Alterations in salivary biochemical composition and redox state disruption induced by the anticonvulsant valproic acid in male rat salivary glands.

OBJECTIVE: To investigate the effects of the anticonvulsant valproic acid (VPA) on salivary glands in male rat using biochemical, functional, histomorphometric, and redox state parameters. MATERIALS AND METHODS: Twenty-four male Wistar rats were randomly distributed into three groups (n = 8 per group): Control (0.9% saline solution), VPA100 (100 mg/kg), and VPA400 (400 mg/kg). After 21 consecutive days of treatment with by intragastric gavage. Pilocarpine-induced saliva was collected to determine salivary flow rate, pH, buffering capacity, and biochemical composition. Analyses of histomorphometric parameters and redox balance markers were performed on the parotid and submandibular glands. RESULTS: Salivary flow rate, pH, buffering capacity, total protein, potassium, sodium, and chloride were similar between groups. However, phosphate and calcium were reduced in VPA400, while amylase was increased in both VPA100 and VPA400. We did not detect significant differences in the areas of acini, ducts, and connective tissue in the salivary glands between the groups. There were no significant changes in the redox status of the submandibular glands. In turn, in the parotid glands we detected reduced total oxidizing capacity and lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARs) and higher uric acid concentration in both the VPA100 and VPA400 groups, and increased superoxide dismutase (SOD) in the VPA400 group. CONCLUSION: Chronic treatment with VPA modified the salivary biochemical composition and caused disruption in the redox state of the parotid gland in rats.

A simple and easy non-derivatization gas chromatography-mass spectrometry method for simultaneous quantification of valproic acid, gabapentin, pregabalin, and vigabatrin in human plasma.

Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.

Use of TLC-Densitometric Method for Determination of Valproic Acid in Capsules.

Determination of valproic acid in the drug was carried out on the aluminum silica gel 60F254 plates and using acetone-water-chloroform-ethanol-ammonia at a volume ratio of 30:1:8:5:11 as the mobile phase, respectively. Two methods of detection of valproic acid were used. The first was a 2% aqueous CuSO4×5H2O solution, and the second was a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system. The applied TLC-densitometric method is selective, linear, accurate, precise, and robust, regardless of the visualizing reagent used for the determination of valproic acid in Convulex capsules. It has low limits of detection (LOD) and limits of quantification (LOQ), which are equal to 5.8 µg/spot and 17.4 µg/spot using a 2% aqueous CuSO4×5H2O solution as visualizing agent and also 0.32 µg/spot and 0.97 µg/spot using a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system as visualizing reagent, respectively. The described analytical method can additionally be used to study the identity of valproic acid in a pharmaceutical preparation. The linearity range was found to be 20.00-80.00 µg/spot and 1.00-2.00 µg/spot for valproic acid detected on chromatographic plates using a 2% aqueous CuSO4×5H2O solution and the 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of valproic acid equal 96.2% and 97.0% in relation to the label claim that valproic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine analysis of valproic acid in pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography and square wave voltammetry in the control of above-mentioned substances, and it can be applied when other analytical techniques is not affordable in the laboratory.

Determination of in vitro metabolic hepatic clearance of valproic acid (VPA) and five analogues by UPLC-MS-QTOF, applicable in alternatives to animal testing.

Laboratory measurements of intrinsic clearance support the development of TK models, with potential relevance to weight of evidence toxicity assessments of xenobiotics, including read-across, the concept of predictive estimation by data extrapolation between chemicals of similar structure (analogues). In this work a procedure with analytical method for determination of in vitro hepatic metabolic clearance, relevant to biotransformation toxicokinetic (TK) modelling, is presented. Cryopreserved primary human hepatocytes represent a suitable cells, due to their biological characteristics, for providing an in vitro model for simulating in vivo metabolic clearance. The experimental part considered an adequate sequential time-frame for collecting samples and controls for all chemicals tested, including centrifugation and aliquoting of the corresponding fractions until the instrumental session. For the first time, in vitro hepatocyte intrinsic clearance was measured for six analogue test chemicals: valproic acid, 2-ethyl caproic acid, octanoic acid, valeric acid, 2-methyl butyric acid and 2-trans pentenoic acid, during incubated cell culture exposure up to 2 h or 3.5 h. The time dependence of any metabolism was determined from analysis of the supernatant at intervals using a new developed analytical method for UPLC coupled with QTOF mass spectrometer. The chemicals could then be ranked by their relative intrinsic clearance. The analyses were reproducible, with coherence of the calculated in vitro intrinsic clearance between experiments.

