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The goal of this investigation was to establish a reliable technique for the quantitation of ponazuril in limited sample volumes. Samples were extracted with chloroform and separation was achieved with a Symmetry RP 18 column. Ultraviolet absorption was measured at 254 nm. A combination of 0.1% formic acid and acetonitrile (50:50) was used as the mobile phase. The calibration curve was linear from 0.1-25 μg/mL, with a lower limit of quantification of 0.1 μg/mL with a 100 μL sample. The precision and accuracy were well within the range set by the Food and Drug Administration and the recovery was over 95%. This technique was used to analyze ponazuril samples and found to be appropriate for pharmacokinetic studies., (© 2021 John Wiley & Sons, Ltd.)

A highly sensitive, specific and simple LC-MS/MS method for quantification of capmatinib (INC280) in rat plasma was presented. The LC-MS/MS method was validated in terms of specificity and selectivity, linearity, accuracy and precision, matrix effect, extraction recovery, dilution integrity, carryover and stability as per the US Food and Drug Administration's bioanalytical method validation guideline. The validated assay was applied for quantification of capmatinib from a pharmacokinetic study in rats following oral administration at the doses of 1.0, 3.0 and 9.0 mg/kg. The calibration curve ranges from 1 to 2000 ng/ml with desirable linearity and r 2  > 0.99. The intra- and inter-batch accuracies were within 99.24-103.59 and 97.76-102.83% with coefficients of variation 5.08-7.36 and 3.18-4.99%, respectively. No significant interference was observed by endogenous peak at the retention time of capmatinib and IS. The assay was free from any matrix effect and showed precise recovery across the calibration curve range, and samples were stable under all experimental conditions. The validated assay was successfully applied to analyze plasma samples of pharmacokinetic study in rat to determine the concentration of capmatinib. In summary, a novel method for analyzing capmatinib in rat plasma has been successfully validated and is now being utilized for quantification of capmatinib from pre-clinical studies., (© 2019 John Wiley & Sons, Ltd.)

Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 μl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow-gradient) and an X-Terra Phenyl column. The UV detection wave length was set at λ max 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20-12.5 μg/ml for EDB and 0.50-12.5 μg/ml for IDB and VDB (r 2  = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice., (© 2019 John Wiley & Sons, Ltd.)

Ethanamizuril is a new triazine compound that has the potential to be a novel anticoccidial drug. Toxicological studies in experimental rats were performed to understand the safety profile of ethanamizuril for drug product development. In this study, a novel, selective and accurate ultra-performance liquid chromatography tandem mass spectrometry method has been developed for the determination of ethanamizuril concentrations in rat plasma. With 4-nitro-o-cresol as an internal standard, sample pretreatment involved a one-step extraction with acetonitrile of 100 μL plasma. The detection was carried out by electrospray ionization mass spectrometry in negative ion mode with selected ion recording. The standard curves were linear (r 2  ≥ 0.999) over the concentration range of 0.1-100 μg/mL. The relative standard deviations of intra- and inter-day precisions were less than 8.4 and 8.87%, respectively. The mean extraction recovery of ethanamizuril from rat plasma was 97.68-102.57%. The method was fully validated and successfully applied to monitor plasma concentrations of ethanamizuril in a short-term toxicity study and two-generation reproduction toxicity study. The result of the study confirmed that the elimination of ethanamizuril in rats is slow., (© 2019 John Wiley & Sons, Ltd.)

Aim: Fotagliptin is a novel dipeptidyl peptidase IV inhibitor under clinical development for the treatment of Type II diabetes mellitus. The objective of this study was to develop and validate a specific and sensitive ultra-performance liquid chromatography (UPLC)-MS/MS method for simultaneous determination of fotagliptin and its two major metabolites in human plasma and urine. Methodology & results: After being pretreated using an automatized procedure, the plasma and urine samples were separated and detected using a UPLC-ESI-MS/MS method, which was validated following the international guidelines., Conclusion: A selective and sensitive UPLC-MS/MS method was first developed and validated for quantifying fotagliptin and its metabolite in human plasma and urine. The method was successfully applied to support the clinical study of fotagliptin in Chinese healthy subjects.

Aim: Recently, polytherapy regimen has been introduced for the treatment of epileptic patients for better seizure control with lesser side effects and better control of multiple seizure types., Methodology: A simple, sensitive and highly specific reversed-phase HPLC method was developed for simultaneous determination of four antiepileptic drugs (AEDs), levetiracetam, lamotrigine, oxcarbazepine and carbamazepine, in real human plasma without interference from endogenous components of plasma., Conclusion: The method was proved to be linear in the range of 0.5-50 µg/ml for all drugs. It was successfully applied for clinical PK study of the AEDs in healthy volunteers following single administration. Also, this method was applied for simultaneous determination of the studied drugs in volunteers' plasma receiving synergistic binary combinations from the four AEDs when used as add-on therapy. The good precision and selectivity of the developed method allow it to be used for routine therapeutic drug monitoring of such drugs as a useful tool in epilepsy management.

Background: Recently, a turbidimetric immunoassay method has been developed for use in the form of a QMS lamotrigine (LTG) commercial immunoassay. This study was designed to evaluate the performance of this immunoassay using a validated high-performance liquid chromatography-ultraviolet (HPLC-UV) method as the reference., Methods: The performance of QMS was initially tested using drug-free plasma spiked with different amounts of LTG and, subsequently, by analyzing 61 trough plasma samples from epileptic patients given the drug as part of their maintenance antiepileptic therapies., Results: The correlation between LTG concentrations measured by QMS and HPLC was good, with a Pearson coefficient of 0.968 (P < 0.0001). The Bland-Altman approach showed that LTG concentrations measured with QMS exceeded HPLC on an average by 15.6% (limits of agreement, -18% to +63%), with a concentration-dependent performance (mean percent bias, 49.5 ± 8.2% and 0.6 ± 12.7% for concentrations less than 2 mg/L and greater than 14.9 mg/L, respectively)., Conclusions: The QMS provided acceptable analytical performance across a wide concentration range for routine LTG measurements, being at least comparable with the other commercial immunoassays. It could be, therefore, considered as a viable alternative to HPLC methods for routine LTG monitoring in the clinical practice, although its suitability for accurate analysis of samples with low concentration is limited.

