Total Valproic Acid Concentrations Research Articles

Association of Valproic Acid and Its Main Metabolites' Plasma Concentrations with Clinical Outcomes among Epilepsy Patients: A 10-Year Retrospective Study Based on Therapeutic Drug Monitoring.

Valproic acid (VPA) is a first-line antiepileptic drug with broad efficacy. Due to significant individual differences in its metabolism, therapeutic drug monitoring is commonly used. However, the recommended therapeutic range (50-100 μg/mL) is inadequate for predicting clinical outcomes. Additionally, the relationship between VPA metabolites and clinical outcomes remains unclear. In this retrospective study, 485 Chinese Southern Han epilepsy patients receiving VPA monotherapy were analyzed after reaching steady-state levels. Plasma concentrations of VPA and its five main metabolites were determined by liquid chromatography-mass spectrometry (LC-MS). We assessed the relevance of the recommended therapeutic VPA range for clinical outcomes and explored the association between VPA/metabolites levels and treatment efficacy/adverse effects. Vitro experiments were conducted to assess 4-ene-VPA hepatotoxicity. The therapeutic range of VPA exhibited no significant correlation with clinical outcomes, and plasma concentrations of VPA failed to serve as predictive indicators for treatment response/adverse effects. Treatment responders had higher 2-PGA concentrations (median, 26.39 ng/mL versus 13.68 ng/mL), with a threshold of 36.5 ng/mL for optimal epilepsy treatment. Patients with abnormal liver function had a higher 4-ene-VPA median concentration (6.41 μg/mL versus 4.83 μg/mL), and the ratio of 4-ene-VPA to VPA better predicted VPA-induced hepatotoxicity (area under the curve, 0.718) than 4-ene-VPA concentration. Vitro experiments revealed that 4-ene-VPA was more hepatotoxic than VPA in HepaRG and L02 cell lines. Total plasma VPA concentration does not serve as a predictor of clinical outcomes. 2-PGA concentrations may be associated with efficacy, whereas the ratio of 4-ene-VPA to VPA may be considered a better biomarker (threshold 10.03%) for VPA-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This was the first and largest observational cohort in China to explore the relationship between patients' parent and metabolites concentrations of VPA and clinical outcomes during the maintenance of VPA monotherapy in epileptic patients. This study provided feasible references of VPA for epilepsy clinical treatment with a larger sample of patients compared with previous studies for a more definitive conclusion based on real-world situations. We found two potential biomarkers in predicting efficacy and liver injury, respectively. This breakthrough has the potential to assist in the rational use of VPA.

Prediction of systemic free and total valproic acid by off-line analysis of exhaled breath in epileptic children and adolescents.

Therapeutic drug monitoring (TDM) of medications with a narrow therapeutic window is a common clinical practice to minimize toxic effects and maximize clinical outcomes. Routine analyses rely on the quantification of systemic blood concentrations of drugs. Alternative matrices such as exhaled breath are appealing because of their inherent non-invasive nature. This is especially the case for pediatric patients. We have recently showcased the possibility of predicting systemic concentrations of valproic acid (VPA), an anti-seizure medication by real-time breath analysis in two real clinical settings. This approach, however, comes with the limitation of the patients having to physically exhale into the mass spectrometer. This restricts the possibility of sampling from patients not capable or available to exhale into the mass spectrometer located on the hospital premises. In this work, we developed an alternative method to overcome this limitation by collecting the breath samples in customized bags and subsequently analyzing them by secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). A total ofn= 40 patients (mean ± SD, 11.5 ± 3.5 y.o.) diagnosed with epilepsy and taking VPA were included in this study. The patients underwent three measurements: (i) serum concentrations of total and free VPA, (ii) real-time breath analysis and (iii) off-line analysis of exhaled breath collected in bags. The agreement between the real-time and the off-line breath analysis methods was evaluated using Lin's concordance correlation coefficient (CCC). CCC was computed for ten mass spectral predictors of VPA concentrations. Lin's CCC was >0.6 for all VPA-associated features, except for two low-signal intensity isotopic peaks. Finally, free and total serum VPA concentrations were predicted by cross validating the off-line data set. Support vector machine algorithms provided the most accurate predictions with a root mean square error of cross validation of 29.0 ± 7.4 mg l-1and 3.9 ± 1.4 mg l-1for total and free VPA (mean ± SD), respectively. As a secondary analysis, we explored whether exhaled metabolites previously associated with side-effects and response to medication could be rendered by the off-line analysis method. We found that five features associated with side effects showed a CCC > 0.6, whereas none of the drug response-associated peaks reached this cut-off. We conclude that the clinically relevant free fraction of VPA can be predicted by this combination of off-line breath collection with rapid SESI-HRMS analysis. This opens new possibilities for breath based TDM in clinical settings.