A case report on fatal intoxication by tapentadol: Study of distribution and metabolism.

We report a case in which a tapentadol acute intoxication was suspected as the cause of death of a 39-year-old man: approximately two days after death, cardiac and femoral blood, as well as urine, bile, gastric content and chest hair, were collected during the autopsy. Tapentadol was detected before and after hydrolysis in femoral (530 ng/mL unconjugated and 1570 ng/mL conjugated) and cardiac (680 ng/mL unconjugated and 3440 ng/mL conjugated) blood, and additionally in bile (3200 ng/mL), urine (9300 ng/mL), chest hair (2850 pg/mg) and gastric content. LC-QTOF screening analysis confirmed the presence of five different tapentadol metabolites (tapentadol-O-glucuronide, tapentadol-O-sulfate, N-desmethyltapentadol, N-desmethyltapentadol-glucuronide and N-desmethyltapentadol-O-sulfate), in urine, bile, cardiac and femoral blood. Positivity of body hairs allowed us to conclude that the man had used tapentadol in the last weeks/months. Autopsy and toxicological results (also positive for clotiapine, diazepam and chlordesmethyldiazepam) suggested that tapentadol could have caused, even at low concentrations, a severe respiratory depression, which contributed to the death of the subject. This is one of the few cases in literature where tapentadol was detected in blood, together with its metabolites, and the only one in which the parent drug was identified in hairs.

Handling deviating control values in concentration-response curves.

In cell biology, pharmacology and toxicology dose-response and concentration-response curves are frequently fitted to data with statistical methods. Such fits are used to derive quantitative measures (e.g. EC[Formula: see text] values) describing the relationship between the concentration of a compound or the strength of an intervention applied to cells and its effect on viability or function of these cells. Often, a reference, called negative control (or solvent control), is used to normalize the data. The negative control data sometimes deviate from the values measured for low (ineffective) test compound concentrations. In such cases, normalization of the data with respect to control values leads to biased estimates of the parameters of the concentration-response curve. Low quality estimates of effective concentrations can be the consequence. In a literature study, we found that this problem occurs in a large percentage of toxicological publications. We propose different strategies to tackle the problem, including complete omission of the controls. Data from a controlled simulation study indicate the best-suited problem solution for different data structure scenarios. This was further exemplified by a real concentration-response study. We provide the following recommendations how to handle deviating controls: (1) The log-logistic 4pLL model is a good default option. (2) When there are at least two concentrations in the no-effect range, low variances of the replicate measurements, and deviating controls, control values should be omitted before fitting the model. (3) When data are missing in the no-effect range, the Brain-Cousens model sometimes leads to better results than the default model.

Quantitation of residual valproic acid in flu vaccine drug substance.

Quantitative measurement of process-related impurities is a critical safety requirement for the production of drug substances of vaccine and therapeutic biologics. A simple and sensitive HPLC method has been developed for separation and quantitation of residual valproic acid (VPA) used in the cell transfection procedure for the manufacturing of an influenza vaccine. The method is comprised of a modified Dole liquid phase extraction followed by a quick pre-column derivatization using 2-bromoacetophenone. Nonanoic acid (NNA) is used as the internal standard (IS) and the quantification is performed by reversed-phase liquid chromatography. This new method can accurately measure as low as 6.8 µg/mL (LOQ) residual VPA in the vaccine drug substance.

Analytical validation of a quantitative method for therapeutic drug monitoring on the Alinity®c Abbott.