To establish the HPLC method to examine plasma concentration of lamotrigine and oxcarbazepine. This study set chlorzoxazone as the internal standard, chromatographic column was Column C18 (200×4.6mm, 5um) of DIKMA company, the mobile phase was methanol, water and trifluoroacetic acid, with rate of 40: 60: 0.0005, at a flow rate of 1 mllmin(-1), the detected wavelength was 240 nm. The plasma concentrations of lamotrigine was 0.5-50ug•mL(-1), the standard curve was excellent for Y=0.5511C-0.5669, r=0.9940, average recovery was 91.40%; The plasma concentrations of oxcarbazepine was 0.5-50ugmL-1, the standard curve was good for Y=0.4026C-0.5895, r=0.9925, and the average recovery was 89.59%; The three plasma concentrations of lamotrigine were respectively 25μg•mL(-1), 10 μg•mL(-1) and 2μg•mL(-1) and its five parallel sample for injection RSD were respectively 4.01%, 6.15% and 4.64%; The three plasma concentration of oxcarbazepine were 25μg•mL(-1)-1(-1), 10μg•mL(-1)-1(-1) and 2μg•mL(-1)-1(-1), and its five parallel sample for injection RSD were respectively 3.05%, 4.27% and 9.01%. This method was easy to operate, high recovery and high precision, and was applicable to the clinical detection for plasma concentration of lamotrigine and oxcarbazepine.

A novel method which involved dispersive liquid-liquid microextraction (DLLME)-back extraction based on ionic liquid (IL) was developed for the determination of three phosphodiesterase-5 (PDE-5) inhibitors, sildenafil (SD), vardenafil (VD) and aildenafil (AD), in human plasma. DLLME based on IL as the extractant solvent and methanol as the dispersive solvent was the first step to extract PDE-5 inhibitors from sample solution; the other step of back extraction was followed by transferring target analytes from the IL to acidified aqueous solution. This two-step extraction ensured the compatibility of the final extractant phase, acidified aqueous solution herein, with the reversed phase high performance liquid chromatography-UV detection, and afforded clean extractant phase. The optimal extraction condition was obtained after systematical optimization. The sample solution (960μL) was extracted by 20μL of 1-octyl-3-methylimidazolium hexafluorophosphate in the presence of 20μL methanol and 300mgmL(-1) NaCl with the assistance of vortex; IL phase enriched with the target analytes was then extracted by 10% acetic acid aqueous solution. Good linearity ranges (SD 1-500ngmL(-1), VD 2-2000ngmL(-1) and AD 2-2000ngmL(-1)) with suitable r(2) (=0.9999) were achieved. Limits of detection (LODs) in pure water were 0.15ngmL(-1), 0.30ngmL(-1) and 0.43ngmL(-1) for VD, SD and AD, respectively. Intra-day and inter-day relative standard deviations were below 6.38%. Finally, this method was applied for the determination of PDE-5 inhibitors in human plasma with satisfactory LODs of 0.92ngmL(-1), 1.19ngmL(-1) and 2.69ngmL(-1) for VD, SD and AD, respectively. Acceptable absolute recoveries were obtained from 100.4% to 103.9%. The developed method afforded a convenient, fast and cost-saving operation with high extraction efficiency for the test analytes. It has potential to be applicable to biological samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A sensitive and fast high-performance liquid chromatographic method coupled with ultraviolet detection is herein reported for the simultaneous determination of human plasma concentration of six antiepileptic drugs frequently used in clinical practice [phenobarbital (PB), primidone (PRM), phenytoin (PHT), carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC)] and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (trans-diol) and licarbazepine (Lic). Sample preparation consisted of a deproteinization step with methanol followed by a solid-phase extraction procedure. Chromatographic separation was achieved in approximately 15 min on a reversed-phase C18 column using a mobile phase composed by water-methanol-acetonitrile-triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at 1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear with regression coefficients greater than 0.992 over the concentration ranges 0.25-100 μg/mL for PB, 0.4-50 μg/mL for PRM, 0.5-50 μg/mL for PHT, 0.1-50 μg/mL for CBZ, LTG and CBZ-E, 0.1-25 μg/mL for OXC, 0.25-10 μg/mL for trans-diol and 0.15-80 μg/mL for Lic. Inter- and intra-day imprecision did not exceed 12.15% and inaccuracy was within ±14.91%. Absolute mean recoveries ranged from 78.49 to 101.04% and no interferences were observed at the retention times of the analytes and internal standard (ketoprofen). This bioanalytical method was successfully applied to real plasma samples from epileptic patients and it seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A simple, sensitive and reproducible isocratic reversed-phase (C(18) ) high-performance liquid chromatography (HPLC) method was developed to determine 7-O-succinyl macrolactin A (SMA) in rat plasma and urine samples using UV detector set at 230 nm. Lamotrigine was used as internal standards (IS) to ensure the precision and accuracy of the method. The retention times of SMA and IS for the plasma sample were 9.2 and 4.4 min, respectively, and those for the urine samples were 7.9 and 4.3 min, respectively. The intra- and inter-day variations of the analytical responses, expressed in terms of relative standard deviation, were less than 14.9%. The accuracy, in terms of average analytical recovery, ranged from 90.4 to 119%. The lower limits of quantification of SMA in rat plasma and urine samples were 0.02 and 0.1 µg/mL, respectively. This method is applicable for the pharmacokinetic studies of SMA in rats., (Copyright © 2012 John Wiley & Sons, Ltd.)