A novel method for predicting the unbound valproic acid concentration

In this study, we constructed a prediction formula for unbound valproic acid (VPA) concentration that was more accurate and widely applicable than previously reported formulae. A total of 136 datasets from 75 patients were analyzed retrospectively. The median of free fraction of VPA was 0.16 (interquartile range: 0.07; range: 0.07–0.45). The parameter that combined total VPA concentration (CtVPA) and serum albumin (SA), (CtVPA [μM] – 2 × SA [μM]), was significantly related to the free fraction of VPA (r = 0.76, p < 0.001). We constructed a combined parameter-based prediction formula for unbound VPA concentration. Analysis using external datasets from patients without severe renal failure showed that the prediction errors of the unbound VPA concentration were lower than those of previously reported formulae. Although the previous formulae showed large prediction errors, especially in the specific range of CtVPA values, the constructed formula showed a weak trend with CtVPA or SA. The formula based on (CtVPA [μM] – 2 × SA [μM]) had high prediction accuracy and wide applicability in predicting the unbound VPA concentration in patients without severe renal failure.

Determination of Free Valproic Acid Concentration in 569 Clinical Samples by LC-MS/MS After Hollow Fiber Centrifugal Ultrafiltration Treatment.

To perform therapeutic drug monitoring of total and free plasma valproic acid (VPA) concentrations in clinical samples and to analyze the related factors. The total VPA concentration in plasma was determined by ultrahigh-performance liquid chromatography with precolumn derivatization with α-bromoacetophenone, and the free VPA concentration was determined by liquid chromatography-tandem mass spectrometry after the plasma was treated by hollow fiber centrifugal ultrafiltration. Regression analysis was performed to examine the associations between free plasma VPA, total plasma VPA, and the plasma protein binding rate. The impact of individual situations, outpatient or inpatient factors, and drug combinations on VPA concentrations were examined. Of the 569 clinical samples, 268 were inpatients and 301 were outpatients, and the total VPA concentration in 138 cases (24.2%) was lower than the effective treatment concentration range; the total and free VPA concentrations in outpatient samples were 11.0% and 26.1% higher than those of inpatients, respectively. There was no linear relationship between the free and total VPA concentrations. The relationship equation between the plasma protein binding rate and free VPA concentrations was as follows: Y = 0.0255X2 - 1.1357X + 97.429 (r = 0.8011). The total and free VPA concentrations were significantly decreased after the coadministration of phenobarbital (83.7% and 64.3% of the control group, P < 0.05) or carbapenem antibiotics (32.0% and 32.7% of the control group, P < 0.05). The total VPA concentrations in patients with epilepsy at our hospital was lower than the effective treatment concentration range, which was inadequate for epilepsy control; the total VPA concentrations of outpatients were higher than those of inpatients; as phenobarbital affects VPA metabolism, therapeutic drug monitoring is recommended. Carbapenem antibiotic coadministration with VPA should be avoided because carbapenem antibiotics can lead to the failure of VPA antiepileptic treatment.

Design and validation of a predictive equation to estimate unbound valproic acid concentration

ObjectivesTotal plasma levels of valproic acid (VPA) may mask an increased risk of adverse effects in hypoalbuminaemic patients since, in these patients, the free fraction is higher. The aim of...

CYP2C9*3/*3 Gene Expression Affects the Total and Free Concentrations of Valproic Acid in Pediatric Patients with Epilepsy.