OBJECTIVE: The aim of this study was to evaluate the analytical performance of the Alinity®c Abbott compared to the Architect® immunoassay system for the determination of drugs having a narrow therapeutic index. METHODS: Valproic acid, amikacin, gentamicin, phenobarbital and vancomycin were analyzed using Particle-Enhanced Turbidimetric Inhibitor Immunoassay (Petinia), phenytoin and theophylline were analyzed using an immunoenzymatic method and a colorimetric method was performed to quantify lithium. The methods were validated according to the total error approach. Seven validation standards were analyzed in quintuplet during four days to establish the limits of the methods. Dilution integrity and interferences (hemolysis and high concentrations of bilirubin and lipids) were also tested. Depending on the analyte, the results obtained for twenty to forty patients on the Alinity® were compared to those obtained on the Architect®. RESULTS: The bias and the coefficients of variation for repeatability and for intermediate precision were lower than 15% for all drugs. Accuracy profiles were acceptable (acceptance limits fixed at 30%) in the validated ranges. The lower limits of quantification (LLOQ) were similar to those determined by Abbott except for gentamicin for which we determined a LLOQ at 1.22 mg/L while Abbott determined it at 0.5 mg/L. All assays diluted linear and analyte concentrations were not affected by interferences. Concentrations obtained for real samples on the Alinity®c are comparable to those obtained on the Architect®ci. CONCLUSIONS: The analytical validation of a method suitable for therapeutic drug monitoring of drugs on the Alinity®c meets the requirements of European Medicines Agency.

Assay of dried blood spot from finger prick for sodium valproate via ink auxiliary headspace gas chromatography mass spectrometry.

Sodium valproate is the most commonly used antiepileptic drug that patients need to keep taking over a long period of time or on a permanent basis. Its blood concentration should be accurately detected to avoid toxicity or side-effects, especially for children and the aged. Dried blood spot (DBS) sampling from finger prick is a minimally invasive and patient-friendly procedure for blood collection. However, there are few studies about rapid detection of sodium valproate in DBS samples in current literatures. In this work, we developed an ink auxiliary headspace gas chromatography mass spectrometry (GC-MS) strategy for direct detection of sodium valproate in DBS from epilepsy patients, which does not need extra solvent extraction or elution. It was discovered that carbon black ink could provide better capacity of heat absorption and dissociation, and higher quality of headspace sampling. The detection sensitivity has been improved with reported headspace GC-MS methods, and the limit of quantitation could reach to 200 ng/mL. Finally, this strategy was practically applied to quantify sodium valproate in DBS samples from 29 epilepsy patients. The result showed higher accuracy with lower relative errors by comparing with the clinical immunoassay results. In conclusion, we developed a direct detection method for DBS samples that is suitable for high-throughput clinical test with great potential for clinical application.

Serum carnitine levels of children with epilepsy: Related factors including valproate.

OBJECTIVE: This study measured the serum carnitine levels in patients with epilepsy and determined the factors contributing to low carnitine levels. METHODS: We measured the serum carnitine levels in 94 consecutive patients with epilepsy, including the free carnitine (FC) and acylcarnitine fractions, using an enzyme cycling method. We defined a low FC as a serum FC level < 36 µmol/L. Age, body mass index (BMI), standard deviation score of BMI (BMI-SDS), use of valproate, cognitive disorder, and feeding problems differed between patients with low and normal FC. In patients taking valproate, the associations of the serum FC level with the platelet count and serum ammonia and amylase levels were analyzed. RESULTS: Univariate analysis showed that a low BMI and BMI-SDS, the use of valproate, and cognitive disorder were more frequent in patients with a low FC. Logistic regression analysis revealed that a low BMI-SDS and cognitive disorders were independently associated with a low FC. Among the patients taking valproate, a low BMI-SDS and age were associated with a low FC. The serum FC and ammonia levels were inversely correlated, whereas no correlation was observed between the serum FC level and platelet count or serum amylase level. CONCLUSION: A low BMI and cognitive disorders were related to a low FC in patients with epilepsy and the serum carnitine levels should be monitored in these patients.

On the plasticizing properties of divalproex sodium: physicochemical and spectroscopic characterization studies.

