Lamotrigine In Human Plasma Research Articles

Normatividad mexicana a la validación de métodos analíticos para cuantificar fármacos en matriz biológica. Ejemplo aplicativo: cuantificación de lamotrigina en plasma humano vía CLAR

Standardization organizations of different natures indicate how to obtain documented experimental evidence that certifies the validity of analytical methods to quantify drugs in the biological matrix for bioequivalence studies. The vast amount of information on this subject highlights its relevance in uniforming the procedures to achieve the desired objective, which can be applied by any laboratory that requires it. However, to avoid discrepancies in the interpretation and / or execution of the various forms of analytical performance evaluation by the authority concerning the international information provided, local regulations are issued as mandatory, highlighting critical situations with discriminatory criteria. This paper presents the minimum analytical performance characteristics and the acceptance criteria required (according to Mexican regulations) to demonstrate that an analytical method is valid to quantify drugs in a biological matrix by HPLC (high-performance liquid chromatography) for a research study at a graduate-level educational institution. It is emphasized that the analytical parameters to be met under this standard consider the specific aspects of the technique and biological sample. For this purpose, a method developed and validated by our research group is used as an example for studying the biological sample. For this purpose, a method developed and validated by our research group to quantify lamotrigine in human plasma for monitoring purposes is used as an example. The performance characteristics of the NOM-177-SSA-2013 applied to the validation of the developed method demonstrate the validity and application of the method for the desired objective.

Determination of lamotrigine in human plasma by HPLC-PDA. Application to forensic samples.

Therapeutic drug monitoring of plasma lamotrigine (LTG) has customarily been carried out in order to prevent some its adverse effects. For forensic purposes, determination of LTG in plasma is an useful tool in cases of accidental overdose or suicidal attempts. Currently, there are several analytical methods available including some based on LC tandem mass spectrometry techniques, but simple and accessible LC-UV methods still can be useful for the purpose. Here we report on a new high-performance liquid chromatography method for the determination of lamotrigine in human plasma which has been developed and validated including selectivity, sensitivity, accuracy, precision and recovery studies. Lamotrigine and the internal standard chloramphenicol were extracted from plasma using liquid-liquid extraction using small volumes of buffer and ethylacetate. Detection was monitored at 305.7 and 276.0nm for lamotrigine and chloramphenicol, respectively. The method was linear concentration dependence within the range of 0.1-10µg/ml, with a mean coefficient of correlation r = 0.993. The limit of detection (LOD) was 0.04µg/ml and the limit of quantification (LOQ) was 0.1µg/ml. Intra and interday precision values were lower than 9.0% at all concentrations studied. The intra and interday accuracy values ranged from - 7.6 to 10.1%. Recovery was found to be 98.9% or higher. The method here described was successfully applied to 11 postmortem blood samples received at the Forensic Sciences Institute of Santiago de Compostela (Spain). A new HPLC method for the determination of lamotrigine in human plasma was developed and validated. A liquid-liquid extraction using small volumes of buffer and ethylacetate was optimized. The proposed method is suitable for forensic toxicological analysis.

A LC-MS3 strategy to determine lamotrigine by Q-Q-trap tandem mass spectrometry coupled with triple stage fragmentation to enhance sensitivity and selectivity.

A high-performance liquid chromatography tandem mass spectrometry cubed (HPLC/MS3) method was developed and validated to quantify lamotrigine in human plasma with carbamazepine as an internal standard. The HPLC/MS/MS system is composed of a Shimadzu UFLC XR high-performance liquid chromatograph coupled with a hybrid linear ion trap triple quadrupole mass spectrometer. Following simple protein precipitation with methanol, the separation of lamotrigine and carbamazepine was performed on an Agilent Poroshell 120 SB-C18 column (4.6 × 50 mm, 2.7 μm) using gradient elution with 0.1% formic acid in water (solvent I) and 0.1% formic acid in methanol (solvent II) at a flow rate of 0.8 mL min-1. The total run time for each sample was 5 min. The method was validated for accuracy, precision, linearity, lower limit of quantification (LLOQ), selectivity, and other parameters. The LC/MS3 method was linear in the concentration range of 0.50-50.0 μg mL-1 (R2 ≥ 0.995). The LLOQ was 0.5 μg mL-1, requiring only 30 μL of human plasma. Intra- and inter-day accuracies were <6.17% and precisions were <11.4% at all concentrations. The absolute recoveries (%) and matrix effect (%) for lamotrigine in human plasma were between 83.8 and 90.7. The developed and validated LC-MS3 assay was successfully applied to monitor the lamotrigine levels in human plasma after the administration of lamotrigine.

