Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation).     

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.     

Effects of common metal ions on the determination of anions by suppressed ion chromatography

A rapid and sensitive method for the determination of domperidone in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometry detection. The samples were rendered basic with 1 M Na2CO3 and the domperidone extracted using tert.-butyl methyl ether, followed by back-extraction into formic acid (2% in water). Chromatography was performed on a Phenomenex Luna C8 (2), 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (300:700, v/v), delivered at 0.2 ml/min. Detection was performed using an Applied Biosystems Sciex API 2000 mass spectrometer set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recovery of domperidone was +/- 100%, with a lower limit of quantification set at 0.189 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric detection resulting in a rapid (extraction and chromatography) and sensitive method for the determination of domperidone in human plasma, which is more sensitive than previously described methods.     

Derivatization of beta-dicarbonyl compound with 2,4-dinitrophenylhydrazine to enhance mass spectrometric detection: application in quantitative analysis of houttuynin in human plasma

Houttuynin (decanoyl acetaldehyde), a beta-dicarbonyl compound, is the major antibacterial constituent in the volatile oil of Houttuynina cordata Thunb. In the present work, detection of houttuynin in human plasma based on the chemical derivatization with 2,4-dinitrophenylhydrazine (DNPH) coupled with liquid chromatography/tandem mass spectrometry was described. The primary reaction products between the beta-dicarbonyl compound and DNPH in aqueous phase were identified as heterocyclic structures, of which the mass spectrometric ionization and fragmentation behavior were characterized with the aid of high-resolution multistage mass spectral analysis. For quantification, houttuynin and internal standard (IS, benzophenone) in plasma were firstly converted to their DNPH derivatives without sample purification, then extracted from human plasma with n-hexane and detected by liquid chromatography tandem mass spectrometry performed in selected reaction monitoring (SRM) mode. This method allowed for a lower limit of quantification (LLOQ) of 1.0 ng/ml using 100-microl plasma. The validation results showed high accuracy (%bias < 2.1) and precision (%CV < 7.2) at broad linear dynamic range (1.0-5000 ng/ml). The simple and quantitative derivatization coupled with tandem mass spectrometric analysis facilitates a sensitive and robust method for the determination of plasma houttuynin in pharmacokinetic studies.     

Quantitative analysis of the farnesyl transferase inhibitor lonafarnib (Sarasartrade mark, SCH66336) in human plasma using high-performance liquid chromatography coupled with tandem mass spectrometry

Lonafarnib is a novel anticancer drug that inhibits farnesyl transferase. To assess its pharmacokinetic properties, we developed a sensitive and quantitative assay using liquid chromatography coupled with tandem mass spectrometry for the determination of lonafarnib levels in human plasma. Sample pretreatment consisted of the addition of an isotopically labeled internal standard and protein precipitation with acetonitrile using 100 microL plasma. Chromatographic separation was performed on an Inertsil ODS-3 analytical column (50 x 2.1 mm i.d., particle size 5 microm) with acetonitrile/water/formic acid (50:50:0.05, v/v/v) as the mobile phase, at a flow rate of 0.2 mL/min. The analytical run time was 8 min. An API365 triple quadrupole mass spectrometer was used for specific and sensitive detection. It was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated using a concentration range of 2.5 to 2500 ng/mL lonafarnib. Validation of the assay was performed according to the most recent FDA guidelines for bioanalytical method validation and all results were within the requirements. The described method was successfully applied to support a clinical phase I trial with lonafarnib.     

Simultaneous quantification of cyclophosphamide, 4-hydroxycyclophosphamide, N,N',N"-triethylenethiophosphoramide (thiotepa) and N,N',N"-triethylenephosphoramide (tepa) in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

The alkylating agents cyclophosphamide (CP) and N, N', N"-triethylenethiophosphoramide (thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens can be complicated by the occurrence of severe and sometimes life-threatening toxicities, pharmacokinetically guided administration of these compounds, to reduce variability in exposure, may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of CP, thiotepa and their respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N, N', N"-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as 4OHCP-SCZ. Sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl of plasma. Chromatographic separation was performed on an Zorbax Extend C18 column (150 x 2.1 mm i.d., particle size 5 microm), with a quick gradient using 1 mM ammonia solution and acetonitrile, at a flow-rate of 0.4 ml min(-1). The analytical run time was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 200-40,000 ng ml(-1) for CP, 50-5000 ng ml(-1) for 4OHCP-SCZ and 5-2500 ng ml(-1) for thiotepa and tepa, using 100 microl of human plasma. These dynamic concentration ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an internal standard. The coefficients of variation were <12% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/- 15%). This robust and rapid LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring of CP, thiotepa and their metabolites in our hospital.     

