqueous solution. MPA was selectively trapped and preconcentrated on the MPA-MIP sorbent using different loading and washing conditions. The good selectivity of MPA-MIP enabled further clean-up of the interfering components in human plasma. For the proposed MISPE-HPLC method, the linearity between responses (peak area) and concentration was found over the range of 1-100microg/ml with a linear regression coefficient (R(2)) of 0.9989. The limit of detection (LOD) and theoretical limit of quantification (LOQ) for MPA in plasma were 0.10 and 0.32microg/ml, respectively. The precisions were 7.3, 3.5 and 4.7% RSD for intra-day assay and 9.2, 4.1 and 5.5% RSD for inter-day reproducibility, respectively, at three concentration levels of MPA in spiked plasma (1, 10 and 100microg/ml). Both recoveries for the extraction (more than 74%) and for the analytical method (more than 87%) were acceptable for screening MPA in plasma samples. Twelve-hour pharmacokinetic profile of MPA for a renal transplant recipient receiving chronic oral dosing of 500mg mycophenolate mofetil (MMF) was investigated. Results indicated that this method could be applied for therapeutic drug monitoring of mycophenolic acid in patient plasma.     

Estimation of carvedilol in human plasma by using HPLC-fluorescence detector and its application to pharmacokinetic study

A simple, precise and sensitive high performance liquid chromatography procedure has been developed for determination of carvedilol in human plasma. The method was developed on Lichrosphere R CN column using a mobile phase of acetonitrile/20 mM ammonium acetate buffer with 0.1% triethylamine (pH adjusted to 4.5) (40/60, v/v). The peaks were detected by using fluorescence detector (excitation wavelength 282 nm and emission wavelength 340 nm). Carvedilol and domperidone (internal standard) were extracted by liquid-liquid extraction procedure using dichloromethane. This method was specific and had a linearity range of 1-128 ng/ml with intra- and inter-day precision (%C.V.) less than 15%. The accuracy ranges from 87.3 to 100.88% and the recovery of carvedilol was 69.90%. The stability studies showed that carvedilol in human plasma was stable during short-term period for sample preparation and analysis. This method was used to assay the carvedilol in human plasma samples obtained from subjects who had been given an oral tablet of 12.5 mg carvedilol.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.     

Enantioselective analysis of praziquantel in plasma samples

A direct enantioselective high-performance liquid chromatography method is described for the quantitative determination of praziquantel enantiomers in plasma samples. The method involves two-step extraction of plasma with toluene, evaporation of the solvent and chromatography on a Chiralcel OD-H column using hexane-ethanol (85:15, v/v) as the mobile phase and detection at 220 nm. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

Isolation of N,N-dialkylated derivatives of valproylglycinamide from dog plasma by active charcoal adsorption and their quantification by high-performance liquid chromatography

A selective assay for quantification of N,N-dimethylvalproylglycinamide (DM-VGD) and N,N-diethylvalproylglycinamide (DE-VGD) in dog plasma utilizing reversed-phase high-performance liquid chromatography and UV detection has been developed. These compounds are derivatives of the potential anticonvulsant drug, valproylglycinamide, which is currently undergoing clinical trials. The method is based on extraction of dog plasma with activated charcoal, separation of the charcoal pallet and extracting it with methanol, evaporation of the solvent and injecting the reconstituted residue onto the column. The active charcoal adsorption method is reliable and reproducible, and it provides a chromatogram free of interfering endogenous plasma compounds. The assay was validated and provided a limit of quantification of 2.3 mmol/l for DE-VGD and 5.3 mmol/l for DM-VGD. Mean recovery of these compounds from plasma averages 75%. This analytical method is suitable for the quantitative determination of DM-VGD and DE-VGD in plasma and it has been applied to a pharmacokinetic study of these compounds in a dog.     

Determination of rifalazil, a potent antibacterial agent, in human plasma by liquid-liquid extraction and LC-MS/MS

A sensitive assay for determination of rifalazil (also known as ABI-1648 and KRM-1648) in human plasma is described. The analytical method utilizes liquid-liquid extraction of plasma with methyl tert-butyl ether, followed by reversed-phase liquid chromatography with a C18 column and a mobile phase gradient utilizing 0.1% formic acid in water and acetonitrile, respectively. Electrospray mass spectrometry in the positive ion mode with selected reaction monitoring of rifalazil and an isotope labeled internal standard, 13C4-rifalazil (ABI-9901) was used for selective and sensitive detection. The calibration range was 0.050-50 ng/mL plasma using 200 microL plasma sample volume. The absolute extraction recovery of rifalazil from K2-EDTA plasma, evaluated at three concentration levels, was 88.6-97.3%, and the recovery for the internal standard was 96.8%. A study of plasma matrix effects showed a peak area response at 90-99% compared to neat solutions for both rifalazil and the internal standard. Stability evaluation of rifalazil in plasma, whole blood and methanol showed that the analyte stability was adequate when stored under study conditions. The precision, as evaluated in three validation batches, was consistent for fortified plasma quality control (QC) samples at four concentration levels, with < or =6% R.S.D. except for at the lowest quality control level where it was 10.7% R.S.D. The accuracy for QC samples (difference between found and nominal concentration) ranged from -2.3% to 5.1%. Similar precision and accuracy values were obtained over 6 months of routine application of this method. It was concluded that the performance improved markedly during routine operation by replacing a closely related structural analog internal standard with the stable isotope internal standard.     

Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.