The objective of the research was to demonstrate the plasticization properties of divalproex sodium, due to its component-valproic acid, on ethyl cellulose, which could prove beneficial for film fabrications or hot melt extrusion based formulations. Films containing 10-50% w/w (DVS/EC) as dry weight were prepared using solvent evaporation method and characterized using texture analyzer, hybrid rheometer, differential scanning calorimetry, thermogravimetry, X-ray diffractometry, polarized microscopy, FTIR, and Raman spectroscopy. It was found that there was a decrease in average peak load, melt viscosity, and glass transition temperature (Tg) while increase in elongation, with increase in concentration of DVS in the films. These results demonstrate the plasticization tendency of DVS on EC, which was attributed to the presence of valproic acid (fatty acid) in DVS. XRD studies showed amorphous nature of the films; however, polarized microscopy revealed the presence of scattered undissolved sodium valproate crystals. The presence of a single Tg established complete miscibility between valproic acid and EC. Films showed reasonable physical stability (similar Tg) at 45 °C/75% RH for 2 weeks (open condition), attributable to the similar solubility parameters of DVS and EC. FTIR and Raman spectroscopy results proved the presence of hydrogen bonding between DVS and EC.

A Validated GC-MS Method for the Determination of Genotoxic Impurities in Divalproex Sodium Drug Substance.

A specific GC-MS method has been developed, optimized and validated for the determination of five genotoxic impurities namely Methyl bromide (Me.-Br), Ethyl bromide (Et.-Br), Isopropyl bromide (Ipr.-Br), n-Propyl bromide (n-Pr.-Br) and n-Butyl bromide (n-But.-Br) in Divalproex sodium (DPS) drug substance. Chromatographic separation of five genotoxic impurities was achieved on DB-1 column (30 m × 0.32 mm, 3.0 µm), consists of 100% dimethyl polysiloxane as stationary phase and passing helium carrier gas. The mass fragments (m/z) were selected for the quantification of Me.-Br (m/z 94), Et.-Br (m/z 108), Ipr.-Br (m/z 122), n-Pr.-Br (m/z 122) and n-But.-Br (m/z 136). Bromide ion (m/z 79) was the qualifier ion for the analytes [(Me.-Br), (Et.-Br), (Ipr.-Br), (n-Pr.-Br) and (n-But.-Br)]. The performance of the method was assessed by evaluating the specificity, linearity, sensitivity, precision and accuracy experiments. The established limit of detection and limit of quantification values for the genotoxic impurities were in the range of 0.005-0.019 µg mL-1. The correlation coefficient values of the linearity experiment were in the range of 0.9947-0.9983. The average recoveries for the accuracy were in the range of 97.6-111.3%. The results proved that the method is suitable for the determination of Me.-Br, Et.-Br, Ipr.-Br, n-Pr.-Br and n-But.-Br contents in divalproex sodium.

Synergistic effect of MoS2 and diamond nanoparticles in electrochemical sensors: determination of the anticonvulsant drug valproic acid.

The authors describe an electrochemical sensor based on the use of diamond nanoparticles (DNPs) and molybdenum disulfide (MoS2) platelets. The sensor was applied to the voltammetric determination of the anticonvulsant valproic acid which was previously derivatized with ferrocene. The MoS2 platelets were obtained by an exfoliation method, and the DNPs were directly dispersed in water and subsequently deposited on a glassy carbon electrode (GCE). The sensor response was optimized in terms of the solvent employed for dispersing the MoS2 nanomaterial and the method for modifying the GCE. Sensors consisting of a first layer of MoS2 dispersed in ethanol/water and a second layer of DNPs give better response. The single steps of sensor construction were characterized by atomic force microscopy and electrochemical impedance spectroscopy. The differential pulse voltammetric response of the GCE (measured at +0.18 V vs. Ag/AgCl) was compared to that of sensors incorporating only one of the nanomateriales (DNPs or MoS2). The formation of a hybrid MoS2-DNP structure clearly improves performance. The GCE containing both nanomaterials exhibits high sensitivity (740 µA ⋅ mM-1 ⋅ cm-2), a 0.27 µM detection limit, and an 8% reproducibility (RSD). The sensor retained 99% of its initial response after 45 days of storage. Graphical abstract Electrochemical sensor by co-immobilization of MoS2 and diamond nanoparticles (DNP). The formation of a hybrid MoS2-DNP structure enhances the performance of the sensor towards valproic acid derivatized with a ferrocene group, when compared with sensors incorporating only DNP or MoS2.