Highly specific and sensitive liquid chromatography-tandem mass spectrometry method for the determination of 3-desmethylthiocolchicine in human plasma as analyte for the assessment of bioequivalence after oral administration of thiocolchicoside

A sensitive method for the determination of 3-desmethylthiocolchicine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The plasma samples were extracted with ethyl acetate and separated on a Phenomenex Luna C18(2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.005% formic acid (350:650, v/v) at a flow rate of 0.35 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recovery for 3-desmethylthiocolchicine was 70%, with a lower limit of quantification set at 0.39 ng/ml. The increased selectivity of mass spectrometric (MS-MS) detection allowed us to distinguish between thiocolchicoside and its primary metabolite 3-desmethylthiocolchicine in human plasma, thereby giving more insight about the pharmacokinetics of the drug in humans.     

Liquid chromatography–electrospray ionization tandem mass spectrometry for the quantification of styraxlignolide A in rat plasma

Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC₁₈ column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r²=0.9978) over the concentration range of 100?10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra‐ and inter‐assays at four QC levels were 1.6–8.3% and from ?12.0 to ?1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague–Dawley rats.     

Determination of ivermectin B(1a) in animal plasma by liquid chromatography combined with electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b).The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.     

High-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Trospium Chloride in Human Plasma and Its Application in a Bioequivalence Study

A selective, rapid, and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated for the quantification of trospium chloride in human plasma. With tramadol chloride as the internal standard, sample pretreatment involved a simple liquid-liquid extraction with chloroform-isopropyl alcohol (40/2, v/v) of 0.5 ml plasma. The analysis was carried out on a Hypsil C₁₈ column (150 mm × 2.1 mm, 5.0 μm) with a flow rate of 0.2 ml/min. The mobile phase was methanol-5% acetic acid-20 mM ammonium methylate (55/30/15, v/v/v). The detection was performed by a selected reaction monitoring (SRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.2–50.0 ng/ml, with a lower limit of quantification of 0.2 ng/ml. The intra- and inter-day precision (RSD) values were below 15%. The method was successfully applied to a clinical bioequivalence study of trospium chloride in Chinese healthy volunteers following oral administration.     

Quantitative analysis of EO9 (apaziquone) and its metabolite EO5a in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the quantitative determination of EO9 and its metabolite EO5a is presented. A 200-microl human plasma aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 ml ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate-methanol (7 : 3, v/v) and 25 microl-volumes were injected into the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide-methanol (gradient system)). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 5 to 2500 ng/ml for EO9 and from 10 to 2500 ng/ml EO5a using 200 microl of human plasma samples. Validation results demonstrate that EO9 and EO5a concentrations can be accurately and precisely quantified in human plasma. This assay will be used to support clinical pharmacologic studies with EO9.     

Determination of nifedipine in dog plasma by high‐performance liquid chromatography with tandem mass spectrometric detection

Nifedipine is a dihydropyridine calcium channel blocker used widely in the management of hypertension and other cardiovascular disorders. In this work, a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated to determine nifedipine in dog plasma using nimodipine as the internal standard. Chromatographic separation was carried out on a C₈ column. The mobile phase consisted of a mixture of acetonitrile, water and formic acid (60:40:0.2, v/v/v) at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode via an atmospheric pressure chemical ionization source. The method has a lower limit of quantification of 0.20 ng/mL with consumption of plasma as low as 0.05 mL. The linear calibration curves were obtained in the concentration range of 0.20–50.0 ng/mL (r = 0.9948). The recoveries of the liquid extraction method were 74.5–84.1%. Intra‐day and inter‐day precisions were 4.1–8.8 and 6.7–7.4%, respectively. The quantification was not interfered with by other plasma components and the method was applied to determine nifedipine in plasma after a single oral administration of two controlled‐release nifedipine tablets to beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.     

Simultaneous quantification of fludarabine and cyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.     

Determination of RSD921 in human plasma by high-performance liquid chromatography-tandem mass spectrometry using tri-deuterated RSD921 as internal standard: application to a phase I clinical trial.

