Gas-chromatographic determination of valproic acid in serum without derivatization.

Valproic acid can be determined by gas-chromatography as the free acid after its extraction (1 ) or derivatization to form methyl, butyl, or phenacyl estars (2-4), the latter methods being more complicated for routine analyses. Other procedures, such as enzyme immunoassay (Syva Co., Palo Alto, CA 94304) or fluoroimmunoassay (Miles Inc., Elkhart, IN 46515), which do not require serum extraction, are very fast but too expensive. We have developed a rapid, simple, and reproducible method for gas-chromatographic determination of valproic acid without derivatization. We used a Model F30 gas-chromatograph equipped with flame ionization detector and a Model 56 chart recorder (all from Perkin-Elmer Corp., Norwalk,CT06856).The2m x 2mm(i.d.) glass column was packed with ic/c SP 1000 on 100/120 Chromosorb WAW (Supelco Inc., Bellefonte, PA 16823). The separation was performed isothermally at 180 #{176}C with a nitrogen flow rate of 30 mL/min. The injector and detector temperatures were respectively 250 and 300 #{176}C. The baseline was manually re-zeroed before each injection. The working solution of valproic acid, 50 mg of authentic valproic acid (Sigma-Tau, Rome, Italy) in 100 mL of drug-free serum, was diluted with vanous volumes of drug-free serum to prepare standard solutions. The internalstandard working solution was 50 L of n-hexanoic acid (Carlo Erba, Milan, Italy) in 100 mL of 0.1 mol/L NaOH. Serum samples were obtained from patients being treated with valproic acid as an anticonvulsant, administered in combination with other drugs. In the procedure, add 250 tL of patient’s serum or standard solution to 100 L of internal-standard solution, then acidify with 100 L of sulfuric acid (18 molIL diluted 1:1 with water); extract the drug by vortex-mixing with 1.0 mL of diethyl ether for about 1 mm and then centrifuging for 5 mm at 1000 x g. Inject 2-3 L ofthe organic layer into the gas chromatograph. (Remember that diethyl ether is highly flammable.) Prepare the calibration curve by measuring the peak height ratio between valproic acid and the internal standard and plotting it vs vaiproic acid concentration. To determine the linearity of peak height (y) vs concentration (x), we made three determination of each of these concentrations of vaiproic acid in serum: 0.0, 31.25, 62.50, 125.0, and 250.0 mg/L. The straight line obtained is expressed by y = 0.35x + 3.47 (r = 0.998). As little as 20 mg of valproic acid per liter of serum, considerably below the therapeutic concentration range (50-100 mg/L), is easily measurable. The efficiency of the extraction step at the concentration 250 mg/L