Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma

A high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed for quantification of aripiprazole and dehydro-aripiprazole, in human plasma. After a simple liquid-liquid extraction, chromatographic separation was carried out on a C18 reversed-phase column, using an ammonium buffer-acetonitrile mobile phase (40:60, v/v). The total run time was only 7 min at a flow-rate of 1.0 ml/min. The precision values were less than 12% and the accuracy values were ranging from 98 to 113% and the lower limit of quantification was 2 ng/ml for both compounds. Calibration curves were linear over a range of 2-1000 ng/ml. The mean trough plasma concentrations in patients treated with aripiprazole were 157 and 29 ng/ml for aripiprazole and dehydro-aripiprazole, respectively.     

Gas chromatography-mass spectrometry determination of matrine in human plasma

A method was developed for the quantification of matrine in human plasma using a liquid-liquid extraction procedure followed by gas-chromatography-mass spectrometry (GC/MS) analysis. Deuterated matrine, an internal standard of the analysis, was spiked into the plasma samples before extraction. Linear detection responses were obtained for matrine concentrations ranging from 10 to 500 ng/ml. The intra-day and inter-day precision ranged from 0.4 to 4.0% and 1.0-3.5%, respectively. The intra-day accuracy was between -7.3 and 4.5%. The limit of quantification for matrine was 23 ng/ml. The extraction efficiency averaged about 38%. The validated GC/MS method will be used to quantify matrine in human plasma samples collected in a clinical trial study.     

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC–MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C₁₈ analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25–1000 ng/ml (r²≥0.996) with accuracy ≤9.8% and precision ≤13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1300 ng/ml for MPA. This HPLC–MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin,a novel highly lipophilic camptothecin derivative,in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2±0.5 min and 8.0±0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at −70°C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Gas chromatographic–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.     

Improved and validated method for the determination of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in serum,and in human liver microsomal preparations using gas chromatography-mass spectrometry

A validated method for the quantification of Delta(9)-tetrahydrocannabinol (THC) and its main metabolites 11-hydroxy-tetrahydrocannabinol (OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in serum is presented. The substances were isolated by solid-phase extraction, derivatised by methylation, and analysed by means of GC-MS in the selected ion monitoring mode. Quantitation was achieved by the addition of deuterated analogues as internal standards. The method was linear up to 10 ng/ml for THC and OH-THC, and up to 50 ng/ml for THC-COOH. The limits of quantification were 0.62 ng/ml for THC, 0.68 ng/ml for OH-THC and 3.35 ng/ml for THC-COOH. The limits of detection for the least intensive ions were 0.52 ng/ml for THC, 0.49 ng/ml for OH-THC and 0.65 ng/ml for THC-COOH. The method was validated according to the requirements of the Journal of Chromatography B. The method has been routinely used on samples from drivers suspected of "driving under the influence". In addition to the forensic application, a cross-validation was carried out by applying the method developed for serum to human liver microsomal preparation samples.     

Simple, sensitive and rapid LC-MS method for the quantitation of indapamide in human plasma--application to pharmacokinetic studies

A sensitive and specific method using liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) has been developed and validated for the identification and quantification of indapamide in human plasma. A simple liquid-liquid extraction procedure was followed by injection of the extracts on to a C18 column with gradient elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different plasma batches. Linearity was established for the concentration range 0.5-100.0 ng/ml, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (RSD%) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.2 ng/ml with 0.2 ml plasma. The proposed method enables the unambiguous identification and quantification of indapamide for pre-clinical and clinical studies.     