PurposeTo perform therapeutic drug monitoring (TDM) of total and free plasma valproic acid (VPA) concentrations in pediatric patients with epilepsy and to analyze related factors.Patients and MethodsPediatric epileptic patients treated in 2015–2019 in our hospital were assessed. Total and free plasma VPA concentrations were obtained by UPLC and LC-MS/MS, respectively. Regression analysis was performed to examine the associations of free plasma VPA with total plasma VPA and plasma protein binding rate. The impacts of individual situation, CYP2C9 genotype, and drug combination on VPA concentration were examined.ResultsOf the 251 patients, 81 had lower total concentrations than effective therapeutic levels; 86 and 31 patients had infections and central nervous system dysplasia, respectively. VPA’s daily doses and free drug concentrations were significantly lower in the CYP2C9 *3/*3 genotype group versus the CYP2C9 *1/*3 and CYP2C9 *1/*1 groups (P<0.05). Free and total VPA concentrations were linked by Y = 0.0004 X2 + 0.042 X + 0.3035 (r=0.6981); VPA plasma protein binding rate and free VPA concentration were related by Y = 0.0003 X2 - 0.0127 X + 0.9777 (r=0.8136). Both total and free VPA concentrations were significantly decreased in patients simultaneously administered phenobarbital, meropenem and biapenem (P<0.05), with therapeutic failure after meropenem/biapenem co-administration.ConclusionFree VPA amounts have nonlinear relationships with total VPA amounts and plasma protein binding rate in epileptic children. Additionally, CYP2C9 *3/*3 expression affects VPA metabolism. Since phenobarbital affects VPA metabolism, TDM is recommended. Meanwhile, carbapenem-co-administration with VPA should be prohibited.

Population pharmacokinetics of unbound valproic acid in pediatric epilepsy patients in China: a protein binding model.

The purpose of this study was to establish a protein binding model of unbound valproic acid (VPA) based on Chinese pediatric patients with epilepsy and provide a reference for clinical medication. A total of 313 patients were included and both their total and unbound VPA concentrations (375 pairs of concentrations) were measured. NONMEM software was used for population pharmacokinetic modeling. The stepwise method was used to screen the potential covariates. Goodness-of-fit plot, bootstrap, and visual predictive check were used for model evaluation. In addition, dose recommendations for typical patients aged 0 to 16years were proposed by Monte Carlo simulations. A one-compartment model of first-order absorption and first-order elimination was used to describe the pharmacokinetic characteristics of unbound VPA, and the linear non-saturable binding equation was introduced to describe the protein binding. Body weight, age-based maturation, and co-medicated with lamotrigine could affect the CL/F of unbound and bound VPA. Model evaluation showed satisfactory robustness of the final model. The dosing regimens for children aged 0 to 16years were proposed based on the final established model. We developed a population pharmacokinetic model of unbound and bound VPA that took account of protein binding. The VPA dosing regimen in pediatric patients with epilepsy needs to be optimized by the body weight, age, and co-medications.

Prediction of Serum-Free and Cerebrospinal Fluid Valproic Acid Levels in Patients With Hypoalbuminemia After Craniotomy.

In patients with hypoalbuminemia after craniotomy, total serum concentrations of valproic acid (VPA) may provide poor clinical insights, owing to saturated protein binding and increased unbound fractions. However, very few clinical laboratories routinely analyze free concentrations of the drug. The aim of this study was to develop a model to predict serum-free and cerebrospinal fluid (CSF) levels of VPA based on its total concentration and to investigate the model's applicability. Total serum and CSF concentrations of VPA in 79 patients were measured using a validated immunoassay between January 2015 and December 2015. The demographic, clinical, and laboratory information of patients were retrieved from medical records. A multiple linear regression analysis was adopted to determine the potential variations and establish the functional relationship between CSF concentration and significant clinical factors. Based on the stepwise multiple linear regression analysis performed using the natural logarithm of the concentration of VPA in the CSF as the dependent variable, serum concentrations of VPA (X1, β' = 0.844), serum albumin concentration (X2, β' = -0.393), and CSF protein concentration (X3, β' = 0.098) were identified as the 3 variables that significantly predicted the dependent variable: (Equation is included in full-text article.), with a coefficient of determination (R) of 0.874. As the CSF protein level is often unavailable, the model was redefined to include 2 variables-serum concentrations of VPA (X1, β' = 0.840) and serum albumin concentration (X2, β' = -0.359): (Equation is included in full-text article.), with R = 0.813. Based on total VPA and serum albumin concentrations, we developed a model to predict serum-free and CSF levels of VPA. This model is useful for correcting dose adjustment in patients with hypoalbuminemia after craniotomy.

