Plasma Valproic Acid Concentrations Research Articles

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs

1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug–drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs.2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change.3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog.4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

Effects of chronic administration of valproic acid to epileptic patients on coagulation tests and primary hemostasis.

Valproic acid (VPA) is an antiepileptic drug that has been associated with impaired hemostasis and increased risk for postsurgical bleeding. However, the published reports provide controversial results. We measured parameters of primary hemostasis in VPA-treated patients with epilepsy, focusing on adenosine nucleotide-dependent platelet responses, which play a central role in primary hemostasis. We enrolled 20 cases (epileptic patients receiving treatment with VPA) and 20 controls (12 epileptic patients receiving treatment with drugs different from VPA and 8 healthy subjects). Measurements included prothrombin time (PT), activated partial thromboplastin time (APTT), platelet count, platelet function analyzer (PFA)-100 closure times, plasma von Willebrand factor levels, platelet content of ADP, ATP, and serotonin (all stored in platelet dense granules), and platelet shape change and aggregation induced by ADP and other platelet agonists, including the ATP analog α,β-methylene-ATP. The plasma concentration of VPA was in the therapeutic range in 17 patients and slightly above the upper limit in 3 patients. There were no statistically significant differences in any of the studied parameters in cases versus controls. Our thorough controlled study failed to show that chronic treatment with VPA induces significant abnormalities of coagulation and primary hemostasis. Therefore, VPA, when present in the circulation in the therapeutic range, does not impair hemostasis.

Association of LEPR and ANKK1 Gene Polymorphisms with Weight Gain in Epilepsy Patients Receiving Valproic Acid.

Background:Weight gain is the most frequent adverse effect of valproic acid (VPA) treatment, resulting in poor compliance and many endocrine disturbances. Similarities in the weight change of monozygotic twins receiving VPA strongly suggests that genetic factors are involved in this effect. However, few studies have been conducted to identify the relevant genetic polymorphisms. Additionally, the causal relationship between the VPA concentration and weight gain has been controversial. Thus, we investigated the effects of single nucleotide polymorphisms (SNPs) in several appetite stimulation and energy homeostasis genes and the steady state plasma concentrations (Css) of VPA on the occurrence of weight gain in patients.Methods:A total of 212 epilepsy patients receiving VPA were enrolled. Nineteen SNPs in 11 genes were detected using the Sequenom MassArray iPlex platform, and VPA Css was determined by high-performance liquid chromatography (HPLC).Results:After 6 months of treatment, 20.28% of patients were found to gain a significant amount of weight (weight gained ≥7%). Three SNPs in the leptin receptor (LEPR), ankyrin repeat kinase domain containing 1 (ANKK1), and α catalytic subunit of adenosine monophosphate-activated protein kinase (AMPK) showed significant associations with VPA-induced weight gain (p < 0.001, p = 0.017 and p = 0.020, respectively). After Bonferroni correction for multiple tests, the genotypic association of LEPR rs1137101, the allelic association of LEPR rs1137101, and ANKK1 rs1800497 with weight gain remained significant. However, the VPA Css in patents who gained weight were not significantly different from those who did not gain weight (p = 0.121).Conclusions: LEPR and ANKK1 genetic polymorphisms may have value in predicting VPA-induced weight gain.

Derivatization oriented strategy for enhanced detection of valproic acid and its metabolites in human plasma and detection of valproic acid induced reactive oxygen species associated protein modifications by mass spectrometry

Valproic acid (VA) is a branch chain fatty acid that is widely used to treat epilepsy and convulsion. Recent studies show that VA can also be used to treat migraine headaches, bipolar disorder, and other diseases such as Alzheimer disease. However, clinical treatment with VA may cause hepatotoxicity, bone marrow suppression, and hyperammonemic encephalopathy. Valproic acid is also a known human teratogen. Because of the potential cytotoxic effects of VA and its major metabolite, 2-propyl 4-pentenoic acid (4-ene VA), VA plasma concentrations must be closely monitored during clinical applications of VA in order to avoid severe side effects.This study developed a derivatization oriented strategy for increasing sensitivity in detecting VA in quantities as low as 20μL and its metabolites in human plasma. After micro-scale liquid-liquid extraction (MLLE) and micro-scale derivatization, VA and 4-ene VA were quantitated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The linear ranges were 10-1000μM for VA and 5-500μM for 4-ene VA. All relative standard deviation (RSD) and relative error (RE) values obtained in intra- and inter-day analyses of VA and 4-ene VA were below 8%. The structures of VA and its metabolite derivatives were further identified by nano ultra performance liquid chromatographic system (nanoUPLC) coupled with tandem mass spectrometry (MS/MS). Since protein modifications induced by VA were also identifiable by nanoUPLC-MS/MS, these modifications may be useful biological indicators of a toxic reaction during clinical applications of VA.

