Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

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

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

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether–n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

CYP3A is responsible for N-dealkylation of haloperidol and bromperidol and oxidation of their reduced forms by human liver microsomes

We studied the biotransformation of haloperidol, bromperidol and their reduced forms by human liver microsomes. Nifedipine oxidation (CYP3A) activity correlated significantly with N-dealkylation rates of haloperidol and bromperidol and oxidation rates of their reduced forms, while neither ethoxyresorufin O-deethylation (CYP1A2) activity nor dextromethorphan O-deethylation (CYP2D6) activity did. In chemical and immunoinhibition studies, only troleandomycin and anti-CYP3A4 serum inhibited both formation rates of 4-fluorobenzoylpropionic acid, a metabolite of haloperidol and bromperidol, and back oxidation rates. Among 10 recombinant isoforms examined, only CYP3A4 showed catalytic activity. The Vmax and Km values of N-dealkylation of bromperidol and reoxidation of reduced bromperidol were similar to those of haloperidol and reduced haloperidol, respectively. The present study indicates that CYP3A plays a major role in N-dealkylation of and oxidation back to bromperidol as well as haloperidol and suggests that modification of in vivo CYP3A activity by inhibition or induction may affect the pharmacokinetics and therapeutic effects of haloperidol and bromperidol.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

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

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

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.     

High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP⁺). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.     

Simultaneous determination of three local anesthetic drugs from the pipecoloxylidide group in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C(18) reversed-phase column (150 x 4.6 mm I.D.) filled with 5-microm particles and attached to a UV detector. The mobile phase was composed of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88-91% for mepivacaine, 87-89% for ropivacaine and 88-91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Determination of betamethasone and betamethasone 17-monopropionate in human plasma by liquid chromatography-positive/negative electrospray ionization tandem mass spectrometry

This study presents a high-performance liquid chromatography-positive/negative electrospray ionization tandem mass spectrometric (LC-ESI(+/-)-MS-MS) method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma using beclomethasone dipropionate as the internal standard (I.S.). Both compounds were extracted from human plasma with ether-cyclohexane (4:1, v/v) and were separated by HPLC on a Hanbon Lichrospher C(18) column with a mobile phase of methanol-water (85:15, v/v) at a flow rate of 0.7ml/min. Calibration curves were linear over the range of 0.10-50ng/ml for BOH and 0.050-50ng/ml for B17P. The inter-run relative standard deviations were less than 14.4% for BOH and 12.3% for B17P. The intra-run relative standard deviations were less than 9.3% for BOH and 7.9% for B17P. The mean plasma extraction recovery for BOH and B17P were in the ranges of 82.7-85.9% and 83.6-85.3%, respectively. The method was successfully applied to study the pharmacokinetics of a new formulation of betamethasone phosphate/betamethasone dipropionate injection in healthy Chinese volunteers.     

Simultaneous analysis of cytochrome P450 probes-dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A "cocktail" approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4'-hydroxy-flurbiprofen and 1'-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify cartridges) extraction procedure from 200 microl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm Lx3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4'-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1'-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: -9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.     

Sensitive determination of omeprazole and its two main metabolites in human plasma by column-switching high-performance liquid chromatography: application to pharmacokinetic study in relation to CYP2C19 genotypes

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether-dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 microm, 150 mm x 4.6mm i.d.) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer-acetonitrile-methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was approximately 25 min. The validated concentration ranges of this method were 3-2000 ng/ml for omeprazole, 3-50 ng/ml for 5-hydroxyomeprazole and 3-1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.     

An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug combination

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile-0.05 M acetic acid adjusted to pH 5.2 with 1.0M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol-acetonitrile-0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30-750 ng/0.5 ml plasma and MQ over a concentration of 75-1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0-1.4% for AS, 0.4-3.4% for DHA and 0.7-1.5% for MQ. The day-to-day coefficients of variation were 1.3-7.6%, 1.8-7.8% and 2.0-3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.     

