First LC-MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.     

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography–tandem mass spectrometry

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC–MS–MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 μm). The mobile phase consisted of methanol–50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode.

The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95±2% and 95±4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5–100 ng/ml concentration range for the two analytes (r²=0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2–4.7% for the metabolite.The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Liquid chromatography-tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration

We describe a liquid chromatography-electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid-liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water-acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5-1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5-1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.     

Application of 13C stable isotope labeling liquid chromatography-multiple reaction monitoring-tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C monoisotopic transition LC-MRM-MS/MS system with D-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The (13)C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography-tandem mass spectrometry method

Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03 min and 4.44±0.01 min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R(2)=0.9714 and R(2)=0.9768) in concentration ranges of 5-50 ng/ml and 25-200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC-MS/MS method are discussed.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Reversed-phase liquid chromatographic-mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions

Reversed-phase HPLC coupled to the atmospheric pressure ionization-electrospray ionization (API-ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide-acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 microg/ml (20 ng per injection) in the scan mode of MS and 0.1 microg/ml (2 ng per injection) in the case of selective ion monitoring.     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C₁₈ solid-phase extraction cartridge, elution from a Spherisorb C₈ reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8±3.5% and 89.1±6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 μg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

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.     

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 microm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.     

Determination of testosterone metabolites in human hepatocytes. I. Development of an on-line sample preparation liquid chromatography technique and mass spectroscopic detection of 6beta-hydroxytestosterone

A rapid and sensitive RP-HPLC assay for determination of 6beta-hydroxytestosterone in human hepatocytes with corticosterone as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20x4 mm I.D.) extraction pre-column and subsequent gradient separation on a Prontosil 60-5 C(18)-H (250x2 mm I.D., 5 micrometer particle size) analytical column in the back-flush mode using a ternary eluent system composed of methanol, tetrahydrofuran and water. Signal monitoring was done by measurement of the responses from liquid chromatography coupled to mass spectroscopy (LC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source conducted in the selected reaction monitoring (SRM) mode. Mean recoveries of 6beta-hydroxytestosterone from an estimate of the biological matrix, i.e., Dulbecco's modified Eagle medium "High Glucose", ranged from 101.8-104.4% for samples containing the target analyte at the 250, 500 and 1000 ng/ml level. The limit of quantitation (LOQ) was 20 ng/ml at an injection volume of 100 microliter determined in the same matrix. Linearity of signal responses versus concentration for all three analytes was accomplished in the range of 100-4000 ng/ml. Mean values of the coefficients of variation (C.V.) for the target analyte obtained for the concentrations 250, 500 and 1000 ng/ml at 5 different days in quintuplicate ranged from 1.5-7.7% (within-day) and 4.8-7.3% (between-day). The corresponding values for the accuracy ranged from 87.7-106.1% for the within-day and from 98.8-102.5% for the between-day measurements. The target analyte was sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.     

Application of C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography–multiple reaction monitoring–tandem mass spectrometry (LC–MRM–MS/MS) method using ¹³C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting ¹³C-labeled peptides in blood sample with a C₁₈ cartridge, the extract was applied to a ¹³C monoisotopic transition LC–MRM–MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5–100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The ¹³C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three ¹³C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each ¹³C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC–MRM–MS/MS method.     

Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry

An analytical method was developed and validated to determine Formoterol in human serum in the range from 0.40 to 100.24 pg/mL by high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) due to the lack of efficient methods to determine very low levels of Formoterol in serum and plasma. Serum was diluted by water and mixed with the internal standard (d6-Formoterol). Formoterol and internal standard were extracted using a cation-exchange solid phase column (SCX-3). After eliminating endogenous serum constituents through washing steps with water and methanol, elution took place using methanol/ammonia. After evaporation of the elution liquid the residue was redissolved and analyzed by HPLC-MS/MS with electrospray ionisation (ESI) in positive mode. A gradient between 10 mM ammonium formate and acetonitrile was used. The inter-batch precision of the calibration standards ranged from 1.55% to 9.01%. The inter-batch accuracy of the calibration standards ranged from 93.37% to 107.30%. The lower limit of quantitation (LLOQ, 0.40 pg/mL) had a precision of 19.67% and an accuracy of 96.78%. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2h at room temperature. Formoterol in human serum was stable for at least 6 months below -20 degrees C. This method has been used widely for quantifying Formoterol after inhalation of 9-36 microg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

High-performance liquid chromatographic–electrochemical assay for the quantitation of BMS-181885 in monkey plasma

A high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantitation of BMS-181885 (I), an anti-migraine agent, in monkey plasma. The assay involved a solid-phase extraction of I and BMY-46317 (internal standard; I.S.) on a 1-ml cyano cartridge using the automatic solid-phase extraction cartridge (ASPEC) system. Immediately following the conditioning of the cyano column (3 ml of methanol and 2 ml of 1% glacial acetic acid), plasma (0.25 ml) was loaded on to the column. The column was then washed with a 3 ml of 0.1 M ammonium acetate buffer (pH 6). The final elution of the analytes was performed using 2 ml of methanol. The eluate was then evaporated to dryness (gentle stream of nitrogen at 40°C) and the residue was dissolved in the mobile phase and injected on to a YMC basic column (15 cm×4.6 mm; 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 0.1 M ammonium acetate at pH 6–acetonitrile–methanol (70:20:10, v/v) was used as the mobile phase. Standard curves, with a lower limit of quantitation of 2 ng/ml of I were linear (r²≥0.998; range: 2–50 ng/ml). Based on the analysis of the quality control (QC) samples, the assay was both accurate and precise. The stability of I was established following freeze–thaw cycles and storage at or below −20°C. The extraction recovery of I from monkey plasma was about 82%. The validated assay method was applied to determine the pharmacokinetics of I in monkeys following a single 1 mg/kg intravenous dose.