Simultaneous determination of metoprolol succinate and amlodipine besylate in human plasma by liquid chromatography-tandem mass spectrometry method and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by simple liquid-liquid extraction with chloroform. The chromatographic separation was performed on a reversed-phase peerless basic C18 column with a mobile phase of methanol-water containing 0.5% formic acid (8:2, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 1-100ng/ml for MPS and 1-15ng/ml AM in human plasma. The MRM transition of m/z 268.10-103.10, m/z 409.10-334.20 and m/z 296.00-205.10 were used to measure MPS, AM and HCTZ (IS), respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of MPS and AM formulation product after an oral administration to Indian healthy human volunteers.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Therapeutic isoniazid monitoring using a simple high-performance liquid chromatographic method with ultraviolet detection

Simultaneous measurement of isoniazid and its main acetylated metabolite acetylisoniazid in human plasma is realized by high-performance liquid chromatography. The technique used is evaluated by a factorial design of validation that proved to be convenient for routine drug monitoring. Plasma samples are deproteinized by trichloroacetic acid and then the analytes are separated on a microBondapak C18 column (Waters). Nicotinamide is used as an internal standard. The mobile phase is 0.05 M ammonium acetate buffer (pH 6)-acetonitrile (99:1, v/v). The detection is by ultraviolet absorbance at 275 nm. The validation, using the factorial design allows one to: (a) test the systematic factors of bias (linearity and matrix effect); (b) estimate the relative standard deviations (RSDs) related to extraction, measure and sessions assay. The linearity is confirmed to be within a range of 0.5 to 8 microg/ml of isoniazid and 1 to 16 microg/ml of acetylisoniazid. This method shows a good repeatability for both extraction and measurement (RSD INH=3.54% and 3.32%; RSD Ac.INH=0.00% and 5.97%), as well as a good intermediate precision (RSD INH=7.96%; RSD Ac.INH=15.86%). The method is also selective in cases of polytherapy as many drugs are associated (rifampicin, ethambutol, pyrazinamide, streptomycin). The matrix effect (plasma vs. water) is negligible for INH (3%), but statistically significant for Ac.INH (11%). The application of this validation design gave us the possibility to set up an easy and suitable method for INH therapeutic monitoring.     

Sensitive liquid chromatographic technique to measure isoniazid in alveolar cells, bronchoalveolar lavage and plasma in HIV-infected patients

The need to monitor the effectiveness of antimicrobial drugs in treating opportunistic infections such as tuberculosis in HIV-infected patients requires the development of sensitive assays. A suitable HPLC method was developed to measure the concentration of isoniazid (INH) in plasma 1 h after a standard 300 mg dose and to detect the low levels typically found in alveolar cells obtained by bronchoalveolar lavage of subjects maintained on a standard regimen of the drug. Following extraction with a chloroform–butanol mixture, the INH was back-extracted into dilute acid which was subsequently analyzed by HPLC using a CN reversed-phase column and an acetonitrile–isopropanol based mobile phase. Another HPLC method was developed using direct injection and a polymer based column to measure minute concentrations of INH in the cell-free lavage. In both systems, detection of the drug was accomplished with a sealed coulometric detector (+0.6 V) capable of giving a consistent daily response without adjustment. When saline, cellular extracts and plasma from untreated subjects were spiked with various amounts of INH and analyzed, the lowest level of quantitation was 10, 25 and 100 ng/ml, respectively. Calibration curves showed good linearity when spiked concentrations were compared to peak areas (r=0.991, 0.993 and 0.998, respectively). Alveolar cell extracts and cell-free bronchoalveolar fluid from HIV-positive patients maintained on a standard INH regimen had detectable levels of INH 4 h after a 300 mg oral dose. The plasma INH at 1 h had a range of 0.3–7.1 μg/ml (n=50). Precision studies with plasma spiked at 0.1, 0.5, 1.0 and 5.0 μg/ml revealed within-run coefficients of variation (C.V.s) of 8.9, 7.2, 4.2 and 4.9%, respectively and analytical recoveries of 97, 108, 108 and 98%, respectively. The day-to-day C.V.s for the plasma method were 7.6, 4.9 and 3.8% at concentrations of 0.5, 1.0 and 3.0 μg/ml, respectively. The results suggest that this rugged HPLC technique can quantitate INH in 1 h plasma with good precision and can be used to estimate the very low INH concentrations found in alveolar cells and cell-free lavage recovered from patients undergoing anti-tuberculosis therapy.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine

Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography-electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5-1000 ng/ml; r > or = 0.9968), intra- and inter-batch precision and accuracy (< or =14.5%), and recoveries (94-106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.     

Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography–tandem mass spectrometry

A method was developed and validated for the quantification of (±)-trans-[2-morpholino-1-(1-naphthaleneethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5–3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of the lipophilic antipsychotic drug chlorpromazine in rat plasma and brain tissue

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for quantification of chlorpromazine in rat plasma and brain tissue. Chlorpromazine was extracted from rat plasma and brain homogenate using liquid-liquid extraction. 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/20 mM ammonium formate (pH 4.25 adjusted with formic acid) with gradient elution. Chlorpromazine was 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. 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 from 0.833 to 833.3 ng/g for brain tissue. The correlation coefficient (R(2)) values were more than 0.998 for both plasma and brain homogenate. The precision and accuracy for intra-day and inter-day were better than 7.54%. The relative and absolute recovery was above 84.9% and matrix effects were lower than 5.6%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of chlorpromazine after chronic treatment.     

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.     