Abstract 272: Dose Optimization of Early High-Dose Valproic Acid for Neuroprotection After Cardiac Arrest

Introduction: High-dose valproic acid (VPA) has been shown to improve the survival and neurologic outcomes in preclinical brain injury models including asphyxial cardiac arrest (CA) in rats. However, its pharmacokinetics, pharmacodynamics, and safety profiles in large animal CA models are unknown. Hypothesis: High-dose VPA can be safely administered after return of spontaneous circulation (ROSC) and cross the blood-brain barrier dose-dependently in a swine CA model. Methods: After 8 minutes of untreated ventricular fibrillation CA, male Yorkshire swine [43.3 (3.0) kg] were resuscitated for up to 16 minutes until ROSC. They were randomized to receive placebo, 75 mg/kg, 150 mg/kg, or 300 mg/kg VPA as 90-minute intravenous infusion (n=5 per group) beginning 20 minutes after sustained ROSC. Animals were monitored for 2 additional hours after infusion ended and then euthanized. Experimental operators were blinded to the treatments. Results: The mean total cardiac arrest duration was 15.0 (1.6) minutes, with no significant differences between groups. At end of infusion, the serum free VPA concentration increased dose dependently, from 139.2 (10.1) mcg/mL [75 mg/kg VPA] to 287.2 (24.6) mcg/mL [150 mg/kg VPA] to 565.6 (36.5) mcg/mL [300 mg/kg VPA] (p&lt;0.05). This corresponded to a dose-dependent decrease in the fraction of serum protein-bound VPA, from 41.8% (1.8) [75 mg/kg VPA] to 23.4% (3.8) [150 mg/kg VPA] to 14.4% (3.8) [300 mg/kg VPA] (p&lt;0.05). Brain total VPA concentrations at end of experiment were 26.1 (3.1) mcg/g [75 mg/kg VPA], 72.5 (18.9) mcg/g [150 mg/kg VPA], and 212.3 (33.4) mcg/g [300 mg/kg VPA] (p&lt;0.05). There was a strong linear correlation between serum free VPA and brain total VPA concentrations (R 2 =0.98). All animals survived until euthanasia, although the 300 mg/kg group required more epinephrine to maintain mean arterial pressure greater than or equal to 80 mmHg and had higher lactic acidosis. Conclusion: Brain VPA concentrations correlate strongly with serum free VPA when high-dose intravenous VPA is given after ROSC in a swine CA model. These results provide the foundation for dose-optimization studies of high-dose VPA as a neuroprotective therapy following resuscitation from CA.

Accuracy of Valproic Acid Concentration Correction Based on Serum Albumin.

Patient-specific factors can alter the pharmacokinetic disposition of valproic acid. Specifically, the free fraction of valproic acid can increase substantially in patients with hypoalbuminemia or as serum drug concentrations rise due to saturable protein binding. Direct measurement of free serum drug concentrations allows for accurate assessment of drug levels, but the assay may not be readily available in all institutions. The effect of hypoalbuminemia on free fraction has been quantified and serves as the basis of an equation used to "correct" measured total valproic acid concentrations. The aim of this study was to evaluate the accuracy of the equation. This retrospective study included adult patients with measurable free and total valproic acid concentrations between July 2014 and June 2017. The primary aim was to assess the relationship between measured and predicted free valproic acid concentrations. Free levels were categorized as subtherapeutic, therapeutic, or supratherapeutic based on the reference range of 7-23mg/L. Concordance was defined as measured and predicted concentrations falling within the same category. The analysis included 174 patients with a median age of 58years and a median albumin of 3g/dL. The majority of patients were hospitalized (88.5%). Concordance occurred in 56.9% of samples. A Spearman's correlation coefficient of 0.60 (p < 0.001) was found between the measured and predicted free valproic acid concentrations. Concordance of concentrations was 42% for ICU patients, 63% for floor patients, and 65% for outpatients. Of those with discordant concentrations, 97% of the predicted concentrations underestimated the measured concentrations. There is discordance between predicted and measured free serum valproic acid concentrations when using the proposed equation. Because of the potential impact of underestimation and variability of free valproic acid concentrations, a measured free level is the ideal option for therapeutic drug monitoring of valproic acid.

