Valproic Acid In Human Plasma Research Articles

A simple and rapid HPLC-UV method for the determination of valproic acid in human plasma using microwave-assisted derivatization with phenylhydrazine hydrochloride

This study presents an efficient high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for monitoring valproic acid (VPA) level in human plasma. This method is distinguished by its simplicity, cost-effectiveness, and rapid execution, addressing the limitations associated with other advanced analytical techniques like liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and immunoassays, which are generally complex and costly for routine application. A challenge in analyzing VPA is its non-linear protein binding profile and the absence of a chromophore in its structure, making direct detection difficult. To overcome this, the study developed an efficient HPLC-UV for VPA determination in human plasma, utilizing a simplified and rapid microwave-assisted derivatization process. Due to the lack of a chromophore in VPA structure, this work developed a microwave-assisted derivatization of VPA using phenylhydrazine hydrochloride (PH HCl). The process optimization was achieved at 450 W for 50 s, facilitating effective HPLC-UV detection. The derivatized product was characterized using 1H nuclear magnetic resonance (NMR) and Fourier transform infrared spectrometer (FT-IR). The derivative, identified as (Z)-N-phenyl-2-propylpentanehydrazonic acid, demonstrated specificity in plasma analysis with no detectable interference. The method exhibited a linear response for VPA concentrations ranging from 30 to 150 μg/mL, with a correlation coefficient exceeding 0.99. Recovery varied between 86.7% and 107%, with a maximum coefficient of variation (CV) of 10.0%. The findings suggest that the microwave-assisted derivatization technique substantially improves the feasibility and cost-effectiveness of HPLC-UV for the analysis of VPA in plasma. This method provides a viable alternative to conventional HPLC methodologies, offering a balance of efficiency and economic practicality for VPA quantification.

Simultaneous determination of eight antiepileptic drugs and two metabolites in human plasma by liquid chromatography/tandem mass spectrometry

AbstractEpilepsy is one of the most prevalent neurological conditions and antiepileptic drugs are the mainstay of epilepsy treatment. High variation in pharmacokinetic profiles of several antiepileptic drugs highlights the importance of therapeutic drug monitoring to estimate pharmacokinetic properties and consequently individualize drug posology. In this work, a simple, rapid and robust liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of carbamazepine and its metabolite carbamazepine-10,11-epoxide, gabapentin, levetiracetam, lamotrigine, oxcarbazepine and its metabolite mono-hydroxy-derivative metabolite, phenytoin, topiramate, and valproic acid in human plasma for therapeutic drug monitoring. d6-Levetiracetam, d4-gabapentin and d6-valproic acid were used as internal standards. After addition of internal standards along with two-step protein precipitation and dilution sample preparation, plasma samples were analyzed on a C18 column using a gradient elution in 5 min without interference. The calibration curves were linear over a 100-fold concentration range, with determination coefficients (r2) greater than 0.99 for all analytes. The limit of quantification was 0.5 μg mL−1 (0.1 μg mL−1 for oxcarbazepine, 2 μg mL−1 for levetiracetam, and 10 μg mL−1 for valproic acid) with precision and accuracy ranging from 3% to 9% and from 94% to 112%, respectively. Intra- and inter-day precision and accuracy values were within 15% at low, medium and high quality control levels. No significant matrix effect was observed in the normal, hemolyzed, lipemic, and hyperbilirubin blood samples. This method was successfully used in the identification and quantitation of antiepileptic drugs in patients undergoing mono- or polytherapy for epilepsy.

A Novel Approach for Therapeutic Drug Monitoring of Valproic Acid Using FT-IR Spectroscopy and Nonlinear Support Vector Regression.