Lamotrigine, an anti-epileptic drug with a phenyltriazine molecular structure, is commonly measured for therapeutic drug monitoring purposes by high performance liquid chromatography (HPLC) or gas liquid chromatography (GLC). A convenient internal standard is the structurally related phenyltriazine compound BWA725C previously obtainable from the Wellcome Foundation, UK. Irsogladine is also structurally similar to lamotrigine and was therefore tested as a possible replacement for BWA725C. A GLC procedure with thermionic detection (NPD) has been utilized routinely for lamotrigine in our drug monitoring facility. Irsogladine was unsuitable, however, because the retention times of irsogladine and a co-prescribed drug, carbamazepine, were very similar. An HPLC method utilizing a Prodigy Phenomenex ODS3 column performed well using either of the internal standards. The pH of the mobile phase had a distinct impact on the spectra of lamotrigine and BWA725C. A mobile phase at pH 3, with detection at 225 nm was required to effectively resolve lamotrigine from sulthiame and irsogladine from phenobarbitone. Comparison of the HPLC and the existing GLC method with routine patient specimens (n = 43) gave an equation, y = 0.9382x + 0.8238, R(2) = 0.9862. Irsogladine was found to be a suitable internal standard for an HPLC analysis of lamotrigine., (Copyright © 2011 John Wiley & Sons, Ltd.)

A simple, fast and sensitive HPLC method with electrochemical detection employing boron-doped diamond electrode (BDD) for the determination of sildenafil (Viagra™), vardenafil (Levitra™) and their main metabolites, N-desmethyl sildenafil and N-desethyl vardenafil in human plasma is presented. The assay involved drug extraction by tert-butyl methyl ether and isocratic reversed-phase liquid chromatography with amperometric detection. Complete separation of all analytes was achieved within 12 min. The mobile phase consisted of 20mM sodium dihydrogen phosphate with 40 mM sodium perchlorate/acetonitrile (70:30, v/v), pH 3.5. The electrode working potential was +1520 mV (vs. Pd/H(2)). Calibration curves were linear over the concentration range of 10-400 ng mL(-1). Phloretin was used as an internal standard. The limits of detection (LOD) and quantification (LOQ) for the studied analytes were within the range of 2-4 ng mL(-1) and 7.0-13.4 ng mL(-1), respectively. The developed method was applied to human plasma samples spiked with analytes at therapeutic concentrations. The study confirms the method's suitability for both pharmacokinetic studies and therapeutic monitoring., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study characterized the concentration-time profile of melamine in the heart, liver, spleen, lungs, kidneys, bladder, feces, urine, and plasma after melamine (MM) administration. Female Sprague-Dawley rats received a single oral dose of 1.0 g/kg body weight. Samples (n=4 per time point) were collected at 12, 24, 48, 72, 96, 120, 144, and 168 h. Based on calculations of the area under the concentration-time curves after dosing, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was used to detect MM concentration in tissues. Peak concentrations of MM are reached in the livers and lungs at 12 h after dosing and in hearts, spleens, kidneys, bladders, feces, urine, and plasma at 24 h after dosing. More than 90% of the ingested MM is excreted in feces and urine within 24 h. These results provided initial understanding of the tissue disposition of MM. Moreover, this study demonstrates that UPLC-MS-MS can be used to detect MM in biological samples.

Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average., (Copyright © 2010 John Wiley & Sons, Ltd.)

This paper reports the development and validation of a methodology for the low-level quantification of melamine and cyanuric acid in limited samples of rat serum. The methodology, based upon ion-exchange solid phase extraction (SPE) and ultra-performance liquid chromatography (UPLC) coupled with electrospray tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode, relies on the use of stable isotope-labeled internal standards and requires only 15 μL samples of serum. The method provides a recovery of 80-110% of melamine with a signal suppression of ca. 55%, and a recovery of 50-90% of cyanuric acid with a signal suppression ca. 40-60%, affording lower limits of quantification (LLOQ) for melamine or cyanuric acid of, respectively, 5 ppb (mean accuracy 109%; CV=4.9%) and 10 ppb (mean accuracy 96%; CV=8.6%). The small sample requirements, excellent sensitivity, accuracy and precision, and high-throughput (5 min of instrument run time) make this methodology optimal for toxicokinetic or exposure assessments studies., (Published by Elsevier B.V.)

A sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) assay has been developed for the quantitative analysis of vardenafil in human plasma. Vardenafil and the internal standard, alprazolam, were extracted from 0.2 mL aliquots of alkalinized plasma by a single solvent extraction into hexane : dichloromethane. Reversed-phase chromatographic separation was affected by gradient elution with mobile phases consisting of 10 mM ammonium formate pH 7.0 (solvent A) and methanol (100%, solvent B), delivered at a flow rate of 0.4 mL/min. The analytes were detected by using an electrospray ion source on a 4000 QTrap triple quadrupole mass spectrometer operating in positive ionization mode. The mass transitions were m/z 489.3 --> 312.2 for vardenafil and m/z 309.2 --> 281.0 for alprazolam. The assay was linear over the concentration range of 0.2-100 ng/mL, with correlation coefficients > or = 0.995. The intra- and inter-day precision was less than 5.4% in terms of relative standard deviation and the accuracy was within 12.7% in terms of relative error. The lower limit of quantitation was set at 0.2 ng/mL. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of plasma samples obtained following the oral administration of vardenafil to healthy male volunteers in a pharmacokinetic study.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of lamotrigine in human plasma using multiplexing technique (two HPLC units connected to one MS-MS). Lamotrigine was extracted from human plasma by solid-phase extraction technique using Oasis Hydrophilic Lipophilic Balance (HLB) or N-vinylpyrrolidone and divinylbenzene cartridge. A structural analog, 3,5-diamino-6-phenyl-1,2,4-triazine, was used as an internal standard (IS). A BetaBasic C(8) column was used for the chromatographic separation of analytes. The mass transition [M+H](+) ions used for detection were m/z 256.0 --> 211.0 for lamotrigine and m/z 188.0 --> 143.0 for IS. The method involved a simple multiplexing, rapid solid-phase extraction without evaporation and reconstitution. The proposed method has been validated for a linear range of 0.025 to 10.000 microg/mL with a correlation coefficient > or = 0.9991. The limit of quantification for lamotrigine was 0.025 microg/mL, and limit of detection was 50.000 pg/mL. The intra-run and inter-run precision and accuracy were within 10.0% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for lamotrigine and IS were 97.9% and 92.5%, respectively. Total MS run time was 1.4 min per sample. The validated method has been successfully used to analyze human plasma samples for applications in pharmacokinetic, bioavailability, bioequivalence, or in vitro in vivo correlation studies.