Determination of lamotrigine in human plasma using liquid chromatography-tandem mass spectrometry.

AimLamotrigine (LTG) is a widely used anti‐epileptic drug that is administered to avoid seizures and to maintain seizure‐free status. However, several factors reportedly cause individual differences of plasma LTG levels, and the therapeutic target range of LTG varies between individuals. Thus, to optimize effective doses of LTG, we developed a rapid and simple method for determining plasma LTG concentrations.MethodsLamotrigine and the internal standard papaverine were extracted from human plasma using solid‐phase extraction. After filtration, 5‐μL aliquots of final samples were injected into the liquid chromatography‐tandem mass spectrometry instrument and LTG and internal standard were separated using a Cadenza CD‐C18 column (100 × 2 mm, 3 μm) with 0.1% formic acid in water/acetonitrile (2/1, v/v).ResultsThe calibration curve was linear from 0.2 to 5.0 μg/mL, and assessments of recovery, intra‐ and inter‐day precision and accuracy, matrix effects, freeze and thaw stability, and long‐term stability demonstrated good reproducibility. Retention times of LTG and internal standard were 1.6 and 2.0 minutes, respectively, and the total run time was 3.5 minutes for each sample.ConclusionWe developed a rapid and simple method for determining plasma LTG concentrations. The present novel system could be used to inform LTG dose adjustments for individual patients.

A simple, rapid and stability indicating validated method for quantification of lamotrigine in human plasma and dry plasma spot using LC-ESI-MS/MS: Application in clinical study.

Lamotrigine (LTZ) is a phenyltriazine derivative which belongs to anti-epileptic drugs (AEDs) class and prescribed as mono- or adjunctive-therapy in treatment of epilepsy. Therapeutic drug monitoring (TDM) of AEDs provides a valid clinical tool in optimization of overall therapy. However, TDM is challenging due to the high biological samples (plasma/blood) storage/shipment costs and the limited availability of laboratories providing TDM services. Sampling in the form of dry plasma spot (DPS) or dry blood spot (DBS) are suitable alternative to overcome these issues. We developed and validated a new method for quantification of LTZ in human plasma and DPS. The extraction of LTZ from plasma and DPS was performed using liquid-liquid extraction with diethyl ether and an extraction solution composed of diethyl ether- methyl tert-butyl ether- acetone (50:30:20, v/v/v), respectively. Lamotrigine- 13C3, d3 was used as internal standard (ISTD) and the chromatographic separation was achieved on Hypurity Advance C18 column (150×4.6mm, 5μm). Quantitative estimation of LTZ and ISTD was performed on a liquid chromatography tandem mass spectrometer coupled with electrospray ionization interface operated under positive mode of ionization. Calibration curves were linear (r2>0.99) over the concentration range of 10-3020ng/mL for both plasma and DPS. Statistical analysis provides insignificant difference between LTZ concentration extracted from plasma and DPS samples. The method is found suitable for application in clinical study and in therapeutic monitoring of LTZ. To the best of our knowledge this is the first report which describing a validated stability indicating assay for quantification of LTZ in dry plasma spot.

Determination of lamotrigine in human plasma and saliva using microextraction by packed sorbent and high performance liquid chromatography–diode array detection: An innovative bioanalytical tool for therapeutic drug monitoring

A ground-breaking high-performance liquid chromatography-diode array detection method based on microextraction by packed sorbent (MEPS) as sample preparation approach is described herein for determination of lamotrigine (LTG), a narrow therapeutic index drug, in human plasma and saliva. MEPS variables and chromatographic conditions were optimized to achieve appropriate selectivity and sensitivity using small sample volumes (100μL). The chromatographic separation of LTG and chloramphenicol [internal standard (IS)] was accomplished in <5min on a C18-column, at 35°C, using a mobile phase composed by acetonitrile/methanol/water-triethylamine 0.3% at pH6.0 (13:13:74, v/v/v) pumped isocratically at 1mL/min. LTG and IS were detected at 215nm. A good linearity was obtained for LTG (r2≥0.9936) in the range of 0.1–20μg/mL in plasma and saliva, with the limit of quantification of 0.1μg/mL. The method was shown to be precise (RSD ≤14.5%) and accurate (bias±13.4%), and the absolute recovery ranged from 64.9% to 73.6%. The stability of LTG was demonstrated in plasma and saliva samples in all studied conditions. The proposed assay was applied to the analysis of real human plasma and saliva samples from epilepsy patients under LTG therapy and the results support its usefulness for therapeutic drug monitoring.