Development of a Simple and Rapid Method to Measure Free Fraction of Valproic Acid in Plasma Using Ultrafiltration and Ultra High Performance Liquid Chromatography–Mass Spectroscopy: Application to Therapeutic Drug Monitoring

Valproic acid (VPA) is widely used as an antiepileptic drug in children, and it is usually coadministered with other antiepileptic drugs. Because of its narrow therapeutic range and large variations in pharmacokinetic/pharmacodynamic behavior in different individuals, therapeutic drug monitoring of the trough total VPA concentration is commonly used to guide dosing. However, as only the free fraction of VPA that exerts pharmacological and toxic effects, it may be more meaningful to determine the unbound VPA concentration in plasma. Free fraction of VPA in plasma was extracted by ultrafiltration. Ultra high performance liquid chromatography coupled with mass spectroscopy was used to measure VPA in the negative ionization mode. This method was validated by studies of its selectivity, linearity, lower limit of quantification, accuracy, precision, recovery, matrix effect, and stability. The method was validated over a linear range of 0.2-25 mcg/mL, and its lower limit of quantification was 0.2 mcg/mL. The method's relative standard deviations for intra- and inter-day precision were <15%, and its accuracy (relative error) was ±3.22%. The recoveries and matrix effect of unbound VPA at 3 different concentrations satisfied our requirements for the analysis of biological samples, and no significant degradation of VPA was observed under different storage conditions. Simple ultra high performance liquid chromatography coupled with mass spectroscopy showed good performance when used to measure unbound VPA concentration, this method may be used to study the relationship between unbound VPA concentrations and its effectiveness by the use of therapeutic drug monitoring.

Monitoring Protein-Unbound Valproic Acid Serum Concentrations in Clinical Practice.

Valproic acid (VPA) is an effective antiepileptic drug and mood stabilizer. A key characteristic of VPA is its high and saturable protein binding at higher concentrations. Although the unbound concentration of VPA is responsible for its pharmacological activity, total drug concentrations are monitored in routine clinical practice. Therapeutic drug monitoring (TDM) of unbound VPA is recommended for specific clinical situations. The goal of this study was to evaluate TDM requests for unbound VPA in clinical practice. All TDM requests at our laboratory for unbound VPA in 2014 and 2015 were evaluated retrospectively. In patients with potentially toxic unbound VPA concentrations (ie, >12 mg/L), we evaluated whether toxicity was noted and whether the dose adjustment advice was followed. Total and unbound VPA concentrations were measured by means of a validated immunoassay. A total of 273 unbound VPA serum concentrations in 132 different patients were analyzed. The main reasons for unbound VPA TDM were decreased renal function (34%) and a low serum albumin (27%). The median (range) unbound VPA concentration was 9.8 (2.5-47.6) mg/L. In 49 patients (37%), the initial unbound VPA concentration was above the threshold of 12 mg/L, potentially resulting in toxicity. Only 6 of these 49 patients had elevated total VPA concentrations. Clinical toxicity was noted in 38 of the 49 patients (77.6%) with elevated unbound VPA concentrations. Toxicities included drowsiness (n = 26), decreased consciousness (n = 4), rigidity (n = 2), and confusion (n = 2). In 36 of the 38 patients with elevated unbound VPA concentrations and clinical toxicity, a dose reduction was applied. In 27 of 36 patients who had their dose reduced, dose reduction was associated with improvement or resolution of VPA toxicity. TDM of unbound VPA is an important tool to manage VPA therapy in selected, vulnerable patients.

Reversible cognitive impairment associated with a high free fraction but subtherapeutic total blood level of valproic acid due to hypoalbuminemia in a bipolar patient

Valproic acid (VPA) is widely used in the treatment of epilepsy and bipolar disorder. It is largely bound to serum proteins (80–95%) in particular albumin, with a saturable binding capacity. Under conditions of hypoalbuminemia, protein binding of VPA will decrease and its pharmacologically-active free fraction will rise, even to toxic levels while measuring subtherapeutic VPA total blood levels [1].We present an elderly bipolar patient with (sub)clinical total levels of VPA and a high free fraction of VPA due to hypoalbuminemia (14–24 g/L) leading to severe reversible cognitive impairment.VPA and the free fraction in particular, was the most likely cause of the cognitive impairment [2]. There was a time-correlation with increasing blood levels of total VPA (68 mg/L, reference 80–120 mg/L [3]), notably the free fraction (37.5 mg/L, reference 5–15 mg/L), and the intoxication.For therapeutic drug monitoring in laboratories, generally, total VPA concentrations (free + protein-bound) are measured instead of free fractions, due to technical difficulties, a lack of established reference ranges [4] and (inter)national guidelines [5,6] not requiring it. This presentation and literature points out that it is clinically relevant to measure the free fraction [7,8], especially in patients with hypoalbuminemia [9–11] to prevent unnecessary side effects and toxicity.We recommend measuring albumin during VPA use; particularly in patients with nephrotic syndrome, liver disease [12] or older adults [13–15]. Hypoalbuminemia demands a free fraction measurement.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Comparison of Serum Total Valproic Acid Levels and %CDT Values in Chronic Alcohol Addictive Patients in an Italian Clinic: A Retrospective Study.