Objective: The purpose of this study was to optimise the HPLC-UV bio-analytical method currently used by the Salamanca University Clinical Hospital for determining lamotrigine plasma levels., Material and Methods: The developed HPLC-UV analytic technique currently in use was shown to be linear, exact and precise, and suitable for use in routine monitoring of lamotrigine levels. The drawback of this method has always been the time required for analysing samples, so our aim was to improve on that elapsed time. That improvement involved using a different chromatographic column from the one used up until now. We replaced the column that was normally used (Kromasil-100C18-5 microm-15*0.4 cm with a LiChroCART-RP18e-3 microm-5.5*0.4 cm); in both cases, a liquid-liquid extraction was performed and the same sample extraction protocol was followed., Results: Both validation methods showed that the two column types are valid for routine lamotrigine monitoring., Conclusion: The decrease in retention time, in addition to a lower quantification limit and better precision and accuracy parameters obtained with the LiChorCART column, suggest that this unit is ideal for use in clinical practice because it enables a large number of determinations to be performed in less time and the greater precision of LTG measurements., (Copyright © 2009 SEFH. Published by Elsevier Espana. All rights reserved.)

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 microL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 microm Hydro-RP, 250 mm x 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2-40 microg/mL for RFN and 2-80 microg/mL for ZNS. The limit of quantitation was 2 microg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM)., (2009 Elsevier B.V. All rights reserved.)

In this study, we describe a method for the analysis of melamine in rat plasma, liver, kidney, spleen, bladder, and brain using trichloroacetic acid precipitation with mixed-mode cation-exchange solid-phase extraction and hydrophilic interaction chromatography coupled to tandem mass spectrometry detection. Method validation was investigated completely, including linearity, precision, accuracy, matrix effect, extraction recovery, and carryover for the determination of melamine. The method exhibited a good linear range covering 20-500 ng/mL, and the overall precision ranged from 1.6 to 16.3%, with the accuracy varying from -7.9 to 15.1%. The mean matrix effects of melamine in rat plasma, liver, kidney, spleen, bladder, and brain ranged from 66.2+/-6.7 to 95.5+/-13.2%, and the mean recoveries for melamine varied from 79.8+/-8.2 to 113.0+/-9.6%. Rat kidney showed the highest level among the organs (192.5% of the plasma melamine level), and the average concentration of melamine in the brain was only 7.5% of the plasma melamine concentration. This work has pointed out that even with the application of two popular preparation procedures (acid precipitation and solid-phase extraction) of melamine, the matrix effect in analyzing biological samples still exists in certain kinds of matrices.

This study describes a simple and sensitive column-switching high-performance liquid chromatographic method with UV detection for the determination of Lamotrigine in 50 microL of serum. After solid-phase extraction of Lamotrigine on an Oasis HLB extraction precolumn (20 x 3.9 mm; dp: 25 microm), chromatographic separation was achieved at 30 degrees C on a Chromolith RP-18e column (50 mm x 4.6 mm i.d.) using a solution of 20% acetonitrile in 15 mM phosphate buffer (pH 7.0) as the mobile phase, at a flow-rate of 2.0 mL/min. The eluant was detected at 215 nm. The retention time for Lamotrigine was 1.28 min. The total analysis time was ca. 5 min. However, the overlap of sample preparation, analysis, and reconditioning of the precolumn increased the overall sample throughput to one injection every 3 min. The method was validated for system suitability, linearity, precision, accuracy, robustness, and limit of quantitation. The linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.9996. Recovery studies achieved from Lamotrigine spiked plasma samples showed values greater than 93%, demonstrating the excellent extraction efficiency of the precolumn. Intra- and inter-day precision were generally acceptable; the coefficient of variation was < 2.3% in all cases. The detection limits for Lamotrigine at a signal-to-noise ratio of 3 was 0.002 microg/mL when a sample volume of 50 microL was injected. However, it was possible to enhance the sensitivity further by injecting larger volumes, up to 200 microL. The method was shown to be robust and the results were within the acceptable range. The method was successfully applied to the determination of Lamotrigine in human serum samples of patients submitted to Lamotrigine therapy.

A simple high-performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 x 4.6 mm i.d. reversed-phase Kromasil KR 100 C(18) (5 microm particle size) column using a mobile phase of acetonitrile-potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10-1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies., ((c) 2009 John Wiley & Sons, Ltd.)

