To the Editor: Valproic acid can be determined by gas-chromatography as the free acid after its extraction. (1 ) or derivatization to form methyl, butyl, or phenacyl ...
(unesterified) rocyte
cholesterol in the eryth-
membrane
exchanges
rapidly
with the unesterified cholesterol in plasma, varying the plasma cholesterol will lead to changes in membrane surface and morphology of the cell (2). How this affects leakage of intra-erythrocytic metabolites into plasma is not known, but deserves further study. References
1. Ladenson JH. Non analytical sources of variation in clinical chemistry results. In Gradwohl’s Clinical Laboratory Methods and Diagiwsis, AC Sonnenwirth, and L Jarrett, Eds, CV Mosby, St Louis, MO, 1980, pp 149-192. 2. Van Deenan LLM, DeGier J. Lipids of the red cell membrane. In The Red Blood Cell, DM Surgenor, Ed, Academic Press, New York, NY, 1974, pp 148-213.
Amin A. Nanji Div. Gun. Chem., Vancouver General Hosp. and Pathol. Univ. British Columbia, Vancouver, Canada V5Z 1M9
un
Dept.
Frohlich
Div. Clin. Chem., Shaughnessy Hosp. and Dept. Pat hol. Univ. British Columbia, Vancouver, Canada V5Z 1M9
ly 250 and 300 #{176}C. The baseline was manually
re-zeroed
tion. The working
before
each
injec-
solution of valproic
acid, 50 mg of authentic (Sigma-Tau, Rome, Italy)
valproic acid in 100 mL of
drug-free serum, was diluted with vanous volumes
of drug-free
serum to pre-
pare 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 patient’s serum or standard 100 L of internal-standard
of
solution to 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. (Re-
member 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 height (y) vs concentration
of peak (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 =
Gas-Chromatographic Determination of Valproic Acid in Serum without Derivatization
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 measur-
able. The efficiency of the extraction step at the concentration 250 mg/L
To the Editor: 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
I... I..
determination derivatization.
(Supelco Inc., Bellefonte,
PA 16823).
990
CLINICAL
CHEMISTRY,
of patients treated with other anticonvulsant drugs. We found no interferences from phenobarbital, phenytoin, carbama.zepine, primidone, ethosuxi-
mide, or endogenous serum constituents with the described experimental conditions
(Figure
1). With
this proce-
dure the retention times are short (3.8 mm for the internal standard and 5.6 mm for vaiproic acid), allowing for rapid analysis without long delays between injections of sample extracts. References 1. Fellenberg AJ, Pollard AC. Rapid and sensitive gas-liquid chrornatographic procedure for the micro-determination of sodium valproate (sodium di-n-propylacetate) in plasma or serum. Clin Chim Acta 81, 203-
208 (1977). 2. Morita Y, Tsuen IR, Mm LL, Atkinson AJ Jr. On a column propylation method for measuring plasma valproate concentrations by gas-chromatography. Ther Drug Monit 3, 193-199 (1981). 3. Huishoff A, Roseboom M. Determination of valproic acid (di-n-propylacetic acid) in plasma by gas-liquid chromatography with pre-column butylation. Clin Chim Acta 93, 9-13 (1979). 4. Chan SC. Monitoring serum valproic acid by gas-chromatography with electron capture detection. Clin Chem 26, 1528-
Sandra Tosoni
Costantino
Alberto 30
L1
We used a Model F30 gas-chromato-
The separation was performed isothermally at 180 #{176}C with a nitrogen flow rate of 30 mL/min. The injector and detector temperatures were respective-
get value 75 mg/L). The mean by our method was 72.48 mgIL (CV 3.43%). To determine the specificity of the assay, we analyzed a commercial sorum standard (Syva Corp.) and serum
Istituto di Chimica Facolt#{224} di Medicina Universit#{224} ole Brescia 25100 Brescia, Italia
of valproic
graph 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
Day-to-day precision, estimated by analysis of the same three controls over a 10-day period, was also