Recent technological progress has bolstered efforts to bring personalized medicine from theory into clinical practice. However, progress in areas such as therapeutic drug monitoring (TDM) has remained somewhat stagnant. In drugs with well-known dose-response relationships, TDM can enhance patient outcomes and reduce health care costs. Traditional monitoring methods such as chromatography-based or immunoassay techniques are limited by their higher costs and slow turnaround times, making them unsuitable for real-time or onsite analysis. In this work, we propose the use of a fast, direct, and simple approach using Fourier transform infrared spectroscopy (FT-IR) combined with chemometric techniques for the therapeutic monitoring of valproic acid (VPA). In this context, a database of FT-IR spectra was constructed from human plasma samples containing various concentrations of VPA; these samples were characterized by the reference method (immunoassay technique) to determine the VPA contents. The FT-IR spectra were processed by two chemometric regression methods: partial least-squares regression (PLS) and support vector regression (SVR). The results provide good evidence for the effectiveness of the combination of FT-IR spectroscopy and SVR modeling for estimating VPA in human plasma. SVR models showed better predictive abilities than PLS models in terms of root-mean-square error of calibration and prediction RMSEC, RMSEP, R2Cal, R2Pred, and residual predictive deviation (RPD). This analytical tool offers potential for real-time TDM in the clinical setting. FTIR spectroscopy was evaluated for the first time to predict VPA in human plasma for TDM. Two regressions were evaluated to predict VPA in human plasma, and the best-performing model was obtained using nonlinear SVR.

Online Column Extraction Coupled with Double-Trap Column System for HPLC Determination of Valproic Acid in Human Plasma Without Derivatization

Online Column Extraction Coupled with Double-Trap Column System for HPLC Determination of Valproic Acid in Human Plasma Without Derivatization

An electromembrane extraction–HPLC‐UV analysis for the determination of valproic acid in human plasma

In this study, an electromembrane extraction (EME) method combined with a simple HPLC‐UV analysis was developed and validated for the determination of valproic acid in human plasma samples. The major parameters influencing EME procedure, namely the solvent composition, voltage, pH of acceptor and donor solutions, salt effect, and time of extraction, were evaluated and optimized. The drug was extracted from the donor aqueous sample solution (pH 5) to the acceptor aqueous solution (pH 13). The donor and acceptor phases were separated by a hollow fiber dipped in 1‐octanol as a supported liquid membrane. A voltage of 60 V during 25 min was applied as the driving force. The drug concentration enrichment factor obtained was >125, which enhanced the sensitivity of the method. The limit of detection and the limit of quantitation were 0.2 and 0.5 μg/mL, respectively. The proposed method was successfully applied to a human plasma sample, with a relative recovery of 75%. The method was linear over the range 0.5–10 μg/mL for valproic acid (R2 > 0.9996) with a repeatability (%RSD) between 0.9 and 3.3% (n = 3). Valproic acid is an anticonvulsant drug with poor UV absorption, and EME can improve the sensitivity of HPLC‐UV for the determination of valproic acid in plasma samples.

Determination of valproic acid in human plasma using dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection.

Dispersive liquid-liquid microextraction coupled with gas chromatography (GC)-flame ionization detector was developed for the determination of valproic acid (VPA) in human plasma. Using a syringe, a mixture of suitable extraction solvent (40 µl chloroform) and disperser (1 ml acetone) was quickly added to 10 ml of diluted plasma sample containing VPA (pH, 1.0; concentration of NaCl, 4% (w/v)), resulting in a cloudy solution. After centrifugation (6000 rpm for 6 min), an aliquot (1 µl) of the sedimented organic phase was removed using a 1-µl GC microsyringe and injected into the GC system for analysis. One variable at a time optimization method was used to study various parameters affecting the extraction efficiency of target analyte. Then, the developed method was fully validated for its accuracy, precision, recovery, stability, and robustness. Under the optimum extraction conditions, good linearity range was obtained for the calibration graph, with correlation coefficient higher than 0.998. Limit of detection and lower limit of quantitation were 3.2 and 6 μg/ml, respectively. The relative standard deviations of intra and inter-day analysis of examined compound were less than 11.5%. The relative recoveries were found in the range of 97 to 107.5%. Finally, the validated method was successfully applied to the analysis of VPA in patient sample. The presented method has acceptable levels of precision, accuracy and relative recovery and could be used for therapeutic drug monitoring of VPA in human plasma.