1. Anticoccidials are widely used as food additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a specific time interval with broilers and pullets, but not for laying hens. 2. This study was set up to develop a new high pressure liquid chromatography (HPLC) method to detect clazuril (CZ: (+/-)-2-chloro-alpha-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-benzeneacetonitrile) in egg yolk and albumen and in plasma; to investigate both the presence of residues of CZ in eggs and its pharmacokinetic behaviour in laying hens. 3. A single oral dose (3 mg/kg BW) and multiple oral doses (3 mg/kg BW for 5 d) were investigated. The analytical method gave very good recovery (64 to 74%) in the three different matrices (yolk, albumen and plasma); precision and accuracy were within 11%. 4. After a single dose no residue was detected in eggs collected for up to 10 d, while following multiple dose treatment, CZ residues were detected until 10 d after the end of treatment. The concentration of the drug was higher in yolk than in albumen with a maximum ratio of 10 : 1. 5. Pharmacokinetics of CZ in laying hens after a single dose showed a detectable concentration of the drug up to 24 h. It reached a steady state after the third administration in multiple dosing. 6. Although further studies are necessary, these results indicate that a single oral dose of CZ could be used as an anticoccidial for laying hens due to the lack of residues in eggs.

An original method based on the use of high-performance liquid chromatography with both coulometric and diode array detection has been developed for the therapeutic drug monitoring of patients with bipolar disorders being treated with olanzapine and lamotrigine. Chromatographic separation was achieved on a reversed-phase C8 column (150 x 4.6 mm internal diameter, 5 microm) using a mobile phase composed of methanol (27%) and a 50.0 mmol/L, pH 3.5 phosphate buffer (73%). For the analysis of olanzapine and its main metabolite, N-desmethylolanzapine, a coulometric detector was used, with electrode 1 set at -200 mV and electrode 2 at +500 mV. Lamotrigine was determined using a diode array detection at 220 nm. The two detectors were connected in series. For the analysis of biological samples, a clean-up procedure was implemented by means of solid-phase extraction using phenyl cartridges and eluting the analytes with methanol; only a small volume of plasma (150 microL) was needed to analyze both olanzapine and lamotrigine. Linear responses were obtained between 0.1 and 50.0 ng mL(-1) for olanzapine, 0.1 and 25.0 ng mL(-1) for N-desmethylolanzapine, and between 0.25 and 10.0 microg mL(-1) for lamotrigine. The extraction yield values were always higher than 90% for all the analytes, with precision (expressed as relative standard deviation values) lower than 3.4%. The method was applied successfully to some human plasma samples drawn from bipolar patients undergoing combined therapy with the two drugs. Satisfactory values for accuracy were obtained, with mean recovery higher than 91%. Thus, the method appears suitable for the investigation of the chemical-clinical correlations in patients receiving therapy with olanzapine and lamotrigine.

A high-performance liquid chromatography (HPLC) assay using UV detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10- hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for the measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders.

A liquid chromatographic method with diode array detection (DAD) has been developed for the analysis of the antiepileptic agent lamotrigine (LTG) and its metabolites, lamotrigine 2-N-glucuronide and 2-N-methylated in plasma samples. The analytes were separated on a C8 RP column, using a mobile phase composed of methanol and a 0.45 mM, pH 3.5 phosphate buffer containing 0.17% triethylamine (24:76 v/v). Melatonin was used as the internal standard (IS). The DAD detector was set at 220 nm for the detection of all the analytes. A simple protein precipitation with methanol guaranteed high extraction yield values (>90%) and good purification from matrix interference. Good linearity was obtained in the 0.1-15.0 microg/mL range for LTG and lamotrigine 2-N-glucuronide and in the 0.1-2.0 microg/mL range for lamotrigine 2-N-methylated. The analytical method was validated in terms of precision, extraction yield, and accuracy. These assays gave RSD% values for precision always lower than 4.3% and mean accuracy higher than 80%. The method seems to be suitable for the analysis of plasma samples from patients treated with Lamictal.

Using isocratic column-switching high-performance liquid chromatography (HPLC) we established a group method for automated quantitative analysis of the antiepileptic drugs lamotrigine, oxcarbazepine and its metabolite 10-monohydroxycarbazepine (MHD) that are also used in psychiatry as mood stabilizers. Samples were cleaned from interfering proteins and lipids by transfer onto a pre-column, using a PerfectBond C-8 material, with 8% acetonitrile in water as a pre-column eluent. Separation was performed by elution onto the analytical column (Betasil C6 5 microm, 250 mm x 4.6 mm) at a flow rate of 1.0 ml/min with potassium dihydrogenphosphate buffer (20 mmol/l, pH3.0)/acetonitrile (70/30; v/v) as analytical eluent. UV-spectrophotometric detection was set to 215 nm for all three compounds. The analytical run was finished within 18 min. Detection limit was 30 ng/ml for lamotrigine, 35 ng/ml for oxcarbazepine and 25 ng/ml for 10-monohydroxycarbazepine. The method was found to be suitable for automated analysis of serum samples of patients treated with lamotrigine and oxcarbazepine.

A new, simple and sensitive high-performance liquid chromatography (HPLC) method with fluorescence detection was developed and validated for the determination of vardenafil in small volumes of rat plasma and bile. The absorbance and fluorescence characteristics of vardenafil were studied and factors that affect the HPLC resolution and fluorescence intensity were examined and optimized. Vardenafil and the internal standard cisapride were extracted using acetonitrile. The separation was achieved on a C18 column at 35 degrees C using acetonitrile-50 mM ammonium acetate aqueous solution (pH 6.8) (40:60) as mobile phase. At a flow rate of 1 ml/min, the total run time was 18 min. Fluorescence was measured with excitation and emission set at 280 and 470 nm, respectively. The calibration curves were linear from 10 to 1000 ng/ml and 0.2-100 microg/ml for plasma and bile samples, respectively. The intra- and inter-day imprecision did not exceed 10.8%, and the accuracy was within 9.6% deviation of the nominal concentration. The method was used successfully to investigate the disposition and biliary excretion of vardenafil in rats.

