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 s...

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Bibliographic Details
Published in:Journal of liquid chromatography & related technologies 2013-06, Vol.36 (16), p.2209-2219
Main Authors: Darlington, Daniel N., Gonzales, Mary D.
Format: Article
Language:English
Subjects:
Acids
Analytical chemistry
Chromatography
Conjugates
Conjugation
DCCH
Fluorescence
HPLC
Hydrazides
Laboratory animals
Mathematical analysis
Plasma
Pools
valproic acid
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Summary: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/H 2 O gradient. Standard curves were generated from the absorbance (λ = 430 nm) and fluorescence (λ ex  = 415 nm, λ em  = 465 nm) that described an exponential curve y-a(1-b x ) with correlation coefficients >0.97. The inter-assay variation measured over 12 days was
ISSN:1082-6076
1520-572X
DOI:10.1080/10826076.2012.717059