Simultaneous determination of zidovudine and lamivudine from rat tissues by liquid chromatography/tandem mass spectrometry

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid, placental, and fetal tissues. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C18 column (5 microm, 150 x 3.9 mm i.d). The mobile phase consisted of 30% methanol and 7.5 mM ammonium acetate (pH 6.5). The method was validated in the range of 0.05-25 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport following co-administration of these compounds at a dose of 25 mg/kg each in a pregnant rat.     

Quantitative determination and sampling of lamivudine and zidovudine residues for cleaning validation in a production area

Lamivudine (3TC) and zidovudine (AZT) are systemic antiviral substances extensively used in human immunodeficiency virus (HIV) infected patients. Nowadays, 3TC, AZT, and several other pharmacologically potent pharmaceuticals are manufactured in the same production area. To assure quality of drug products and patient safety, properly validated cleaning methodology is necessary. A carefully designed cleaning validation and its evaluation can ensure that residues of 3TC and AZT will not carry over and cross contaminate the subsequent product. The aim of this study was to validate a simple analytical method for verification of residual 3TC and AZT in equipment used in the production area and to confirm the efficiency of the cleaning procedure. The liquid chromatography method was validated using a Nova-Pak C18 column (3.9 x 150 mm, 4 microm particle size) and methanol-water (20 + 80, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Ultraviolet detection was made at 266 nm. The calibration curve was linear over a concentration range of 2.0-22.0 microg/mL with a correlation coefficient of 0.9998. The detection and quantitation limits were 0.36 and 1.21 microg/mL, respectively. The intra-day and interday precision expressed as relative standard deviation were below 2.0%. The mean recovery of the method was 99.19%. The mean extraction recovery from manufacturing equipment was 83.5%.     

A rapid and sensitive method for simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry detection

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lamivudine (3TC) and zidovudine (AZT) in human serum, using didanosine (ddI) as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 230.0 --> 111.8 for 3TC, m/z 268.1 --> 126.8 for AZT, and m/z 237.2 --> 136.8 for ddI. The limits of detection and quantitation were 3 and 10 ng/mL for 3TC, and 5 and 15 ng/mL for AZT. The method was linear in the studied ranges (10-1500 ng/mL for 3TC and 15-3000 ng/mL for AZT), with r(2) > 0.99 for each drug, and the run time was 4 min. The intra-assay precisions (%) were in the ranges 1.9-8.7 (3TC) and 2.2-8.9 (AZT), the inter-assay precisions were in the ranges 2.6-9.0 (3TC) and 4.2-8.1 (AZT), and the intra- and inter-assay accuracies were >97% for both drugs. The absolute recoveries were 95-99% for 3TC (45, 600 and 1200 ng/mL) and 104-112% for AZT (45, 1000 and 2400 ng/mL). The analytical method was applied to a bioequivalence study in which 24 healthy adult volunteers received single oral doses of the reference formulation and two test combined AZT/3TC tablets, in an open, three-period, balanced, randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (extrapolated area under the serum concentration vs. time curve from time zero to infinity), it was concluded that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of absorption of both 3TC and AZT.     

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for simultaneous quantification of olanzapine, clozapine, ziprasidone, haloperidol, risperidone, and its active metabolite 9-hydroxyrisperidone, in rat plasma using midazolam as internal standard (IS). The analytes were extracted from rat plasma using a single step liquid-liquid extraction technique. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 0.1 ng/mL and correlation coefficient (R(2)) values for the linear range of 0.1-100 ng/mL were 0.997 or greater for all the analytes. The intra-day and inter-day precision and accuracy were better than 8.05%. The relative and absolute recovery was above 77% and matrix effects were low for all the analytes except for ziprasidone. This validated method has been successfully used to quantify the plasma concentration of the analytes after chronic treatment with antipsychotic drugs.     