A New Derivatization Method to Enhance Sensitivity for the Determination of Low Levels of Valproic Acid in Human Plasma

A novel and sensitive high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of valproic acid (VPA) in human plasma. The method was based on derivatization of VPA using 2-bromo-2'-acetonaphthone as a new derivatization reagent. Caprylic acid was used as an internal standard (IS). Under the optimized extraction and derivatization conditions, the method showed good linearity in the range of 0.05-200 μg mL(-1) and the limit of detection was as low as 0.01 μg mL(-1). The relative standard deviation for intra-day and inter-day (n = 5) was <5%. The recovery ranged from 95.2 to 101.4%. The proposed method is proved to be highly sensitive, simple and rapid, and was successfully applied to the analysis of VPA in plasma samples from patients with generalized epilepsy.

Simultaneous determination of asenapine and valproic acid in human plasma using LC–MS/MS: Application of the method to support pharmacokinetic study

Combination of asenapine with valproic acid received regulatory approval for acute treatment of schizophrenia and maniac episodes of bipolar disorders. A simple LC–MS/MS method was developed and validated for simultaneous quantification of asenapine and valproic acid in human plasma. Internal standards were added to 300μL of plasma sample prior to liquid–liquid extraction using methyl tertiary butyl ether (MTBE). Chromatographic separation was achieved on Phenomenex C18 column (50mm×4.6mm, 5μm) in isocratic mode at 40°C. The mobile phase used was 10mM ammonium formate–acetonitrile (5:95, v/v) at a constant flow rate of 0.8mL/min monitored on triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The injection volume used for LC–MS/MS analysis was 15μL and the run time was 2.5min. These low run time and small injection volume suggest the high efficiency of the proposed method. The method was validated over the concentration range of 0.1–10.02ng/mL and 10–20,000ng/mL for asenapine and valproic acid respectively. The method recoveries of asenapine (81.33%), valproic acid (81.70%), gliclazide (78.45%) and benzoic acid (79.73) from spiked plasma samples were consistent and reproducible. The application of this method was demonstrated by a pharmacokinetic study in 8 healthy male volunteers with 5mg asenapine and 250mg valproic acid administration.

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid–liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10mM histidine, 10mM 3-(N-morpholino)propanesulfonic acid and 10μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4–300μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7–3.5%, n=3, for the concentration range from 1 to 150μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

DETERMINATION OF TOTAL AND UNBOUND CONCENTRATIONS OF VALPROIC ACID IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

An analytical method for the determination of valproic acid in human plasma and ultrafiltrated human plasma has been developed and validated. The analytical method lies in the solid phase extraction of valproic acid (VPA), followed by its quantification through an LC-MS method, using benzoic acid as the internal standard. The intra-assay and inter-assay precisions of this technique ranged between 4.29% and 9.91% and its accuracy ranged between 1.00% and 7.73%, for VPA spiked in plasma matrix. For VPA spiked in ultrafiltrated plasma, matrix precisions were from 6.29% to 9.84% and accuracies, from −1.17% to 5.38%. The limit of quantification was set at 1 µg/mL (precision 3.20%, accuracy 11.31%). The linear working range of concentrations was established between 10 and 250 µg/mL for the plasma matrix and between 1 and 100 µg/mL for the ultrafiltrated plasma matrix. The absolute recovery of valproic acid was over 82% in the studied range of concentrations in both matrixes. Valproic acid was found to be stable in both matrixes at the assayed conditions. This method has shown to be suitable for the analysis of free and total valproic acid concentrations in human plasma samples.

Analisis Asam Valproat dalam Plasma Secara Kromatografi Gas

Valproic acid is an anticonvulsant drug that works by increasing the levels of γ-aminobutyric acid (GABA). Determination of valproic acid is quite difficult because it has no chromophore groups in its structure. Aa analytical method using gas chromatography (GC) with flame ionization detector for the determination of valproic acid in human plasma has been developed and optimized. Valproic acid was extracted from plasma by liquid-liquid extraction method using diethyl ether. The optimum analysis conditions for valproic acid in plasma were achieved by regulated gas chromatography injector and detector at a temperature of 250oC and temperature programming with an initial temperature of 70oC and 5oC temperature increasing per minute until a temperature of 100oC, then held for 1 minute. Then the temperature was increased by 2°C per minute until the column temperature to 150oC. The optimum conditions of analysis took 32 minutes. In the concentration range from 40.0 to 100.0 µg/mL yielded a linear calibration curve with correlation coefficient (r) of 0.9894. Accuracy (% diff) of this method was -13.67% to 12.33% with precision (CV) between 9.33% to 14.92%, and relative recovery test was 86.33% to 112.33%. Keywords : gas chromatography, optimation, plasma in vitro, valproic acid