Risk factors of hyperammonemia in patients with epilepsy under valproic acid therapy.

Hyperammonemia has been reported to be associated with patients who receive valproic acid (VPA) therapy. This study aimed to determine the risk factors for hyperammonemia in patients with epilepsy treated with VPA. One hundred and fifty-eight adult patients with epilepsy aged older than 17 years who received VPA therapy were enrolled into this study. Blood samples were taken during the interictal state and analyzed for the blood level of ammonia. Statistical analysis was conducted between different groups of patients. The results showed that the frequency of hyperammonemia associated with VPA therapy was 27.8% (ammonia level >93 µg/dL), and 5.1% of the patients had severe hyperammonemia (ammonia level >150 µg/dL). The blood ammonia level was significantly correlated with the dosage of VPA and the plasma concentration of VPA. An increase of 1 mg in the dosage of VPA increased the risk of hyperammonemia by 0.1%. In addition, combination treatment with liver enzyme inducing antiepileptic drugs (AEDs) and antipsychotic drugs increased the risk of hyperammonemia. In conclusion, the use of VPA in adult patients with epilepsy was associated with a dose-dependent increase in blood concentrations of ammonia. Combination treatment with liver enzyme-inducing AEDs and antipsychotic drugs increased the risk of VPA-induced hyperammonemia. Most of the patients with VPA-induced hyperammonemia were asymptomatic; however, if patients taking VPA present with symptoms such as nausea, fatigue, somnolence, ataxia, and consciousness disturbance, the blood ammonia level should be measured.

Influence of Uridine Diphosphate Glucuronosyltransferase 2B7 −161C&gt;T Polymorphism on the Concentration of Valproic Acid in Pediatric Epilepsy Patients

Valproic acid (VPA) is widely used to treat various types of epilepsy. Interindividual variability in VPA pharmacokinetics may arise from genetic polymorphisms of VPA-metabolizing enzymes. This study aimed to examine the relationships between plasma VPA concentrations and the -161C>T single nucleotide polymorphism in uridine diphosphate glucuronosyltransferase (UGT) 2B7 genes in pediatric epilepsy patients. This study included 78 pediatric epilepsy patients carrying the cytochrome P450 (CYP) 2C9*1/*1 genotype and who were not treated with the enzyme inducers (phenytoin, phenobarbital, and carbamazepine), lamotrigine, and/or topiramate. CYP2C9*3 and UGT2B7 -161C>T polymorphisms were identified using methods based on polymerase chain reaction-restriction fragment length polymorphism. Blood samples were drawn from each patient under steady-state conditions, and plasma VPA concentrations were measured. Significant differences in adjusted plasma VPA concentrations were observed between carriers of CC, CT, and TT genotypes in the UGT2B7 -161C>T polymorphism (P = 0.039). Patients with the CC genotype had lower adjusted plasma VPA concentrations than those with CT or TT genotype (P = 0.028). These data suggest that the UGT2B7 -161C>T polymorphism in pediatric epilepsy patients carrying the CYP2C9*1/*1 genotype affects VPA concentration.

The effect of hyperglycemia on the pharmacokinetics of valproic acid studied by high-performance liquid chromatography with electrochemical detection

The effects of hyperglycemia on the pharmacokinetics of valproic acid (VPA) were examined by time-concentration profiles of plasma VPA accompanied with blood glucose (BG) changing. In addition, time-concentration profiles of plasma free fatty acids (FFAs) were also obtained to examine the interaction between VPA and FFAs in vivo. For the experiments in vivo, normal rats, given multiple doses of maltose orally, and diabetic rats, which were made to maintain hyperglycemia, were used. Plasma VPA and FFA were determined by high-performance liquid chromatography with electrochemical detection (HPLC–ECD) systems based on the reduction of quinone for the selective determination of acids, respectively. BG was determined by pocket-size glucose meter. The maximum plasma concentrations (Cmax) of VPA in normal rats given multiple doses of maltose orally and in diabetic rats were remarkably decreased in comparison with those in the control rats. From the present study, it was shown that the metabolism of plasma VPA is accelerated under hyperglycemia. Moreover, we also found that VPA was preferentially metabolized in comparison with the plasma FFA in vivo.

Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

Sex related differences on valproic acid pharmacokinetics after oral single dose

Plasma concentration-time data obtained after an oral single dose of 500-mg valproic acid (VPA) in a delayed release formulation was used to model enterohepatic cycling of the drug. Fourteen healthy subjects (seven women and seven men) were enrolled, food intake was standardized, blood samples were withdrawn up to 48h post dosing and VPA plasma concentrations were analyzed by HPLC-UV method. Secondary peaks of VPA were observed in almost all subjects. A population pharmacokinetic analysis with sex, weight, age and contraceptive therapy as possible covariates was performed. Women without contraceptive therapy presented a longer lag time, a lower VPA hepatic output and a higher reabsorbed fraction than men. Weight affected both volume of the central compartment and the absorption rate constant. Women under contraceptive therapy showed similar disposition parameters to men. Reabsorbed fraction of bioavailable dose was 46.2% in women and 21.8% in men, revealing important sex differences in drug hepatobiliar output. These results showed that VPA displays sex-related pharmacokinetic differences due to different metabolic and transporter-mediated disposition.

HPLC DETERMINATION OF VALPROIC ACID IN PLASMA BY CONJUGATION TO A HYDRAZIDE

We developed a method to measure plasma levels of valproic acid (VA) using pre-column conjugation of VA to the fluorescent probe, 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH). Swine plasma was spiked with varying concentration of VA. 2-phenylbutyric acid (2PB) was used as an internal standard. VA and 2PB were extracted from plasma (10 µL) using NaCl, MeCN, and ethyl acetate. Extraction efficiency ranged from 85–92%. After conjugation to DCCH, the VA and 2BP, conjugates were easily separated on reverse phase HPLC using a 55–70% MeCN/H2O gradient. Standard curves were generated from the absorbance (λ = 430 nm) and fluorescence (λex = 415 nm, λem = 465 nm) that described an exponential curve y-a(1-bx) with correlation coefficients >0.97. The inter-assay variation measured over 12 days was <3% for the high pool (5 mg/mL) and ≤10% for the low pool (0.5 mg/mL). The intra-assay variation measured over 5 replicates/day was <3.5% for both the high and low pools. The calculated standard curves predicted the actual values of the standards. This assay can be used to measure plasma concentrations of VA in patients or laboratory animals being treated with VA.

Circadian variation of Valproic acid pharmacokinetics in mice

Valproic acid (VPA) is currently one of the most commonly used antiepileptic drugs. This study aims to investigate whether VPA pharmacokinetics varied according to circadian dosing-time. A single dose of VPA (350mgkg−1) was administered by intraperitonally (i.p.) route to a total of 132 mice synchronized for 3weeks to 12h light (rest span) and 12h dark (activity span). Four different circadian times (1, 7, 13 and 19 HALO) of drug injection were used (33 mice/circadian time). At each circadian time, blood samples were withdrawn at (0h) and at 0.083, 0.166, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2 and 3h after VPA injection. Plasma VPA concentrations were determined by an EMIT method.There were no significant differences in Tmax of VPA whatever the circadian-time of injections (Tmax=0.166h). However, there were relevant differences in Cmax between the four circadian groups (p<0.005), it varied between 386±30.86mgL−1 in mice treated at 7 HALO and 824±39.85mgL−1 in mice treated at 19 HALO. The AUC0–∞ was significantly two times higher when VPA was administered at 19 HALO as compared to the injection at 7 HALO. Drug dosing at 7 HALO resulted in highest ClT value: 0.405±0.006Lh−1kg−1, whereas ClT was significantly slower when VPA was administered at 19 HALO (0.157±0.009Lh−1kg−1) (p<0.0001). The AUC0–∞ was significantly 2-fold higher when VPA was administered at 19 HALO (2216.65±138.91mgh−1L−1) as compared to the injection at 7 HALO (864.09±16.82mgh−1L−1) (p<0.0001).Cosinor analysis showed circadian rhythm in different pharmacokinetic parameters. Cmax and AUC0–∞ have a significant circadian rhythm with an acrophase located at 20.16 HALO±0.16h (the middle of the active span) (p<0.001), whereas ClT and Vd showed a significant circadian rhythm with an acrophase located respectively at 7.86 HALO±0.57h and 6.13 HALO±0.07h (the middle of the rest span) (p<0.001).The large circadian variation of VPA pharmacokinetic processes might be involved in the mechanisms of circadian rhythm in murine toxicity since the optimal tolerance corresponded to the time which induces lowest Cmax and AUC values.

Removal of Valproic Acid by Plasmapheresis in a Patient Treated for Multiple Sclerosis

We present a case of a patient with multiple sclerosis who was treated with plasmapheresis and valproic acid. We used therapeutic drug monitoring to determine whether plasma concentrations of valproic acid were kept within the therapeutic window and to determine the amount of valproic acid that was removed by plasmapheresis.