Development and validation of an HPLC/MS/MS method for determining the thiazolidinone PG15 in rat plasma

A rapid, sensitive, and simple HPLC/MS/MS method was developed and validated for the determination of (5Z,E)-3-[2-(4-chlorophenyl)-2-oxoethyl]-5-(1H-indol-3-ylmethylene)-thiazolidine-2, 4-dione (PG15) in rat plasma using chlortalidone as an internal standard (IS). Analyses were performed using a C18 column and isocratic elution with acetonitrile-water (90 + 10, v/v) containing 10 mM ammonium hydroxide (pH 8.0) as the mobile phase pumped at 0.3 mL/min. Detection was performed by MS with negative ion mode electrospray ionization. Rat plasma samples were prepared by deproteinizing with acetonitrile. Detected fragments were 395.1 > 171.9 for PG15 and 337.3 > 189.9 for the IS. Calibration curves were linear from 10 to 1000 ng/mL, with the determination coefficient > 0.99. The intraday and interday precisions were less than 12.2 and 11.3%, respectively. The applicability of the HPLC/MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after oral administration of PG15 to rats, and it provided the necessary sensitivity, linearity, precision, accuracy, and specificity.     

Determination of Abacavir in maternal plasma, amniotic fluid, fetal and placental tissues by a polarity switching liquid chromatography/tandem mass spectrometry method

A rapid and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of concentrations of the carbocyclic nucleoside antiviral Abacavir in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. All tissue samples were homogenized in water prior to analysis and all samples were prepared by acetonitrile precipitation followed by dilution with HPLC-grade water. Separation of the analyte and internal standard from the matrices was achieved on a C(8) analytical column (2.1 x 150 mm, 5 microm particle size). The mobile phase consisted of 10 mM ammonium acetate/acetonitrile using a gradient method at a flow rate of 0.25 mL/min for all matrices. The method yields retention times of approximately 3.4 and 5.1 min for the internal standard (Azidouridine) and Abacavir, respectively. For all matrices the limit of detection was approximately 1 ng/ml. Recoveries from the different matrices ranged from 53-87% for Abacavir and from 69-84% for Azidouridine. Within- and between-run precision (%RSD) and accuracy (%Error) were under 15% for all matrices.     

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 μm RP-Aqueous C(30) 140? column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.     

Determination of lamivudine/stavudine/efavirenz in human serum using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch

A high-performance liquid chromatography/tandem mass spectrometry (LC-MS-MS) method with ionization polarity switch was developed and validated in human serum for the determination of a lamivudine (3TC)/stavudine (d4T)/efavirenz combination HIV therapy. Solid phase extraction (SPE) was used to extract these anti-HIV drugs and internal standard aprobarbital. A gradient mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer with pH adjusted to 4.5 using glacial acetic acid was utilized to separate these drugs on a hexylsilane column (150 x 2.0 mm i.d.). The total run time between injections was 18 min. The precursor and major product ions of these drugs were monitored on a triple quadrupole mass spectrometer in the multiple reactions monitoring (MRM) mode. Ionization polarity was switched in the middle of the LC run allowing these anti-HIV drugs with different physicochemical properties to be detected simultaneously. The effect of ion suppression from human serum was studied and no interference with the analysis was noted. The method was validated over the range of 1.1-540 ng/mL for 3TC, 12.5-6228 ng/mL for d4T and 1.0-519 ng/mL for efavirenz. The method was shown to be accurate, with intra-day and inter-day accuracy less than 14.0% and precise, with intra-day and inter-day precision less than 13.1%. The extraction recoveries of all analytes were higher than 90%.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of lamivudine,stavudine and nevirapine in human plasma by LC–MS/MS and its application to pharmacokinetic study in clinic

A new high‐throughput LC–MS/MS method for the simultaneous determination of lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma is presented, with zidovudine as an internal standard. The analytes were extracted from plasma by protein precipitation and only 150 μL plasma was needed. Chromatographic separation was achieved on a Shiseido C₈ column (150 × 2.0 mm, 5 μm) with a total run time of 6 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was developed and validated over the concentration range of 25–5000 ng/mL for 3TC and NVP and 20–4000 ng/mL for d4T. The method was validated in terms of intra‐ and inter‐day precision (≤8.6%), accuracy (within ± 8.4%), linearity and specificity. The method has been successfully applied to the pharmacokinetic study of a combination treatment of 300 mg lamivudine, 30 mg stavudine and 200 mg nevirapine in 22 healthy male volunteers under fasting conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of chlordiazepoxide and selected antidepressants using CZE