A high-performance liquid chromatography assay with ultraviolet detection was developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 1-30 microg/mL for lamotrigine, 2-20 microg/mL for carbamazepine, and 1-40 microg/mL for zonisamide. Within- and between-run precision studies demonstrated coefficient of variation <10% at all tested concentrations. Other anti-epileptic medications tested did not interfere with the assay. The method is appropriate for determining lamotrigine, carbamazepine and zonisamide serum or plasma concentrations for therapeutic monitoring.

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of lamotrigine (LTG) in serum is reported. The method involves extraction of the drug by ethyl acetate followed by separation on TLC silica plates using a mixture of toluene-acetone-ammonia (7:3:0.5), as eluting solvent. Densitometric analysis was carried out at 312 nm with lamotrigine being detected at Rf of 0.54. The analytical method has excellent linearity (r=0.998) in the range of 20-300 ng/spot. This assay range is adequate for analyzing human serum, as it corresponds to lamotrigine concentrations measured in human serum from epileptic patients. The method was validated for sensitivity, selectivity, extraction efficiency, accuracy and intra and inter-day reproducibility. The limit of detection and limit of quantification were found to be 6.4 and 10.2 ng, respectively. Good accuracy is reported in the range of 92.06-97.12% and high precision with %CV in range of 0.53-2.59. The method was applied for determination of serum lamotrigine levels in epileptic patients and in pharmacokinetic study of lamotrigine administered orally to rabbits.

A simple reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous estimation of the antiepileptic drugs (AEDs) lamotrigine (LTG), phenobarbitone (PB), carbamazepine (CBZ) and phenytoin (PHT) in human serum. The procedure involves extraction of the AEDs by mixing 200 microl of serum with 200mul of acetonitrile containing 10 microg/ml of pentobarbitone as internal standard (IS). After centrifugation, 10 microl of the supernatant was injected onto a NOVA PAK C-18 column (250 mm x 4.6mm, 5 microm Hypersil ODS) and eluted with a mobile phase consisting of phosphate buffer (10 mM)-methanol-acetonitrile-acetone in the ratio of 55:22:12:11 (v/v) adjusted to pH 7.0. A UV detector set at 210 nm was employed for detection. The AEDs were well resolved from the human serum constituents and the internal standard. The method can quantify LTG, PB, CBZ, and PHT at concentrations as low as 0.2 microg/ml. The method was quantitatively evaluated in terms of linearity, accuracy, precision, recovery, selectivity, sensitivity, and specificity. The method is simple, convenient, and suitable for the analysis of AEDs from human serum.

A selective and sensitive HPLC method is described for assay of the new antiepileptic drug pregabalin in serum/plasma. Following acetone precipitation of the sample, the drug was derivatized with picryl sulfonic acid (PSA) before chromatography on a C8 column. No interference from coadministered antiepileptics has been observed. The assay is calibrated over the range 0.5 mg/L to 8 mg/L. In addition to qualitative and quantitative validation data for the assay, concentration measurements in predose samples from a group of patients with dose escalated to 600 mg/d pregabalin are presented. The drug concentrations measured were in the range 2.8-8.2 mg/L at steady state. The method is robust and will be suitable for monitoring pregabalin therapy.

High performance liquid chromatography coupled with liquid-liquid-liquid microextraction was developed for the simultaneous determination of sildenafil and vardenafil in human plasma. The effects of extraction solvent, the volume of organic solvent, dropsize of acceptor phase, stirring rate and extraction time on the enrichment factors of analytes were investigated. The optimized experimental conditions, 300 microL toluene as the organic phase, 2 microL 0.2 mol/L HCl as the acceptor phase, 600 r/min of the stirring rate, and 40 min of the extraction time, were gotten. Under these conditions, high enrichment factors were obtained. The linear range of studied analytes was from 5 microg/L to 1.0 mg/L. The relative standard deviation was lower than 5%. The limits of detection were 1 microg/L for sildenafil and 0.5 microg/L for vardenafil at signal-to-noise ratio of 3. The method with little solvent consumption may provide high analyte preconcentration and excellent sample clean-up, and it is a sensitive and suitable method for simultaneous determination of the above two substances in human plasma.

Lamotrigine is a broad-spectrum antiepileptic agent. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of lamotrigine in 50 microl of plasma. Lamotrigine and the internal standard guanabenz were extracted with 1.2 ml of diethyl ether, after the samples alkalinized with 10 microl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a silica column with the mobile phase of acetonitrile-water containing 0.2% phosphoric acid and 0.3% triethylamine (pH 2.7) (84:16, v/v), at a flow-rate of 1 ml/min. The eluant was detected at 225 nm. The retention time was about 6 min for lamotrigine and 7 min for guanabenz. No endogenous substances and concomitant anticonvulsants were found to interfere. Calibration curves were linear from 0.1 to 5 microg/ml. The relative recovery of lamotrigine averaged about 80%. The limit of quantitation was 0.1 microg/ml. The intra- and inter-day precision (expressed as coefficient of variation, CV) was 8.1%, or less, and the accuracy was within 11.5% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring lamotrigine concentration.

Lamotrigine is an anticonvulsant drug recently approved in Italy for clinical use. Therapeutic monitoring of lamotrigine is relevant for patient management and avoidance of toxicity. The authors describe a simple, sensitive, and highly selective high-performance liquid chromatography method that does not involved extraction for analysis of serum lamotrigine. Serum (20 microL) with internal standard (BW 725 C) was injected directly into a column (25 cm x 4.6 mm) with an internal surface reversed phase (ISRP). The mobile phase consisted of 0.01 mol/L potassium phosphate bibasic (pH 6.0) and acetonitrile (82:18), the flow rate was 1.0 mL/min, and UV detection was optimized at 330 nm. The overall between-run coefficient of variance ranged from 1.89% to 3.25% and the lowest limit of detection was 0.05 mg/L. High linearity (r = 0.9996) in a wide range of concentrations (0.1-20.0 mg/L) and no interference with other antiepileptic drugs, benzodiazepines, and tricyclic antidepressants were the other characteristics of the method. The innovation of this method is the use of ISRP column and the choice of detection wavelength, which allow a shorter analysis time (5-6 minutes). The possibility of direct injection of plasma samples into the column permits a reduction in reagent consumption and in analytic steps, and hence in analytic error.