Analysis of [3',3'-d(2)]-nicotine and [3',3'-d(2)]-cotinine by capillary liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive LC/MS/MS assay was developed for the quantification of d(2)-nicotine and d(2)-cotinine in plasma of current and past smokers administered d(2)-nicotine. After solid phase extraction and liquid-liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d(2)-nicotine (0.03-6.0 ng/ml plasma) and d(2)-cotinine (0.15-25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d(2)-nicotine and d(2)-cotinine, respectively. The coefficient of variation was 3.7% for d(2)-nicotine and 2.5% for d(2)-cotinine. The method was applied to two ongoing studies of d(2)-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

Determination of captopril in human plasma, using solid phase extraction and high-performance liquid chromatography, coupled to mass spectrometry: application to bioequivalence study

A specific high performance liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed for the determination of captopryl in plasma. The retention time was 1.45 and 1.37 min for captopril and enalapril, respectively. The overall mean recovery, using SPE extraction with OASIS HLB cartridges, was found to be 107.2+/-9.5 and 100.04+/-2%, respectively. Calibration curves were linear in the concentration range of 10.00-2000.00 ng/ml, and the lower limit of quantification (LLOQ) was 10.00 ng/ml. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 0.88 to 1.95%. Intra-batch accuracy ranged from 97.15 to 105.77%, while intra-batch precision ranged from 2.49 to 5.66% at concentrations of 30.00, 760.00 and 1500.00 ng/ml. The developed method was applied to study bioequivalence of captopril in a group of 25 human subjects at a single oral dose of a 50mg tablet.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Optimisation of an extraction method for the determination of prostaglandin E2 in plasma using experimental design and liquid chromatography tandem mass spectrometry

A new extraction method has been developed for the extraction of prostaglandin E(2) (PGE(2)) from human plasma of patients suffering chronic inflammatory disorders. The extraction solvents were optimised systematically and simultaneously by using a central composite design. The optimised method involves precipitation of the protein fraction, centrifugation, evaporation and dissolution of the supernatant in the mobile phase, screening to confirm the presence of the analyte, and quantification of the positive samples by liquid chromatography tandem ion-trap mass spectrometry. Tandem mass spectrometry in negative mode was performed by isolating and fragmenting the ion [PGE(2)-H](-) signal m/z 351. Identification and quantification was carried out by extracting the ion fragment chromatograms at 333, 315 and 271 m/z. The quantitative determination was linear for the low nanogram (1-50 ng/ml) and upper picogram (400-1000 pg/ml) range studied, using 15 and 0.5 ng/ml of internal standard, respectively. The lower limit of detection was 2.5 pg for an injection volume of 25 microl. The optimised extraction method showed high reproducibility (coefficients of variation<4%) and recovery values, estimated from standard addition experiments, ranging from 96 to 98%.     

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%.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Automated online dual-column extraction coupled with teicoplanin stationary phase for simultaneous determination of (R)- and (S)-propranolol in rat plasma using liquid chromatography-tandem mass spectrometry

An automated online sample extraction method for rat plasma was developed and validated for the quantification of (R)- and (S)-propranolol following the intravenous administration of either the racemate or the individual enantiomers at 5 mg/kg. A dual-column extraction system coupled to a chiral stationary phase (CSP) was used in conjunction with liquid chromatography-tandem mass spectrometry. In this method, two Oasis HLB extraction columns (50x1.0 mm) in parallel were used for online plasma sample purification and teicoplanin CSP (Chirobiotic T) was used for the enantiomeric separation. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for re-conditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a relatively shorter run time and higher throughput. The lower limit of detection was 0.5 ng/ml and the lower limit of quantification was 2 ng/ml for each enantiomer using 25 microl of rat plasma. The method was validated with a linear calibration curve between 2 and 2000 ng/ml for (R)- and (S)-propranolol, respectively. The intra- and inter-day precision (C.V.) was no more than 7.6% and the accuracy of the assay was between 92 and 103%. The teicoplanin CSP proved to be rugged with excellent reproducibility of chromatographic parameters.     

Direct gas chromatographic determination of dechloroethylcyclophosphamide following microsomal incubation of cyclophosphamide

A method for the sensitive determination of dechloroethylcyclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C₈ cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.     

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).