A fast, sensitive and specific method is presented for the quantification of RSD921 in human plasma by liquid chromatography coupled with tandem mass spectrometry using tri-deuterated RSD921 (3d-RSD921) as an internal standard. A single-step liquid/liquid extraction was performed with diethyl ether/hexane (80 : 20, v/v) using 0.5 ml of plasma. The plasma calibration curves were linear from 0.1 to 20 ng ml(-1) (r > 0.999). Between-run precision, based on the percent relative deviation for replicate (n = 40) quality controls, was < or =7.27% (0.5 ng ml(-1)), < or =7.39% (5.0 ng ml(-1)), and < or =5.06% (20.0 ng ml(-1)). Between-run accuracies, based on the relative error, were +/-2.59%, +/-1.23% and +/-1.64% respectively. The method was developed to evaluate the pharmacokinetic profile after 15 min of intravenous stepwise-ascending infusion dose of RSD921 in 18 healthy volunteers. A dissociation study of protonated RSD921 and 3d-RSD921 by collision-induced dissociation using in-source fragmentation and tandem mass spectrometry is also presented.     

Simultaneous determination of a new phosphodiesterase-5 inhibitor DA-8159 and its active metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometry

Summary An LC-MS-MS method for the simultaneous determination of DA-8159 and its active metabolite, DA-8164 in human plasma was developed. DA-8159, DA-8164 and the internal standard, sildenafil were extracted from human plasma with dichloromethane at basic pH. A reversephase HPLC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-ammonium formate (10 mM, pH 6.0) (60:40,v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode. The method showed a satisfactory sensitivity (lower limits of quantification, 2.0 ng mL¹), precision, accuracy, recovery and selectivity. The successful determination of DA-8159 and DA-8164 in the plasma of a volunteer who ingested a single dose of 100 mg DA-8159 confirms that the present method can be used for plasma analysis for clinical trial.     

Determination of limonin in dog plasma by liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A highly sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of limonin in beagle dog plasma using nimodipine as internal standard. The analyte and internal standard (IS) were extracted with ether followed by a rapid isocratic elution with 10 mm ammonium acetate buffer–methanol (26:74, v/v) on a C₁₈ column (150 × 2.1 mm i.d.) and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 469.4 → 229.3 and m/z 417.2 → 122.0 were used to measure the analyte and the IS. The assay was linear over the concentration range of 0.625–100 ng/mL for limonin in dog plasma. The lower limit of quantification was 0.312 ng/mL and the extraction recovery was >90.4% for limonin. The inter‐ and intra‐day precision of the method at three concentrations was less than 9.9%. The method was successfully applied to pharmacokinetic study of limonin in dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of lisinopril in human plasma by high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine lisinopril in human plasma. Sample pretreatment involved a one-step protein precipitation with methanol of 0.1 mL plasma. Analysis was performed on an Inertsil ODS-3 column (2.1 × 50 mm i.d., 3 μm) with mobile phase consisting of methanol-water (containing 0.2% formic acid; 55:45, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode via an electrospray ionization source. Each plasma sample was chromatographed within 2.5 min. The linear calibration curves for lisinopril were obtained in the concentration range of 1.03-206 ng/mL (r(2) ≥ 0.99) with a lower limit of quantification of 1.03 ng/mL. The intra- and inter-day precisions (relative standard deviation) were not higher than 11%, and accuracy (relative error) was within ±6.8%, determined from quality control samples for lisinopril, which corresponded to the guidance of the Food and Drug Administration. The method described herein was fully validated and successfully applied to the pharmacokinetic study of lisinopril tablets in healthy male volunteers after oral administration.     

Determination of the lipophilic antipsychotic drug ziprasidone in rat plasma and brain tissue using liquid chromatography-tandem mass spectrometry

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LCESI-MS/MS) method with low matrix effects was developed and validated for the quantification of the lipophilic antipsychotic ziprasidone from rat plasma and brain tissue. Ziprasidone was extracted from rat plasma and brain homogenate using a single-step liquid-liquid extraction. Ziprasidone was separated on an Agilent Eclipse XDB C8 column (150 x 2.1 mm i.d., 5 microm) column using a mobile phase of acetonitrile-0.02% ammonia in water (pH 7.20 adjusted with formic acid) using gradient elution. Ziprasidone was detected in the positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recovery, matrix effects and stability were determined. The LLOQ was 0.2 ng/mL for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/mL for plasma and 0.833-833.3 ng/g for brain tissue. The correlation coefficient (R2) values were more than 0.996 for both plasma and brain homogenate. The precision and accuracy intra-day and inter-day were better than 8.13%. The relative and absolute recovery was above 81.0% and matrix effects were lower than 5.2%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of ziprasidone after chronic treatment.     

Sensitive liquid chromatographic-tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane-isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex Luna C18 (2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.