The authors have developed a simple isocratic high-pressure liquid chromatographic (HPLC) assay for the simultaneous determination of lamotrigine and other frequently coadministered antiepileptic drugs in serum samples. Lamotrigine extraction was performed on a reversed-phase Oasis HBL preparation column. The eluates containing butalbital as internal standard were separated with a 7-microm Chromsystems C18 250 x 4.0 mm I.D. reversed-phase column at a temperature of 40 degrees C using a mobile phase consisting of pH 3.8 phosphate-acetonitrile buffer (55:45, v/v), at a flow rate of 0.8 mL/min. Ultraviolet detection was carried out at 210 nm. Measurement of the peak:height ratio allowed quantitative determination of the samples. The method was linear over a concentration range of 0.2 to 20 microg/mL for lamotrigine. Recovery was >90%. Within-day and between-day coefficients of variation ranged from 1.8% to 6.7%. The mean lamotrigine concentration was 8.01 +/- 5.63 microg/mL. After studying sera from 130 patients treated with lamotrigine the authors confirmed that associated antiepileptic therapy affected the serum lamotrigine levels, which were significantly higher in patients under valproic acid treatment.

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78-46.95 micromol/l, with a mean coefficient of correlation (r)> or =0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 micromol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.

A rapid high-performance liquid chromatographic method was developed using a short silica column (30 mm x 4.6 mm) with an aqueous methanol mobile phase consisting of methanol-water-NH4H2PO4 (94:5.96:0.04) adjusted to a final apparent pH of 5.0 and pumped at a flow-rate of 1 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. Within-day variation of the method was 4.46% at 0.75 microg/ml and 2.37% at 6.0 microg/ml, and day-to-day variation was 9.10% at 0.75 microg/ml and 7.28% at 6.0 microg/ml.

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 microm Zorbax C8 column. Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm. The method was linear in the range of 1-20 microg/ml, with a mean coefficient of determination (r2=0.998). The limit of detection was 0.6 microg/ml and the lower limit of quantitation was 1 microg/ml using 200 microl of plasma. Within- and between-day accuracy and precision were below 6% at all analysed concentrations except at the limit of quantitation. No interfering peaks were found by commonly monitored antiepileptic drugs. Recovery was found to be > or =99%. Satisfactory performance was obtained in the evaluation of epileptic patient samples, whose results of plasma concentration measurements are briefly discussed. We conclude that this is a reliable method for the routine monitoring of lamotrigine concentration in plasma in the clinical setting.

An improved method is presented for the determination of proguanil, cycloguanil and 4-chlorophenylbiguanide in 100-microl capillary blood samples applied to sampling paper. This method also utilises a solid-phase extraction technique and high-performance liquid chromatography. Different kinds of sampling paper, such as ion-exchange and cellulose sampling paper were tested. The best elution recovery (70-80%) was obtained after treatment of cellulose sampling paper with a quaternary ammonium compound. The limit of determination was 50 nmol/l for cycloguanil and 4-chlorophenylbiguanide and 125 nmol/l for proguanil using 100 microl capillary blood. The stability of the analytes and elution performance from sampling paper was validated at different temperature and storage time. Venous blood and capillary blood concentrations of proguanil and metabolites were found to be similar.

We have extended automated sequential trace enrichment of dialysates and high performance liquid chromatography to include the simultaneous assay of lamotrigine with other anticonvulsant drugs: phenobarbitone, carbamazepine and phenytoin. The procedure is reliable and precise (between-batch coefficients of variation < 2.8%) with no interference from other commonly prescribed drugs. Patients on lamotrigine presented with serum concentrations up to 67 mumol/L with no adverse effects, suggesting that the therapeutic range may need re-appraisal.

A rapid sensitive and simple high-performance liquid chromatographic (HPLC) method for the determination of lamotrigine in plasma is described. The drug was extracted from 100 microliters of plasma with chloroform:isopropanol (95:5% v/v) in the presence of 100 microliters of phosphate buffer (10 mM). The extract was evaporated and the residue was reconstituted with mobile phase and injected onto the HPLC system. The drug and the internal standard (chloramphenicol) were eluted from a Symmetry C18 stainless steel column at ambient temperature with a mobile phase consisting of 0.01 M potassium-acetonitrile-methanol (70.20:10% v/v/v), adjusted to pH 6.7, at a flow rate of 1.3 ml min-1 and the detector was monitored at 214 nm. Quantitation was achieved by measurement of the peak-area ratio of the drug to the internal standard and the lower limit of detection for lamotrigine in plasma was 20 ng ml-1. The intraday precision ranged from 3.34 to 6.12% coefficient of variation (CV) and the interday precision ranged from 2.15 to 8.34% CV. The absolute and relative recoveries of lamotrigine ranged from 86.93 to 90.71% and from 95.18 to 107.13%, respectively. The method was applied in studying the pharmacokinetics of lamotrigine administered orally to rabbits. This reliable micro-method would have application in pharmacokinetic studies of lamotrigine where only small sample sizes are available, e.g. paediatric patients.