Macrophage-Specific Targeting of Isoniazid Through Mannosylated Gelatin Microspheres

Active targeting of drug molecules can be achieved by effective attachment of suitable ligands to the surface of carriers. The present work was attempted to prepare mannosylated gelatin microspheres (m-GMs) so as to achieve targeted delivery of isoniazid (INH) to alveolar macrophages (AMs) and maintain its therapeutic concentration for prolonged period of time. Microspheres were prepared by emulsification solvent extraction method and evaluated for physicochemical characteristics, drug release, ex vivo drug uptake by AMs and pharmacokinetic characteristics. Fourier transform infrared spectroscopy and nuclear magnetic resonance spectral analysis confirmed that mannosylation took place through Schiff base formation between aldehyde and amino groups of mannose and gelatin, respectively. Prepared microspheres offered suitable physicochemical characteristics for their delivery to AMs. Their average size was about 4 μm and drug entrapment efficiency of 56% was achieved with them. Ex vivo uptake results indicated that in comparison to plain microspheres, m-GMs were selectively uptaken and were found to be associated with phago-lysosomal vesicles of AMs. Pharmacokinetic studies showed the formulation could maintain the therapeutic concentration of INH for prolonged period of time even with a reduced clinical dose. m-GMs were found to be stable in broncheo-alveolar lavage fluid. The study concluded that ligand decorated carriers could be a potential strategy to improve the therapeutic properties of INH.     

High-performance liquid chromatographic analysis of the antituberculosis drugs aconiazide and isoniazid

Reversed-phase HPLC methods are described for determining the stability and concentration certification of the antituberculosis prodrug aconiazide (ACON) in aqueous dosing solution and for assessing the concentrations of ACON and isoniazid (INH) in plasma from ACON-treated male and female Fischer-344 rats. ACON was analyzed in plasma by direct injection; it was separated on a 250 × 4.6 mm I.D. 5 μm C₁₈ column using a 40% aqueous methanol mobile phase containing 5 g/l ammonium formate, and detected at 313 nm. INH was determined in the plasma of treated rats after a two-step precipitation of plasma proteins; it was separated on a 250 mm × 4.6 mm I.D. 5 μm CN column, eluted with 5% aqueous isopropanol containing 5 g/l ammonium formate, and detected with an electrochemical detector at +0.8 V. These methods allow a simple, rapid, and reliable determination of ACON and INH in plasma down to 0.1 μg/ml.     

Determination of endocannabinoid receptor antagonist SR141716 (rimonabant) in plasma by liquid chromatograph tandem mass spectrometry

SR141716 (rimonabant) is an endocannabinoid receptor antagonist. Endocannabinoids are a class of chemicals that affect neurotransmission via G-protein coupled CB1 (brain) and CB2 (peripheral tissue) receptors. Numerous animal studies have shown that SR141716 binds with the CB1 receptor in the brain, resulting in several biological consequences including reduced alcohol intake and reward as well as reduced food consumption. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the quantitative measurement of SR141716 in both human and rat plasma to support the investigation of this compound. A suitable internal standard (AM251) has been chosen and the experimental conditions have been optimized for the separation and detection of singly charged positive ions of SR141716 and the internal standard. A protein precipitation protocol has been developed for extraction of SR141716 and the internal standard from plasma samples. Quantitation was achieved using multiple-reaction-monitoring (MRM) mode for SR141716 (m/z 463-->m/z 363) and the internal standard (m/z 555-->m/z 455) and calibration curve over the concentration range of 5.00-1000 ng/ml was plotted using the peak-area ratio versus the concentration of SR141716 with a LOD and LLOQ of 1.09 and 3.62 ng/ml, respectively. The method developed has been used to analyze SR141716 in rat plasma samples from an animal study.     

Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5-100 ng/ml with correlation coefficient, R(2) > or = 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.     

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.     

The oral gold compound auranofin triggers arachidonate release and cyclooxygenase metabolism in the alveolar macrophage

We examined the effect of in vitro incubation with the oral gold compound auranofin (AF) on arachidonic acid (AA) release and metabolism by rat alveolar macrophages (AMs). AF stimulated dose- and time-dependent release of 14C-AA from prelabeled AMs, which reached 4.7 +/- 0.3% (mean +/- SEM) of incorporated radioactivity at 10 micrograms/ml for 90 min, as compared to 0.5 +/- 0.1% release following control incubation for 90 min (p less than 0.001). Similar dose- and time-dependent synthesis of thromboxane (Tx) A2 (measured as TxB2) and prostaglandin (PG) E2 was demonstrated by radioimmunoassay of medium from unlabeled cultures, reaching 18-fold and 9-fold, respectively, of the control values at 10 micrograms/ml AF for 90 min (p less than 0.001 for both). AF-induced TxB2 and PGE2 synthesis was inhibited by indomethacin as well as by pretreatment with methylprednisolone. No increase in the synthesis of immunoreactive leukotrienes (LT) B4 or C4 was noted at any dose or time of AF. High performance liquid chromatographic separation of 14C-eicosanoids synthesized by prelabeled AMs confirmed that AF induced the release of free AA and its metabolism to cyclooxygenase, but not 5-lipoxygenase, metabolites. The ability of AF to trigger macrophage AA metabolism may be relevant to the exacerbation of certain inflammatory processes which sometimes accompany gold therapy.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile-methanol-20 mM sodium sulfate-acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0-102.4% and 99.0-108.7%) with calibration curve ranges 10.0-2000 ng/ml and 30.0-6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.