Reliable GC Method for Related Substances in Divalproex Sodium Drug.

A specific GC method has been developed, optimized and validated for the determination of seven related substances namely N,N-dimethyl valpronamide, valeric acid, 2-methyl valeric acid, 2-ethyl valeric acid, 2-isopropyl valeric acid, 2-n-butyl valeric acid and 2-propyl-2-pentenoic acid in divalproex sodium (DPS) drug substance. Chromatographic separations of these seven impurities were achieved on DB-FFAP column (30 m × 0.53 mm, 1.0 µm), which consists nitroterephthalic acid modified polyethylene glycol material as stationary phase. DPS is a coordination complex of the sodium valproate and valproic acid (VPA). Nonanoic acid is used as internal standard. All the seven related substances, VPA and nonanoic acid were extracted into dichloromethane and monitored by GC with flame ionization detector. The performance of the developed method was assessed by evaluating specificity, linearity, sensitivity, precision, accuracy and robustness. Forced degradation experiments were conducted to evaluate the degradation behavior of DPS. The established limits of detection (LODs) and limits of quantification (LOQs) values for the related substances were in the ranges of 4-5 and 12-15 µg mL-1, respectively. Further, for VPA, LOD and LOQ values were 4 and 12 µg mL-1, respectively. The correction factors of these related substances with respect to VPA and lie between 0.92 and 1.44. The average recoveries were in the range of 92.4-108.4%.

Real-Time Quantitative Analysis of Valproic Acid in Exhaled Breath by Low Temperature Plasma Ionization Mass Spectrometry.

Real-time analysis of exhaled human breath is a rapidly growing field in analytical science and has great potential for rapid and noninvasive clinical diagnosis and drug monitoring. In the present study, an LTP-MS method was developed for real-time, in-vivo and quantitative analysis of γ-valprolactone, a metabolite of valproic acid (VPA), in exhaled breath without any sample pretreatment. In particular, the effect of working conditions and geometry of the LTP source on the ions of interest, protonated molecular ion at m/z 143 and ammonium adduct ion at m/z 160, were systematically characterized. Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) was carried out in order to identify γ-valprolactone molecular ions (m/z 143), and the key fragment ion (m/z 97) was used for quantitation. In addition, the fragmentation of ammonium adduct ions to protonated molecular ions was performed in-source to improve the signal-to-noise ratio. At optimum conditions, signal reproducibility with an RSD of 8% was achieved. The concentration of γ-valprolactone in exhaled breath was determined for the first time to be 4.83 (±0.32) ng/L by using standard addition method. Also, a calibration curve was obtained with a linear range from 0.7 to 22.5 ng/L, and the limit of detection was 0.18 ng/L for γ-valprolactone in standard gas samples. Our results show that LTP-MS is a powerful analytical platform with high sensitivity for quantitative analysis of volatile organic compounds in human breath, and can have potential applications in pharmacokinetics or for patient monitoring and treatment. Graphical Abstract ᅟ.

Validated spectrophotometric methods for determination of sodium valproate based on charge transfer complexation reactions.

