High Pressure Liquid Chromatographic Determination of Hydrochlorothiazide in Human Serum and Urine M. J. Cooper,* A. R. Sinaiko, M. W. Anders, and B. L. Mirkin Departments of Pharmacology and Pediatrics, Division of Clinical Pharmacology, University of Minnesota, 105 Millard Hall, Minneapolis, Minn. 55455
on a Vortex mixer for 1 min and centrifuging for 5 min, 1 ml of the organic layer was transferred to another tube and 0.5 ml of a 1N sodium hydroxide solution added. This solution was mixed and allowed to stand for 1min at room temperature, after which the upper layer was discarded. Ether (1ml) was then added to the sodium hydroxide solution,followed by mixing. After standing for 1min, the ether layer was discarded and traces of ether were removed with a gentle stream of air. An aliquot of the aqueous layer was injected into the liquid chromatograph. Since initial studies indicated that hydrochlorothiazide was unstable in strongly alkaline media for prolonged periods, the back-extraction step into sodium hydroxide solution was performed just prior to the liquid chromatographic analysis. Liquid Chromatography. A Waters 6000 pumping system (Waters Associates) coupled to a Spectroflow SF 770 variable wavelength detector (Schoeffel Instrument Corp.) was used for these studies. A reverse phase system consisting of a 30 cm X 4 mm FBondapak CIS column (Waters Associates) was utilized with methanol-0.01 M sodium dihydrogen phosphate solution (1:4)as the eluent, at a flow rate of 0.6 ml/min. The detector was operated at 271 nm. For urine samples, a sensitivity setting of 0.1 A (full scale deflection) was used, but for the lower levels present in serum, a sensitivity of 0.01 A (full scale Hydrochlorothiazide (6-chloro-3,4-dihydro-7-sulfamyl- deflection) was required. Peak heights were used for quantitation. All standard curves were linear and passed through the origin. 2H- 1,2,4-benzothiadiazine 1-1-dioxide), I, A new method has been developed for the quantitatlve estlmatlon of the diuretic and antlhypertensive agent, hydrochlorothlazide, in human serum and urlne. The drug is extracted into ethyl acetate from biological materlal and then back-extracted into sodium hydroxide solutlon. This aqueous solution Is chromatographed on a reverse phase system, uslng a pBondapak C,S column and methanol-0.01 M sodium dihydrogen phosphate as eluent and detection at 271 nm. The recovery from serum was 98.2 f 3.7% (mean f SE, n = 9) over the concentration range of 200-800 ng/ml. In urine, the recovery was 91.5 f 2.5% (mean f SE, n = 8) over the range of 20-100 pg/ml. No interference was observed in this assay from the following drugs which may be administered concurrently durlng hydrochlorothlazlde therapy: azathloprine, chlorthalidone, guanethidine, methyldopa, mlnoxidll, prednlsone, and spironolactone.
RESULTS AND DISCUSSION
has been one of the most widely used diuretic and antihypertensive agents over the past decade. It is administered in dosages in the range of 25-250 mg/day. Because of the incidence of various side effects such as hyperglycemia, hyperuricemia, hyponatremia, and hypokalemia, it may prove useful to monitor serum and urine levels of hydrochlorothiazide in individual cases in order t o obtain optimal therapeutic response and minimal side effects and also to assure compliance with therapy. Few methods have been reported for the determination of hydrochlorothiazide in biological specimens. Sheppard et al. (1) reported a colorimetric procedure applicable to urine and Anderson e t al. (2) used 14C-labeled drug t o study its disposition in adults. Osborne ( 3 ) and Sohn e t al. ( 4 ) developed thin-layer chromatographic procedures suitable for screening for the presence of various thiazide diuretics in urine. More recently, Vandenheuvel e t al. ( 5 )analyzed the drug in human blood and plasma using gas chromatography with electron capture detection. Honigberg e t al. (6) used high pressure liquid chromatography (HPLC) t o analyze various antihypertensive dosage forms, including hydrochlorothiazide. A new method for the determination of hydrochlorothiazide in human serum and urine using solvent extraction and HPLC is described here. The method is simple, rapid, sensitive, and requires only microliter volumes of biological material.
Representative chromatograms of serum and urine samples are shown in Figures 1and 2. Hydrochlorothiazide added t o serum over the range of 200-800 ng/ml was recoverable to the extent of 98.2 f 3.7% (mean f SE, n = 9). Control samples of serum showed no interfering peaks (Figure 1).A recovery of 91.5 f 2.5% (mean f SE, n = 8) was obtained when hydrochlorothiazide (20-100 wg/ml) was added to urine. No inter-
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EXPERIMENTAL
Flgure 1. HPLC of (A) blank serum and (B) serum containing 800 ng hydrochlorothiazide/ml.
Extraction Procedure. Solid sodium bicarbonate (200 mg) and ethyl acetate (2 ml) was added to 1ml serum or urine. After mixing
Conditions: Column 30 cm X 4 mm FBondapak Cj8: eluent, methanol-0.01 M sodium dihydrogen phosphate (1:4): flow rate, 0.6 ml/min. Detection at 271 nm
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Flgure 2. HPLC of (A) blank urine and (B) urine containing 80 drochlorothiazide/ml.
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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 (