Comparison of Two Formulations of Sodium Divalproate Plasma Concentrations after a Single 500 mg Oral Dose in Healthy Subjects, and Stochastic Sub-analysis of the Individual ?Clinical Perceptible? Levels

A new generic formula containing 500 mg of sodium divalproate in enteric releasing coated tablets was compared to the reference product in a pharmacokinetic, randomized, cross-over 2×2 study performed in 24 healthy adult males. After administering a single dose, it was demonstrated that the maximum plasma concentration of valproic acid varies within the 98.4-113.0 range for 90% CI (test/reference); and the area under the curve of plasma levels over the 24 h period following the intake varies within the 91.1-99.5 range; therefore, it can be seen that both parameters are within the range of acceptance for the interchangeability of the products. Individual results show the typical inter- subject and inter -treatment variations of divalproate. Since the latter have an impact on the prescription decision, results are sub-analyzed considering a plasma threshold of valproic acid as effective or stochastically clinical perceptible (Cper).Complete duration of plasma levels above Cper is comparable for both formulations (p<0.81); nevertheless, the test product reaches the Cper value faster than the reference compound (p<0.04). Therefore, we consider that the individual perception of efficacy of the generic product should not be inferior to that of the reference. In conclusion, the tested formula is bioequivalent and the further sub-analysis of individual variations above a plasma level of stochastic clinical perception of effects shows non-relevant differences between both brands.

Reduced Valproic Acid Serum Concentrations Due to Drug Interactions With Carbapenem Antibiotics

The plasma concentrations of valproic acid (VPA) are known to decrease during the concomitant administration of carbapenem antibiotics, such as meropenem, imipenem, and ertapenem. This study summarizes 6 cases of drug-drug interactions between VPA and carbapenem antibiotics. To investigate the onset and severity of the reductions in the concentration of VPA in patients with or without the coadministration of carbapenem antibiotics, the authors performed a retrospective evaluation of therapeutic drug monitoring (TDM) reports that described a decrease in the serum concentrations of VPA during the concomitant use of carbapenem antibiotics from January 2008 to December 2010 in the Seoul National University Hospital. The evaluated TDM reports included 6 cases. The decrement ratio of the VPA serum concentration was calculated from the TDM reports, and the change in the half-life of the VPA was also estimated. Six cases presented with changes in the VPA serum concentration before and after the administration of carbapenem antibiotics. (Three cases were treated with meropenem, 2 were treated with ertapenem, and 1 was treated with imipenem.) The VPA concentrations reduced by (mean ± SD) 88.7 ± 5.3% (3 cases of meropenem), 74.0 ± 9.8% (2 cases of ertapenem), and 73.3% (1 case of imipenem), respectively, and the half-life of VPA reduced by 80.1 ± 9.0%, 64.4 ± 24.2%, and 50.6%, respectively. The interaction between VPA and carbapenem antibiotics caused decreases in the VPA serum concentrations; the extent of this decrease was greater in the meropenem-treated patients than in the imipenem-treated or ertapenem-treated cases. Because the therapeutic effect of VPA depends on its serum concentration, it should be recognized that there may be a loss of seizure control in patients using VPA with carbapenem antibiotics.

The effect of combination therapy on the plasma concentrations of traditional antiepileptics

The present study aimed at determining the differences in plasma concentrations of traditional antiepileptics such as phenytoin, carbamazepine, and valproic acid in patients receiving monotherapy and combination therapy. In addition, the effect of gender and age on plasma drug concentration was evaluated in these patients. For this purpose, plasma trough concentrations obtained during routine therapeutic monitoring of these drugs were assessed retrospectively. The average plasma concentrations reached the apparent therapeutic ranges, except for the average plasma concentration of phenytoin, which was below the therapeutic range in patients who received only phenytoin or in combination with the other agents. Phenytoin when combined with carbamazepine or valproic acid significantly decreased the average plasma concentrations of these drugs to subtherapeutic concentrations. The results showed that plasma carbamazepine concentrations were higher in men than in women, whereas plasma concentrations of valproic acid and phenytoin were higher in women than in men. The difference in this regard between men and women was found to be statistically significant for phenytoin. The difference between the average plasma concentrations of carbamazepine, phenytoin, and valproic acid among age groups was not significant. In conclusion, our study measured the average plasma antiepileptic drug concentrations in patients with epilepsy who were receiving monotherapy and combination therapy and were routinely monitored, and has thus shown the importance of drug monitoring in the evaluation of the effectiveness of these drugs.