This work presents the development, validation and application of four simple and direct spectrophotometric methods for determination of sodium valproate (VP) through charge transfer complexation reactions. The first method is based on the reaction of the drug with p-chloranilic acid (p-CA) in acetone to give a purple colored product with maximum absorbance at 524nm. The second method depends on the reaction of VP with dichlone (DC) in dimethylformamide forming a reddish orange product measured at 490nm. The third method is based upon the interaction of VP and picric acid (PA) in chloroform resulting in the formation of a yellow complex measured at 415nm. The fourth method involves the formation of a yellow complex peaking at 361nm upon the reaction of the drug with iodine in chloroform. Experimental conditions affecting the color development were studied and optimized. Stoichiometry of the reactions was determined. The proposed spectrophotometric procedures were effectively validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. Calibration curves of the formed color products with p-CA, DC, PA and iodine showed good linear relationships over the concentration ranges 24-144, 40-200, 2-20 and 1-8µg/mL respectively. The proposed methods were successfully applied to the assay of sodium valproate in tablets and oral solution dosage forms with good accuracy and precision. Assay results were statistically compared to a reference pharmacopoeial HPLC method where no significant differences were observed between the proposed methods and reference method.

Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48µM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods.

Involvement of p38 MAPK in the Drug Resistance of Refractory Epilepsy Through the Regulation Multidrug Resistance-Associated Protein 1.

Increased expression of multidrug-resistance associated protein 1 in brain tissue has been reported which lead to multidrug resistance of refractory epilepsy. However, the mechanism of up-regulated expression is still unclear. In our previous study, we have found that the MAPK signaling pathway mediated the expression of P-glycoprotein. So in this study, we used a rat model of refractory epilepsy to examine whether p38 MAPK affect the expression of MRP1 and the concentrations of AEDs in the brain. The expression of MRP1 and p38 MAPK was detected by immunofluorescence, Western-blot and real time-PCR, while the concentration of AEDs was measured by microdialysis and HPLC. The result showed that SB202190, the specific inhibitor of p38 MAPK, could down-regulate the expression of MRP1, while increase the concentrations of valproate and lamotrigine in hippocampus extracellular fluid of refractory epileptic rat. We demonstrate that p38 MAPK signaling pathway may be involved in drug resistance of refractory epilepsy by regulating MRP1.

A simple validated RP-HPLC bioanalytical method for the quantitative determination of a novel valproic acid arylamide derivative in rat hepatic microsomes.

A simple and specific bioanalytical method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet detection was developed and validated for the determination of a novel valproic acid arylamide, N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) in rat hepatic microsomes (a subcellular fraction containing phase I enzymes, especially cytochrome P450). The chromatographic separation was achieved using a reversed-phase Zorbax SB-C18 column and a mobile phase of acetic acid in water (0.2% v/v) and acetonitrile (40:60 v/v) with a flow rate of 0.5 mL/min. The calibration curve was linear over the range of 882-7060 ng/mL (r(2) = 0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99 ng/mL, respectively. The method was validated with excellent sensitivity, and intra-day accuracy and precision varied from 93.79 to 93.12%, and from 2.12 to 4.36%, respectively. The inter-day accuracy and precision ranged from 93.29 to 97.30% and from 0.68 to 3.60%, respectively. The recovery of HO-AAVPA was measured between 91.36 and 97.98%. The assay was successfully applied to the analysis of kinetic metabolism and pharmacokinetic parameters in vitro by a substrate depletion approach.

Correlation of saliva and serum free valproic acid concentrations in persons with epilepsy.

PURPOSE: Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) in serum is frequently used in clinical settings however saliva could be an alternative to measure free concentration of drugs. In the present study, we observed the possible correlation of VPA concentration between serum and saliva in persons with epilepsy (PWE). METHODS: A total of 59 paired serum and saliva samples were assayed from 65 consecutive PWE (51 males and 14 females; age range 9-65 years). Patients were subjected to either VPA monotherapy or its combination with other AEDs for at least three months. Steady state trough concentration of unbound VPA drug was quantified using HPLC. The correlation between serum and saliva free VPA concentration was evaluated. RESULTS: Out of 65 patients, 27 were on monotherapy of VPA and 38 were on VPA with other antiepileptic drugs. Saliva VPA concentration significantly correlated with serum free VPA concentration (p<0.05). Poor correlation was observed between serum and saliva VPA concentration with the daily dose (p>0.05) respectively. CONCLUSIONS: Our study reveals that serum and saliva VPA concentrations are significantly associated in PWE. These associations may facilitate monitoring and evaluation of VPA levels non-invasively for PWE.