Quantification of lamotrigine in patient plasma using a fast liquid chromatography-tandem mass spectrometry method with backflush technology.

Recent concerns have been raised by neurologists and patients with epilepsy regarding the bioequivalence of generic lamotrigine to the brand Lamictal. Bioequivalence studies require the quantification of lamotrigine in human plasma, including in the presence of other drugs, for studies that will use patients with epilepsy rather than healthy volunteers. Lamotrigine was extracted from plasma through a simple protein precipitation and analyzed by fast liquid chromatography coupled to heated electrospray ionization with tandem mass spectrometric detection. A backflush step to remove interferent accumulation on column was included, and a stable isotope-labeled lamotrigine was used as an internal standard. The method was validated for accuracy, precision, interday and intraday coefficient of variation, specificity, lower limit of detection, lower limit of quantification, linearity, range, instrument precision, freeze-thaw, dilution integrity, and sample stability. Specificity evaluation included consideration of the impact of other antiepileptic drugs. The described method has a linear range of 8-10,000 ng/mL of lamotrigine (r = 0.9999) and a lower limit of detection of 1 ng/mL and a lower limit of quantification of 8 ng/mL. Intraday and interday reproducibility were less than 10.0% relative SD and 10.4% relative SD, respectively, and the percent recovery varied from 96.6% to 109.3% at various lamotrigine concentrations. A backflush step reduced matrix effects and no interference peak from plasma or other antiepileptic drugs were observed. A liquid chromatography-heated electrospray ionization-tandem mass spectrometry method including a backflush step was developed and validated to measure lamotrigine concentration in patient plasma. The method will be applied to bioequivalence studies that compare brand versus generic lamotrigine.

Liquid chromatography tandem mass spectrometry method for the estimation of lamotrigine in human plasma: Application to a pharmacokinetic study

A reliable, selective and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of lamotrigine in human plasma using lamotrigine-13C3, d3 as an internal standard. Analyte and internal standard were extracted from human plasma by solid-phase extraction and detected in positive ion mode by tandem mass spectrometry with electrospray ionization (ESI) interface. Chromatographic separation was performed on a Chromolith® SpeedROD; RP-18e column (50−4.6mm i.d.) using acetonitrile: 5±0.1mM ammonium formate solution (90:10, v/v) as the mobile phase at a flow rate of 0.500mL/min. The calibration curves were linear over the range of 5.02–1226.47ng/mL with the lower limit of quantitation validated at 5.02ng/mL. The analytes were found stable in human plasma through three freeze (−20°C)-thaw (ice-cold water bath) cycles and under storage on bench-top in ice-cold water bath for at least 6.8h, and also in the mobile phase at 10°C for at least 57h. The method has shown good reproducibility, as the intra- and inter-day precisions were within 3.0%, while the accuracies were within ±6.0% of nominal values. The validated LC–MS/MS method was applied for the evaluation of pharmacokinetic and bioequivalence parameters of lamotrigine after an oral administration of 50mg lamotrigine tablet to thirty-two healthy adult male volunteers.

A sensitive and selective HPLC method for estimation of lamotrigine in human plasma and saliva: application to plasma-saliva correlation in epileptic patients

A sensitive and selective high performance liquid chromatographic (HPLC) method was developed and validated for estimation of lamotrigine (CAS 84057-84-1) in human plasma and saliva. The chromatographic separation was achieved with a reversed phase column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer pH 6.5 (30:70) with a flow rate of 1 mL/min. The calibration curve was linear within the working range for both plasma and saliva. The validated method has been successfully applied for a study of lamotrigine in human plasma and saliva to establish the correlation between these two matrices. A scatter plot of plasma versus salivary lamotrigine concentrations showed a gold linear relationship between them (Pearson correlation coefficient, r = 0.6832, p < 0.001).

Rapid and sensitive LC‐MS/MS method for quantification of lamotrigine in human plasma: application to a human pharmacokinetic study

A highly sensitive, specific and fully validated LC-MS/MS method as per general practices of industry has been developed for estimation of lamotrigine (LAM) with 100 μL of human plasma using flucanozole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using electrospray ionization. A simple liquid-liquid extraction process was used to extract LAM and IS from human plasma. The total run time was 2.0 min and the elution of LAM and IS occurred at 1.25 and 1.45 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (20:40:40, v/v) at a flow rate of 0.50 mL/min on a Discovery CN (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for LAM. A linear response function was established for the range of concentrations 0.1-1500 ng/mL (r > 0.998) for LAM. The intra- and inter-day precision values for LAM met the acceptance as per Food and Drug Administration guidelines. LAM was stable in the set of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

Rapid Quantification of Lamotrigine in Human Plasma by Two LC Systems Connected with Tandem MS

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.