Influence of UDP-glucuronosyltransferase polymorphisms on valproic acid pharmacokinetics in Chinese epilepsy patients

The aim of this study was to investigate the genetic polymorphisms of UGT1A3, UGT1A6, and UGT2B7 in Chinese epilepsy patients and their potential influence on the pharmacokinetics of valproic acid (VPA). The genetic architectures of UGT1A3, UGT1A6, and UGT2B7 in 242 epilepsy patients were detected by DNA sequencing and PCR-restriction fragment length polymorphism. Steady-state plasma concentrations of VPA in 225 patients who had received VPA (approx. 250-1,000mg/day) for at least 2 weeks were determined and associated with UGT polymorphisms. The allelic distribution of UGT1A3 in our Chinese epilepsy patients was significantly different from that in healthy subjects based on reference data. The standardized trough plasma concentration (C(S)) of VPA was much lower in our patients with the UGT1A3*5 variant than in the wild type carriers (3.24 ± 1.05 vs. 4.68 ± 1.24μg·kg·mL(-1)·mg(-1), P < 0.01). UGT polymorphisms had no influence on the pharmacokinetic interactions between carbamazepine and VPA. Our results suggest that UGT1A3*5 may be an important determinant of individual variability in the pharmacokinetics of VPA and that it may be necessary to increase VPA dose for UGT1A3*5 carriers to ensure its therapeutic range of 50-100μg/mL.

Relationship between Patient Demographic Characteristics, Valproic Acid Dosage and Clearance in Adult Iranian Patients.

As there are conflicting findings regarding the clearance-dose and patient characteristics relationships for valproic acid (VPA), this study was conducted to investigate the relationship between patient demographic characteristics, VPA dosage and the drug clearance in adult Iranian patients. Patients (N= 47) were either on monotherapy with VPA or were under co-treatment with drugs that have no effect on VPA pharmacokinetic (PK) profile. All of the patients received VPA at therapeutic dose. Steady state trough plasma concentrations of VPA were determined by Fluorescence Polarization Immunoassay (FPIA) and VPA apparent clearance (CL/F) were calculated in each patient. Mean VPA dose and VPA CL/F were 8.93±2.2 mg/kg/day and 0.65±0.55 l/hr respectively. No significant correlations were found between VPA CL/F and patients' age, TBW and VPA dose. VPA CL/F values of male and female patients were compared and no significant difference between these two groups was noted (P> 0.05). Significant correlation between VPA dose and total trough plasma concentration was found (P= 0.001). Mean total VPA plasma concentration was 54.51±23.74 mg/l. Our study showed PK of VPA was not affected by age, sex, TBW and VPA dose. However, for detailed results and construction of VPA PK model in Iranian patients, it is necessary to evaluate VPA PK in a larger sample size with different VPA doses, age and TBW ranges.

Effects of UGT1A6, UGT2B7, and CYP2C9 genotypes on plasma concentrations of valproic acid in Chinese children with epilepsy

Effects of UGT1A6, UGT2B7, and CYP2C9 genotypes on plasma concentrations of valproic acid in Chinese children with epilepsy

Effects of UGT1A6, UGT2B7, and CYP2C9 Genotypes on Plasma Concentrations of Valproic Acid in Chinese Children with Epilepsy

Valproic acid (VPA) is one of the most commonly prescribed drugs for the treatment of epilepsy. Interindividual variability in VPA dose and plasma concentration may reflect functional consequences of genetic polymorphisms in genes encoding drug-metabolizing enzymes. The aim of this study was to determine the relationship between plasma concentrations of VPA and single nucleotide polymorphisms (SNPs) involving uridine diphosphate glucuronosyltransferase (UGT) 1A6 (UGT1A6), UGT2B7, and cytochrome P450 2C9 (CYP2C9) genes in Chinese children with epilepsy. UGT1A6, UGT2B7, and CYP2C9 polymorphisms were identified by the polymerase chain reaction-restriction fragment length polymorphism approach or direct automated DNA sequencing in 98 epileptic patients treated with VPA monotherapy. Patients with double heterozygosities at nucleotide positions T19G, A541G and A552C in the UGT1A6 gene, were associated with higher VPA doses compared to those with wild type or single heterozygosity (p = 0.010). Lower adjusted plasma VPA concentrations were also observed in patients with UGT1A6 double heterozygosities than those with single heterozygosity (p = 0.027). There were no differences in VPA dose or adjusted plasma VPA concentrations among the UGT2B7*2 or CYP2C9*3 genotypic groups. These results suggest that UGT1A6 mutations affect VPA metabolism in epileptic children. It needs to be further investigated in a larger cohort of patients.

LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC–MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C 8 column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol-10 mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.