A 2021 study evaluated the agreement between an enzyme-multiplied immunoassay technique (EMIT) and Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) for routine therapeutic drug monitoring of valproic acid (VPA) in pediatric patients with epilepsy. The study included 774 plasma samples from 711 children, with outcomes assessing assay correlation, absolute and relative bias, and diagnostic concordance across therapeutic VPA ranges. Although a strong correlation was observed between methods (r2 = 0.9281), EMIT systematically overestimated VPA concentrations compared with LC-ESI-MS/MS, with a mean absolute bias of 14.5 mcg/mL and a relative overestimation of 27.8%, resulting in diagnostic mismatch in 32.9% of samples. The authors noted that this difference may be related to antibody cross-reactivity with valproate conjugated metabolites, including valproate glucuronide (VPAG), which were not evaluated during immunoassay validation. The study did not assess individual metabolite concentrations or clinical toxicity outcomes. [1]
The Handbook of Drug Monitoring Methods describes anticonvulsants as drugs for which therapeutic drug monitoring is appropriate, as blood concentrations correlate with pharmacodynamic effects, while dose–concentration relationships are variable. The text notes that multiple assay methodologies are used, including chromatographic methods (GC, HPLC, mass spectrometry) and immunoassays, with different sources of analytic interference depending on method. For immunoassays, cross-reactivity with structurally related drugs or metabolites is a recognized issue and is well documented for phenytoin and carbamazepine, where metabolites and prodrugs can significantly interfere with measured concentrations. In contrast, the handbook reports no documented clinically significant interferences with immunoassays for measurement of valproic acid, noting only minor cross-reactivity. [2]
A 2020 study evaluated the performance of an enzyme-multiplied immunoassay technique (EMIT) for routine therapeutic drug monitoring of valproic acid using the Emit 2000 Valproic Acid Assay. The study assessed assay calibration, quality control performance, and clinical applicability in both therapeutic monitoring and acute overdose settings, demonstrating that EMIT can reliably quantify serum valproic acid within its calibrated range. The assay uses monoclonal antibodies reactive to valproic acid and is intended to measure the parent drug; however, the authors did not perform or report formal analytical cross-reactivity testing with valproate metabolites or glucuronide conjugates, so definitive conclusions regarding metabolite interference cannot be drawn from this study. The article describes that valproic acid undergoes extensive hepatic metabolism, producing unsaturated metabolites such as 2-en-VPA and 3-keto-VPA, which have been implicated in toxicity mechanisms including hyperammonemia, hepatotoxicity, and pancreatitis. Based on these findings, the study supports that valproate metabolites contribute to clinical toxicity, but it does not establish that these metabolites are directly measured or meaningfully contribute to serum valproic acid concentrations reported by CPT 80164 immunoassays. [3]
A 1980 study evaluated an enzyme-multiplied immunoassay technique (EMIT) for the measurement of serum valproic acid by comparison with gas-liquid chromatography. Using 80 patient samples over a wide range of concentrations, the authors reported a significant correlation coefficient (r= 0.979) and state that the correlation between the two methods examined is satisfactory for routine measurement of valproic acid. The EMIT assay demonstrated high analytical specificity, with no antibody cross-reactivity detected when tested against other commonly prescribed antiepileptic drugs, including phenobarbitone, primidone, phenytoin, carbamazepine, and ethosuximide. The study did not evaluate cross-reactivity with valproic acid metabolites or conjugates and did not assess the contribution of metabolites to valproic acid toxicity. [4]
Valproic acid (VPA) toxicity is primarily linked to its metabolites, which can cause significant harm to the human body. These toxic effects are dose-dependent and can impact systems like the central nervous system or specific organs like the liver. VPA metabolites arise as intermediates, byproducts, or end products of VPA metabolism, with hepatocytes playing a role in organizing these metabolic processes to mitigate or reduce the accumulation of toxic compounds. Specific metabolites like 3-hydroxy, 4-hydroxy, and 5-hydroxy valproic acids are known for their hepatotoxic effects, being products of P-oxidation and involving several cytochrome P450 isoenzymes. 3-Oxovalproic acid, while suspected to be toxic, is still under study for its full toxic potential. Valproyl-Coenzyme A is a significant mitochondrial metabolite of VPA that can inhibit succinate-CoA ligase, potentially leading to mitochondrial DNA depletion and subsequent liver failure in patients with mitochondrial deficiencies. 2-N-Propyl-4-oxopentanoic acid can cause teratogenic effects and microvesicular steatosis. 4-Ene-valproic acid is highly hydrophobic, contributing to hepatotoxicity and hyperammonemia and is under study for therapeutic potential in various conditions. 2-Propyl-2,4-pentadienoic acid, through glucuronidation and inhibition of β-oxidation, leads to impaired urea production and associated hyperammonemia. 2-Ene-valproic acid is neurotoxic, associated with neurological adverse drug reactions (ADRs), such as diplopia, seizures, and cognitive and behavioral disorders. Valproylcarnitine, by reducing L-carnitine concentrations through urinary excretion, can lead to lipid myopathy, hypoglycemia, fatty liver disease, and hyperammonemia. [5]