Optimisation of a high-efficiency liquid chromatography technique for measuring lamotrigine in human plasma

ObjectiveThe 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 methodsThe 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−100C18−5μm−15*0.4cm with a LiChroCART-RP18e−3μm−5.5*0.4cm); in both cases, a liquid-liquid extraction was performed and the same sample extraction protocol was followed. ResultsBoth validation methods showed that the two column types are valid for routine lamotrigine monitoring. ConclusionThe 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.

Development of an Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Micromethod for Quantification of Lamotrigine in Human Plasma and its use in a Bioequivalence Trial

The aim of the present work was to develop a chromatographic technique coupled with mass spectrometry for the measurement of lamotrigine in plasma. Lamotrigine and guanabenz (internal standard) were measured by selected reaction monitoring. The method was validated and applied in a bioequivalence trial on 26 female volunteers. Lamotrigine chewable tablets (100 mg) were administered and monitored for up to 96 h. The method was linear between 0.05 and 5.0 µg/ml, with acceptable stability, accuracy and precision. Mean maximum plasma concentration was 1.37 µg/ml and was reached at 1.6 h postdose. Elimination half-life was 32.7 h. Lamotrigine tablets were bioequivalent. Ultra-performance liquid chromatography with tandem mass spectrometry represents a powerful tool in terms of sensitivity, specificity and high-throughput analysis.

LC method for the therapeutic drug monitoring of lamotrigine: Evaluation of the assay performance and validation of its application in the routine area

An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of lamotrigine in human plasma has been developed and validated for use in pharmacokinetic studies. A validation strategy based on the accuracy profiles was used to select the most appropriate regression model and to determine the limits of quantitation as well as the concentration range. On the other hand, the present paper also shows this validation approach as a suitable tool to guaranty the quality of the results obtained by the use of the analytical validated methodology for plasma lamotrigine determination in a routine setting and to ensure the risk of obtaining the future measurements outside the previously fixed acceptance limits.

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

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 μmol/l, with a mean coefficient of correlation ( r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/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.

Determination of lamotrigine in human plasma by high-performance liquid chromatography

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 μm 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 μg/ml, with a mean coefficient of determination ( r 2=0.998). The limit of detection was 0.6 μg/ml and the lower limit of quantitation was 1 μg/ml using 200 μl 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 ≥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.

New micromethod for the determination of lamotrigine in human plasma by high-performance liquid chromatography.

An extraction procedure and reversed-phase high-performance liquid chromatographic assay is described and validated for the determination of lamotrigine in human plasma. The method involves extraction with chloroform-isopropanol after alkalinization with a carbonate buffer, back-extraction into 0.05% phosphoric acid and separation by reversed-phase HPLC using a 5-micron Supelco diphenyl column (150 x 4.6 mm I.D.). Quantitation was performed by measurement of the UV absorbance at a wavelength of 265 nm. This method was carried out on 50-200 microliters samples of plasma, depending on whether they were pediatric or adult samples. The lower limit of quantitation was 0.2 microgram/ml using 200 microliters of plasma. A linear response was tested from 0.5 to 20 micrograms/ml. Within- and between-day accuracy and precision were always below 10.0% at all analysed concentrations. The method selectivity towards the most used antiepileptic drugs has been proven. Satisfactory performances were obtained in the evaluation of samples from epileptic patients.

A Rapid and Specific Assay for the Determination of Lamotrigine in Human Plasma by Normal-Phase HPLC

A rapid and specific high-performance liquid chromatography assay of lamotrigine in human plasma is described. Lamotrigine is extracted with dichloromethane from buffered plasma to which an internal standard has been added. The solvent is directly injected into a 250 x 4.6-mm Spherisorb Silica column and the drug is eluted by using a mixture of methanol, n-heptane, dichloromethane, and 28-30% ammonium hydroxide (20:40:40:0.3 vol/vol) at a flow rate of 1 ml/min. The eluates are detected at 240 nm. The assay requires 250 microliters of sample, and concentrations as low as 0.4 microgram/ml can be measured accurately. The method is linear in the range of 0.4-16 micrograms/ml, with a mean coefficient of correlation (r) > or = 0.997. Within- and between-day relative standard deviations at three different concentration levels (1, 4, and 8 micrograms/ml) are < or = 8.6%.