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Flgure 2. HPLC of (A) blank urine and (B) urine containing 80 drochlorothiazide/ml.
Kg
hy-
Conditions: same as Figure 1
fering peaks were seen in control samples of urine (Figure 2). The following drugs which may be administered concurrently with hydrochlorothiazide therapy, did not interfere with the assay: azathioprine, chlorthalidone, guanethidine, methyldopa, minoxidil, prednisone, and spironolactone. The extraction into ethyl acetate is facilitated by salting out the drug with an excess of solid sodium bicarbonate. Since hydrochlorothiazide is a weak acid, it is not ionized a t this pH. In the final back extraction procedure, sodium hydroxide solution is used; this extracts the ionized form into the aqueous layer and serves as a further purification step. Hydrochlorothiazide has a uv absorption maximum a t 226 nm, but this wavelength was found unsuitable for quantitative analyses since interfering peaks were observed. Another absorption maximum occurs a t 271 nm and, though less intense, was used for routine assay since it was free of interfering peaks.
The lower limit of detection was determined to be 50 ng/ml serum. In their spectrophotometric assay, Sheppard et al. (I) reported a sensitivity of the order of 10 pg/ml using 1 ml of urine. More recently, Meyer et al. (7) modified the above method and found a lower level of sensitivity of less than 2 pg/ml using 4 ml of urine. Honigberg et al. (6) in their work on the HPLC of hydrochlorothiazide and other diuretic-antihypertensive preparations, investigated various parameters for optimal chromatographic conditions for multidrug formulas. Typically 100-pg amounts were chromatographed, and no biological specimens were analyzed. In their gas chromatographic assay using electron capture detection, Vandenheuvel et al. ( 5 ) also reported a sensitivity of about 0.05 Wg/ml in human blood and plasma. However, their procedure involves derivatization and is, overall, lengthier than the procedure described here. Moreover, their studies with labeled material showed a recovery of about 50% of the drug. Although 1ml of biological fluid was used in the present study, the same extraction procedure can be performed with much lower volumes (