Human plasma quantification of droperidol and ondansetron used in preventing postoperative nausea and vomiting with a LC/ESI/MS/MS method

An analytical method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the simultaneous identification and quantification of droperidol and ondansetron in human plasma. The two drugs were isolated from 0.5 mL of plasma using a basic liquid-liquid extraction with diethyl ether/heptane (90/10, v/v) and tropisetron and haloperidol as internal standards, with satisfactory extraction recoveries. They were separated on a 5-μm C(18) Highpurity column (150 mm×2.1 mm I.D.) maintained at 30°C. The elution was achieved isocratically with a mobile phase of 2 mM HCOONH(4) pH 3.8 buffer/acetonitrile (60/40, v/v) at a flow rate of 200 μL/min. Data were collected either in full-scan MS mode at m/z 100-450 or in full-scan MS-MS mode, selecting the [M+H] (+) ion at m/z=294.0 for ondansetron, m/z=285.2 for tropisetron, m/z=380.0 for droperidol and m/z=376.0 for haloperidol. The most intense daughter ion of ondansetron (m/z=212.0) and droperidol (m/z=194.0) were used for quantification. Retention times for tropisetron, ondansetron, droperidol and haloperidol were 2.50, 2.61, 3.10 and 4.68 min, respectively. Calibration curves were linear for both compounds in the 0.50-500 ng/mL range. The limits of detection and quantification were 0.10 ng/mL and 0.50 ng/mL, respectively. The intra- and inter-assay precisions were lower than 6.4% and intra- and inter-assay recoveries were in the 97.6-101.9% range for the three 3, 30 and 300 ng/mL concentrations. This method allows simultaneous and rapid measurement of droperidol and ondansetron, which are frequently co-administrated for the prevention of postoperative nausea and vomiting.     

High-performance liquid chromatographic method with diode array detection to identify and quantify atypical antipsychotics and haloperidol in plasma after overdose

For toxicological purposes, an HPLC assay was developed for the simultaneous determination of haloperidol and atypical antipsychotics (risperidone, 9-hydroxyrisperidone, olanzapine, clozapine) in human plasma. After a double-step liquid-liquid extraction, compounds were separated on a C(8) column eluted with a gradient of acetonitrile and phosphate buffer 50 mM pH 3.8. A sequential ultraviolet detection was used (260, 280 and 240 nm). Calibration curves were linear in the range 10-1000 ng/ml. The limits of quantification were 5 ng/ml for all drugs. Average accuracy at four concentrations ranged from 93 to 109%. Both inter- and intra-day variation coefficients were lower than 11% for all drugs. This simple and rapid method (run time<15 min) is currently used for poison management.     

Determination of a novel cognitive enhancer,X9121, and its mono N-oxide metabolite,XG696, in dog plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A selective and sensitive high-performance liquid chromatographic assay for a novel cognitive enhancer, X9121 (I), and its mono N-oxide metabolite, XG696 (II), in dog plasma has been developed. Compounds I, II and internal standard (I.S.) were first extracted from dog plasma using a solid-phase Bond Elut Certify I 10-ml LRC reservoir extraction cartridge. Chromatographic separation of I, II and I.S. was conducted on a reversed-phase Zorbax Stable Bond cyano column. Ammonium acetate buffer (0.05 M, pH 6)-acetonitrile-triethylamine (75:25:0.1, v/v) was used as the mobile phase. Detection of all three compounds was by UV light absorbance at 313 nm. Using 0.5 ml of dog plasma for extraction, the minimum quantifiable limit was 10 ng/ml and the assay was linear from 10 to 5400 ng/ml. The coefficients of variation for intra-day precision ranged from 2.2 to 8.5% for I and from 2.5 to 9.8% for II. The coefficients of variation for the inter-day precision for these two compounds ranged from 2.6 to 9.0% and from 3.6 to 16.2%, respectively. The absolute percent differences for the accuracy results were within 11.0% of the spiked concentrations. Compounds I and II were stable in frozen plasma at −20°C for at least 67 days.