BackgroundValproate is a broad-spectrum anticonvulsant that is effective in the treatment of tonic-clonic, myoclonic and absence seizures as well as in partial seizures as a second-line drug. It has been widely demonstrated in the literature that the effect of valproate on type-A γ-aminobutyric acid (GABA-A) receptors may reduce relapse to ethanol abuse. This retrospective study evaluated a 3-year period in which 42 patients from the Department of Alcoholism and Substance Abuse (DASA) were treated with valproate.ObjectivesWe compared different serum total valproic acid (VPA) concentrations, and the effectiveness of this drug in maintaining alcohol abstinence was evaluated by percentage of carbohydrate deficient transferrin (%CDT) values.MethodCDT is a biochemical marker used for identifying regular high alcohol consumption and monitoring abstinence in outpatients during treatment. Serum concentrations of valproate were divided into four groups: <10, 10–30, 31–50, and >50 µg/mL.ResultsThis study shows that a mean serum total VPA concentration >30 µg/mL is more effective in maintaining alcohol abstinence than a lower one (p < 0.05). In this study, mean serum total VPA concentrations between 31 and 50 µg/mL showed the same effectiveness as higher ones (>50 µg/mL); in fact, there was no significant difference in mean %CDT values between these two groups (p > 0.05). After at least 12 months’ treatment with valproate, mean platelet counts increased by 12 × 103/μL compared with baseline (254 ± 63 vs 242 × 103/μL, p > 0.05, respectively) in patients with mean serum total VPA levels <10 μg/mL; increased by 8 × 103/μL from baseline (253 ± 59 vs 245 × 103/μL, p > 0.05, respectively) in patients with levels between 10 and 30 μg/mL; decreased by 2 × 103/μL from baseline (265 ± 63 vs 267 × 103/μL, p > 0.05, respectively) in patients with levels between 31 and 50 μg/mL, and decreased by 48 × 103/μL from baseline (215 ± 56 vs 263 × 103/μL, p < 0.05, respectively) in patients with levels >50 μg/mL.ConclusionA mean serum total concentration lower than the currently accepted therapeutic level (50–100 µg/mL) may have the same effectiveness in maintaining alcohol abstinence with a lower risk of presenting side effects.

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.

Correlation between Plasma Ammonia Level and Serum Trough Concentration of Free Valproic Acid in Patients with Epilepsy

Therapeutic drug monitoring of valproic acid (VPA) is essential to prevent toxicity, but the correlation between plasma ammonia level and serum VPA concentration remains unclear. We examined the correlation of plasma ammonia level with VPA dose and serum trough concentrations of total and free VPA in Japanese patients with epilepsy. Thirty-eight data sets from 19 Japanese patients with epilepsy were analyzed. The relations of VPA dose and serum total and free VPA concentrations with plasma ammonia level, and the breakpoints of VPA parameters predicting hyperammonemia (plasma ammonia higher than 60 µmol/L) were analyzed. A significant positive correlation was observed between plasma ammonia level and VPA dose (r(s)=0.56, p=0.00062), serum trough total VPA concentration (r(s)=0.55, p=0.00086) and serum trough free VPA concentration (r(s)=0.58, p=0.00041). The breakpoints predicting hyperammonemia were VPA dose of 30.4 mg/kg, serum trough total VPA concentration of 90.9 µg/mL, and serum trough free VPA concentration of 8.65 µg/mL, with impurity reductions at 1.35, 1.35 and 2.02, respectively. These findings suggest that serum trough concentration of free VPA is the most reliable predictor for hyperammonemia, and that the risk of developing hyperammonemia may increase in patients with serum trough free VPA concentrations higher than 8.65 µg/mL.