N-(1-Naphthyl)ethylenediamine, a New UV Labeling Reagent Used for LC Determination of Valproic Acid in Human Plasma

A high performance liquid chromatography method is presented for the determination of valproic acid levels in human plasma. The method was based on pre-column derivatization using N-(1-naphthyl)ethylenediamine as a new labeling agent. The calibration curve was linear in the investigated concentration range between 0.1 and 100 μg mL−1 and showed good accuracy and reproducibility. The assay provided a limit of quantification of 0.1 μg mL−1 for valproic acid and a limit of detection of 10 ng mL−1, respectively. The presented method was successfully applied to the determination of valproic acid levels in plasma after oral administration of 600 or 800 mg of sodium valproate.

A New High-Throughput LC-MS/MS Assay for Therapeutic Level Monitoring of Valproic Acid in Human Plasma

A new high-throughput liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay for the quantification of valproic acid in human plasma was developed and validated. The separation was performed on a Zorbax SB-C18 column under isocratic conditions using a 48:52 (v/v) mixture of acetonitrile and 0.1% (v/v) acetic acid in water at 45 °C with a flow rate of 0.8 mL/min. The detection of valproic acid was performed in SIM mode (m/z 143.1). The human plasma samples (0.2 mL) were deproteinized with methanol and aliquots of 2 μL from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r > 0.9972), precision (CV > 7.8 %) and accuracy (bias > 5.7 %) over the range of 5-200 μg/mL plasma. Lower limit of quantification (LLOQ) was 5 μg/mL and the recovery was between 98-106 %. The method is not expensive, it needs a minimum time for plasma sample preparation and has a run-time of 2.4 min for instrument analysis (retention time of valproic acid was 1.8 min). The developed and validated high-throughput method is very simple, rapid and efficient, with wide applications in clinical level monitoring, pharmacokinetics and bioequivalence studies.

A simple and rapid determination of valproic acid in human plasma using a non‐porous silica column and liquid chromatography with tandem mass spectrometric detection

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay was developed and validated to determine valproic acid in human plasma. The method involved a solid-phase extraction of valproic acid and betamethasone valerate, an internal standard, from plasma and detection using an LC-MS/MS system with electrospray ionization source in negative ion mode. Separation was achieved within 3 min on a non-porous silica column with mobile phase containing ammonium acetate and methanol. Multiple reaction monitoring was utilized for detection monitoring at 142.89-142.89 for valproic acid and 457.21-457.21 for the internal standard. The calibration curve for valproic acid was linear over the range of 0.5-150 microg/mL. The limit of detection was 0.17 microg/mL and the lower limit of quantification was 0.5 microg/mL, when 0.2 mL plasma was used for extraction. The percentage coefficient of validation for accuracy and precision (inter- and intra-day) for this method was less than 9.5% with recovery ranging from 82.3 to 86.9% for valproic acid.

Development of water‐phase derivatization followed by solid‐phase microextraction and gas chromatography/mass spectrometry for fast determination of valproic acid in human plasma

In this study, a simple, rapid, and sensitive method was developed and validated for the quantification of valproic acid (VPA), an antiepileptic drug, in human plasma, which was based on water-phase derivatization followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS). In the proposed method, VPA in plasma was rapidly derivatized with a mixture of isobutyl chloroformate, ethanol and pyridine under mild conditions (room temperature, aqueous medium), and the VPA ethyl ester formed was headspace-extracted and simultaneously concentrated using the SPME technique. Finally, the analyte extracted on SPME fiber was analyzed by GC/MS. The experimental parameters and method validations were studied. The optimal conditions were obtained: PDMS fiber, stirring rate of 1100 rpm, sample temperature of 80 degrees C, extraction time of 20 min, NaCl concentration of 30%. The proposed method had a limit of quantification (0.3 microg/mL), good recovery (89-97%) and precision (RSD value less than 10%). Because the proposed method combined a rapid water-phase derivatization with a fast, simple and solvent-free sample extraction and concentration technique of SPME, the sample preparation time was less than 25 min. This much shortens the whole analysis time of VPA in plasma. The validated method has been successfully used to analyze VPA in human plasma samples for application in pharmacokinetic studies. All these results show that water-phase derivatization followed by HS-SPME and GC/MS is an alternative and powerful method for fast determination of VPA in biological fluids.