A simple CE method was developed and validated for the simultaneous determination of chlordiazepoxide (CHL), amitriptyline, and nortriptyline (mixture I) or the determination of CHL and imipramine (mixture II) using the same BGE. Sertraline and amitriptyline were used as internal standards for the first and second mixtures, respectively. The method allows amitriptyline to be completely separated from its impurity and main metabolite nortriptyline, which can be quantified from 0.2 μg/mL. The separation was achieved using 20 mM potassium phosphate buffer pH 5 containing 12 mM β‐cyclodextrin and 1 mM carboxymethyl‐β‐cyclodextrin. UV detection was performed at 200 nm and a voltage of 15 kV was applied on an uncoated fused‐silica capillary at 25°C. These experimental conditions allowed separation of the compounds to be obtained in 7 min. Calibration graphs proved the linearity up to 40 μg/mL for CHL, up to 100 μg/mL for amitriptyline and imipramine, and up to 5 μg/mL for nortriptyline. The accuracy and precision of the method have been determined by analyzing synthetic mixtures and pharmaceutical formulations. The analytical results were quite good in all cases indicating that the method was linear, sensitive, precise, accurate, and selective for both mixtures.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

Simultaneous quantitation of the diastereoisomers of scholarisine and 19‐epischolarisine,vallesamine, and picrinine in rat plasma by supercritical fluid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Dengtaiye tablet has been used to treat chronic bronchitis cough. Scholarisine, 19‐epischolarisine, vallesamine, and picrinine are the representative constituents of Dengtaiye. A rapid and sensitive assay based on supercritical fluid chromatography with tandem mass spectrometry has been developed and validated for the determination of the diastereoisomers of scholarisine and 19‐epischolarisine, vallesamine, and picrinine in rat plasma using lamotrigine as internal standard. The analysis in a run time of only 6 min was performed on an ACQUITY UPC² Trefoil^TM BEH 2‐EP column (3.0 × 150 mm, 2.5 μm) at 50ºC. The mobile phase consisting of carbon dioxide and methanol (2 mM ammonium formate) was performed as follows: 15% methanol (2 mM ammonium formate) maintained at 0–2 min, 15–19% methanol (2 mM ammonium formate) at 2–4 min, 19–15% methanol (2 mM ammonium formate) at 4–6 min. The flow rate was 1.50 mL/min. The assay was linear over the concentration ranges 50–10000 pg/mL for scholarisine, 19‐epischolarisine, vallesamine, and picrinine with corresponding lower limits of quantitation of 50 pg/mL. Intra‐ and interday precisions were in the range 1.42–12.85% with accuracies in the range –11.71–2.48%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 108 mg/kg Dengtaiye tablet to rats.     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.     

Determination of the enantiomeric impurity in S-(-)pantoprazole using high performance liquid chromatography with sulfobutylether-beta-cyclodextrin as chiral additive

A new and accurate HPLC method using sulfobutylether-beta-cyclodextrin (SBE-beta-CD) as chiral mobile phase additive (CMPA) was developed and validated for the determination of R-(+)pantoprazole in S-(-)pantoprazole. The influences of type and concentration of CD, ACN content and buffer pH of mobile phase on the resolution and retention of enantiomers were investigated. A baseline resolution of pantoprazole enantiomers was achieved on a Spherigel C18 column (150 mm x 4.6 mm, 5 microm) using ACN and 10 mM phosphate buffer (pH 2.5) containing 10 mM SBE-beta-CD (15:85 v/v) as mobile phase with a flow rate of 0.9 mL/min at 20 degrees C. The detection wavelength was set at 290 nm. The method was extensively validated in terms of accuracy, precision and linearity according to the International Conference on Harmonisation (ICH) guidelines and proved to be robust. The LOD and LOQ for R-(+)pantoprazole were 0.2 and 0.5 microg/mL, respectively, with 5 microL injection volume. A good linear relationship was obtained in the concentration range of 0.5-6.0 microg/mL with r(2) >0.999 for R-(+)pantoprazole. The percentage recovery of the R-(+)pantoprazole ranged from 92.1 to 101.2 in bulk drug of S-(-)pantoprazole. The method is capable of determining a minimum limit of 0.05% w/w of R-enantiomer in S-(-)pantoprazole bulk samples.     

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.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.