Apparent clearance of valproic acid in elderly epileptic patients: estimation of the confounding effect of albumin concentration

Background. Valproic acid (VPA) apparent clearance (CL) estimated from total serum concentrations is analogous in elderly and non-elderly adult patients. As drug–protein binding decreases in old age, the aim of our study was to evaluate the confounding effect of the serum albumin concentration on the VPA apparent CL in elderly patients.Methods. In 102 epileptic out-patients treated with VPA in monotherapy, serum total steady-state trough concentrations (Css) were determined. Css concentrations were normalized for a 42 g/L albumin concentration (CssN), and the apparent CL and normalized apparent CLN were calculated.Results. A poor concordance of 53% was found in the classification of Css and CssN levels of VPA as subtherapeutic, therapeutic, or supratherapeutic dose. In the elderly (≥65 years) and non-elderly adult patients, the VPA apparent CL was similar; however, normalized apparent CLN was significantly lower in older patients (P < 0.01), with a 40% median decrease.Conclusions. Total VPA concentrations should be interpreted with caution, mainly in older patients, in which determination of unbound or normalized total drug concentrations may be clinically useful. Normalization of total concentrations permits an estimation of the masking effect of serum albumin concentrations on the VPA apparent CL in elderly patients.

Comparison of Two Binding Equations for Prediction of the Concentration of Unbound Valproic Acid in the Serum of Adult Epileptic Polytherapy Patients

Because binding of valproic acid to plasma proteins affects the efficacy of the drug in the treatment of epilepsy (only the unbound fraction of the drug is effective) we have compared two methods which use different binding parameters to predict the in-vivo concentration of unbound valproic acid in serum. The study was performed on 46 serum samples from 29 polytherapy adult patients with epilepsy. Mean prediction error, mean absolute prediction error and root mean squared error were calculated for each method; these values served as a measure of prediction bias and precision. The mean absolute prediction errors and root mean squared errors for the two methods were similar in magnitude (Method 1, mean absolute prediction error = 10.0 microM, root mean squared error = 15.0 microM; Method 2, mean absolute prediction error = 10.3 microM, root mean squared error = 13.5 microM). Method 2 had a general tendency to over-predict unbound valproic acid; both methods had a tendency to over-prediction for total concentrations above 500 microM. Method 1 had a tendency to under-prediction at total concentrations below 250 microM. Within the total concentration range of valproic acid investigated, Method 1 was superior to Method 2 for prediction of unbound serum valproic acid. Our approach using Method 1 may be useful for prediction of unbound serum valproic acid concentration in patients with total valproic acid concentrations ranging from 250 to 500 microM; Method 2 may be useful for patients with total valproic acid below 500 microM. Our results suggest that there is wide and unpredictable variability in valproic acid binding to serum proteins among study populations.

High Unbound Fraction of Valproic Acid in a Hypoalbuminemic Critically Ill Patient on Renal Replacement Therapy

To describe a hypoalbuminemic critically ill patient with subtherapeutic total valproic acid serum concentrations but unbound valproic acid concentrations within normal limits. During an intensive care unit admission, a 61-year-old woman with urosepsis and multiorgan dysfunction syndrome developed tonic-clonic seizures with respiratory failure, and tracheal intubation was performed. An intravenous loading dose of valproic acid 1500 mg (25 mg/kg) was administered and therapy was continued with valproic acid 750 mg (12.5 mg/kg) twice daily. Because of progressive renal failure, continuous venovenous hemofiltration was started on day 3 of valproic acid therapy. On day 7 of valproic acid therapy, routine testing of serum valproic acid trough concentration returned as undetectable. Subsequent determinations of trough serum concentrations of total valproic acid showed values below the therapeutic range. Data from a full pharmacokinetic curve (multiple blood samples during a dosing interval) showed that the free fraction of valproic acid was >60%. Although total valproic acid concentrations were still low, the unbound concentrations were considered therapeutic. Serum albumin was 1.2 g/dL on the multiple sampling day. The patient's hypoalbuminemia probably explains the remarkably high free fraction of valproic acid. Our hypothesis is that the low albumin level was associated with high plasma clearance of valproic acid, leading to extremely low total drug concentrations. To our knowledge, this high percentage of free valproic acid has not been previously described. Health-care professionals should be aware of the need of early determination of both total and free fraction valproic acid serum concentrations in hypoalbuminemic critically ill patients. Increasing the dose of valproic acid purely based on total valproic acid serum concentrations in this patient population should be avoided.