Development and validation of a sensitive assay of valproic acid in human plasma by high-performance liquid chromatography without prior derivatization

Sensitive and selective determination of valproic acid in plasma by high-performance liquid chromatography (HPLC) is usually achieved with pre-column derivatization. In the present work, the derivatization is omitted due to using a simple but highly selective plasma extraction procedure and an optimized chromatographic condition. Valproic acid and the internal standard octanoic acid were extracted from plasma samples with n-hexane under acidic condition followed by back-extraction into diluted triethylamine. Chromatography was performed on a CN column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (30:70, v/v), pH 3.5. Detection was made at 210 nm and analyses were run at a flow-rate of 1 ml/min. The method was specific and sensitive with a quantification limit of 1.25 microg/ml and a detection limit of 0.1 microg/ml in plasma. The mean absolute recovery for valproic acid using the present plasma extraction procedure was 75.8%. The intra- and inter-day coefficient of variation and percent error values of the assay method were all in acceptable range. Calibration curves were linear (r>0.999) from 1.25 to 320 microg/ml in plasma.

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high‐performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).

Liquid chromatography/electrospray ionization mass spectrometry method for the quantification of valproic acid in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the quantification of valproic acid, an antiepileptic drug, in human plasma using benzoic acid as internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the single ion monitoring mode using the respective [M-H]- ions, m/z 143 for valproic acid and m/z 121 for the IS. The assay exhibited a linear dynamic range of 0.5-60 microg/mL for valproic acid in human plasma. The lower limit of quantification was 500 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of valproic acid and the IS from spiked plasma samples were 96.1+/-4.2 and 95.6+/-2.7%, respectively. A run time of 4.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.

Simple and sensitive fluorimetric liquid chromatography method for the determination of valproic acid in plasma

A simple and sensitive liquid chromatographic method is described for the analysis of valproic acid in human plasma. The method is based on the derivatization of valproic acid extracted from acidified plasma with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative is highly responsive to a fluorimetric detector (excitation at 230 nm and emission at 350 nm), giving a low detection limit of 0.6 μM (S/N = 3, 10 μl injected). The relative standard deviations of the method for intra- and inter-day analyses ( n = 5) are below 3.3 and 4.1%, respectively. Toluene was used for the extraction of valproic acid from plasma and the toluene extract obtained was subjected to subsequent derivatization without solvent replacement. The simple method was applied to the analysis of valproic acid in plasma of dosed patients using only small amount of sample (10–50 μl plasma).

DETERMINATION OF VALPROIC ACID IN HUMAN PLASMA BY HPLC WITH FLUORESCENCE DETECTION

ABSTRACT A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of valproic acid in human plasma. The method is based on pre-column derivatization using a new fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The internal standard for the assay procedure was cyclohexanecarboxylic acid. The optimum monitoring conditions and the stability of the derivatives were investigated. The derivatization reaction proceeds in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30C with a reaction time of 30 min. The resulting derivatives were separated under isocratic conditions (acetonitrile–water, 60 : 40, v/v) on a LiChrospher RP-18 column (125 × 4.0 mm, i.d. 5 μm) and were detected fluorimetrically at a wavelength of 435 nm with an excitation of 345 nm. All chromatographic experiments were carried out at a flow rate 1.0 mL/min at ambient temperature. The method was fully validated and applied for the determination of valproic acid over the concentration range of 6.0–150.0 mg/mL. The detection limit for valproic acid added to plasma sample was 0.13 μg/mL plasma (3 pg on column). The recovery of valproic acid averaged 100.4%. The accuracy of the assay ranged from −0.5% to 1.3%. The method was shown to be highly reproducible and it seems to be adequate for routine therapeutic drug monitoring.