An improved and validated method is presented for the determination of proguanil, cycloguanil, and 4-chlorophenylbiguanide in plasma, whole blood, and urine using solid-phase extraction (SPE) technique and reversed-phase high-performance liquid chromatography (HPLC). The HPLC method uses isocratic elution with acetonitrile:phosphate buffer 0.1 mol/l, pH 2.6 (21.5:78.5 vol/vol) at a flow rate of 1.0 ml/min for the separation. The recovery of proguanil and metabolites ranged from 82% to 104%. The limit of determination was 20 nmol/l for proguanil and its metabolites in plasma and approximately 50 nmol/l for proguanil and metabolites in whole blood. Different stationary phases for HPLC and SPE were tested and the best chromatographic separation from endogenous constituents and other antimalarial drugs was achieved with cyanopropyl stationary phases.

Lamotrigine (LTG), a newly introduced antiepileptic drug, appears to have potential therapeutic advantages for the treatment of patients with partial-onset seizures. Increasing clinical application and research of LTG demand a simpler and more rapid analytical procedure to determine LTG concentration in body fluids and tissues. The authors have developed an effective one-step procedure for sample preparation followed by high-performance liquid chromatography (HPLC) to quantitate LTG in plasma, urine, and brain tissues. Body fluids and brain homogenates were treated with cold acetonitrile to precipitate protein. The samples were fractionated on a 250 x 4.6 mm C18 reversed-phase column with an isocratic mobile system consisting of potassium phosphate buffer, acetonitrile, and methanol (70:16:14). The method had a LTG detection limit of 0.02 microg/ml in plasma and 0.03 microg/ml in urine. The coefficients of variation were <2.7% for intraday and 4.2% for interday analyses. The recovery of LTG added to plasma, urine, and brain homogenate ranged from 98% to 100%. The method was applied to a clinical study to determine plasma and urine concentrations of LTG in subjects receiving a single oral dose of LTG. The calculated pharmacokinetic parameters were comparable to those previously reported. The method proved to be simple, fast, reproducible, and useful in clinical investigation and monitoring of LTG concentrations.

Lamotrigine is a phenyltriazine anticonvulsant recently approved for clinical use. A high-performance liquid chromatographic (HPLC) method was developed using a silica column (5 microm) with an aqueous methanol mobile phase consisting of 94% methanol, 5.92% water, and 0.08% NH4H2PO4 adjusted to a final apparent pH of 4.0 and pumped at a flow rate of 1.0 ml/minute. Ultraviolet detection was carried out at a wavelength of 280 nm, and plasma samples were prepared for HPLC analysis by extraction into ethyl acetate after basification. Retention times for lamotrigine and its internal standard (BWA725C) were 10.3 and 11.2 minutes, respectively, and there was no chromatographic interference from other commonly coadministered anticonvulsants. Calibration curves were linear over a concentration range of 0.5 to 30 mg/l, with intra-assay and interassay coefficients of variation less than 8%. Assessment of assay performance in an international quality assurance program showed an average bias of 0.3% compared with the consensus mean. A review of 52 patient specimens showed that, if patients were grouped according to coadministered anticonvulsants, a significant correlation between lamotrigine dosage and concentration was evident in those coadministered valproate (in the absence of metabolic inducers) and in those coadministered a combination of valproate and inducers, but not in patients coadministered inducers alone. Mean (SD) trough concentrations were 9.2 (5.2), 2.8 (1.3), and 3.8 (2.8) mg/l in the valproate, inducer, and combination groups, respectively.

The use of the system, automated sequential trace enrichment of dialysates (ASTED), to prepare plasma samples for the estimation of lamotrigine, its glucuronide and methylated metabolites in plasma prior to gradient high-performance liquid chromatography (HPLC) is described. Using this technique the procedure was observed to be specific for all three compounds and linear over the range 0.04 to 10 microg/ml for lamotrigine and the glucuronide metabolite and 2 to 500 ng/ml for the methylated metabolite. The within-run precision (C.V.) at four different supplemented plasma lamotrigine concentrations of 0.04, 0.10, 2.5 and 10.0 microg/ml was 6.21, 5.17, 1.29 and 0.73%, respectively, and the between-run precision (C.V.) estimated to be 13.49, 6.08, 1.95 and 1.78%, respectively. The overall accuracy (% bias) of the procedure was estimated to be 12.50, 0.00, 2.80 and 1.80%, respectively. The glucuronide and methylated metabolites in plasma showed similar assay performance.

Lamotrigine (lamictal) is a new anticonvulsant drug approved by the FDA for clinical use. Therapeutic monitoring of lamotrigine is useful for patient management and avoidance of toxicity. The suggested therapeutic range is 1 to 4 micrograms/ml. The authors describe a simple high-performance liquid chromatographic (HPLC) method for analysis of lamotrigine from serum. Serum (0.5 ml) was alkalinized with borate buffer (pH 9.8). Lamotrigine and the internal standard thiopental were extracted with 10 ml of chloroform. After evaporation of the extract, the residue was reconstituted in the mobile phase (prepared by mixing 750 ml of potassium dihydrogen phosphate, 550 ml of deionized water, 430 ml of methanol, and 100 microliters of triethylamine as an ion pairing reagent) and injected into an LC-18 column (15 cm x 4.6 mm). The authors use this HPLC system routinely in their laboratory for the analysis of barbiturates. They demonstrated that the same system can be used for the analysis of lamotrigine. The within-run and between-run precisions of the lamotrigine assay were 1.63% (mean = 3.05, SD = 0.05 microgram/ml, n = 6) and 3.7% (mean = 2.97 micrograms/ml, SD = 0.11, n = 8). The assay was linear for serum lamotrigine concentrations of 0.5 microgram/ml to 20 micrograms/ml with a detection limit of 0.5 microgram/ml. The authors observed excellent correlation between serum lamotrigine concentrations measured by their assay and a reference laboratory in six patients receiving lamotrigine. Their assay is free from interferences from common tricyclic antidepressants, benzodiazepines, other common anticonvulsants, salicylate, and acetaminophen.