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Anal. Chem. 1981, 53, 604-606
Determination of Sulfinalol Hydrochloride in Human Plasma and Urine by Liquid Chromatography with Amperometric Detection George B. Park," Raymond F. Koss, Scott K. O'Nell, Gerard P. Palace, and Jerome Edelson Department of Drug Metabolism and Disposition, Sterling- Winthrop Research Institute, Rensselaer, New York 12 144
The analysis of plasma and urlne for sulfinalol hydrochloride, an experimental antlhypertensive drug, Is described. the method involves a solvent extractlon followed by HPLC wlth amperometric detection employing a moblle phase of 93 % acetonltrlle/7 % aqueous ammonium acetate. The precision and accuracy of the method are both better than f5 %, The minimum quantlflable levels in plasma and 1 to 10 dlluted urlne are 2.6 and 2.1 ng/mL, respectlvely. The method has been used for the analysls of plasma and urlne samples from humans, dogs, cats, and monkeys.
Reverse-phase liquid chromatography with amperometric detection has been widely used for the analysis of easily oxidizable compounds in pharmaceutical formulations and various biological tissues (1). A majority of the applications have employed aqueous mobile phases. Several applications of the amperometric detector using mobile phases containing high percentages of nonaqueous solvents have been published, including the analysis of urine for mescaline, methadone, and phencyclidine (mobile phase of 80% acetonitrile) (2), the determination of morphine in blood (mobile phase of 90% methanol) (3), and the detection of several pharmaceuticals in solutions by use of various alcohols, acetonitrile, and methylene chloride as mobile phases (4). In general, however, such mobile phases have not been employed to a great extent because of their high resistivities which lead to nonlinearity of response, low sensitivity, and high signal-to-noise ratios. An advantage of using nonaqueous solvents is that they can improve chromatographic selectivity and peak shape. Sulfinalol HCl, 4-hydroxy-CY-[[ [3-(4-methoxyphenyl)-lmethylpropyl]-amino]methyl]-3-(methylsulfinyl)benzenemethanol hydrochloride, (Figure 1 (I)) is a new antihypertensive agent undergoing clinical evaluation. The low anticipated therapeutic dose of sulfinalol HC1, less than 25 mg/day, necessitated the development of a method to quantitate drug levels as low as 10 ng/mL. This report describes a method involving solvent extraction and reverse-phaseliquid chromatography with amperometric detection by using a mobile phase which is 93% acetonitrile. EXPERIMENTAL SECTION Apparatus. Chromatographic separations were performed on a modular system composed of a Constametric I1 pump (Laboratory Data Control), a Model 7120 sample injector (Rheodyne, Berkeley, CA), and either a 30 cm X 3.9 mm i.d. fi-Bondapak NH2 (Waters ASSOC.,Milford, MA) or a 25 cm X 4.6 mm i.d. Zorbax NH2 (DuPont, Wilmington, DE) column. The detector was a Model LC-4 amperometric detector (Bioanalytical Systems, West Lafayette, IN) and a TL-5 glassy carbon electrode with a 5-mil (ca. 127 pm) spacer. All connecting tubing before the column was 0.2 mm i.d. stainless steel tubing. The tubing connecting the outlet of the column and the detector was 0.5 mm i.d. low-pressure plastic tubing. Reagents. Sulfinalol HC1 and the internal standards, (a-[ [ [ 2-(3,4-dimethoxyphenyl)ethyl]amino]methyl]-4hydroxy-3-(m~thylsulfinyl)benzenernethanol hydrochloride 0003-2700/81/0353-0604$01.25/0
(11) and l-[4-(hydroxy)-3-(methylsulfinyl)phenyl]-2-[[3-(4methoxypheny1)-l-methylpropyl]amino]ethanonehydrochloride (111)(Figure l), were synthesized at Sterling-Winthrop Research Institute. Omnisolv acetonitrile and reagent grade chloroform were freshly distilled in glass at atmospheric pressure. All other reagents were analytical reagent grade and used without further purification. A saturated, pH 8.9, ammonium chloride solution was prepared by adjusting the pH of a saturated ammonium chloride solution to 8.9 with concentrated ammonium hydroxide; this solution will be referred to as saturated ammonium chloride. Mobile Phase. The chromatographic mobile phase was 93:7 (v/v acetonitrile:0.2 mol/L ammonium acetate adjusted to an apparent (measured with a glass electrode containing aqueous KC1 and standardized against aqueous buffers) pH of 6.6 with glacial acetic acid. The mobile phase was filtered and degassed before use. The mobile phase was prepared, with freshly distilled acetonitrile, on the day it was used, to minimize the residual current of the detector. Standards and Prepared Samples. Stock solutions of sulfinalol HC1 and the internal standard were prepared in methanol on the day of analysis. Standards were prepared by adding appropriate aliquots of the stock solutions to 1mL of control plasma (oxalate anticoagulant) or l-mL aliquots of human urine diluted 1 to 10 with distilled water. Fresh standards were prepared on each day of analysis. Samples were prepared for single-blind analysis in the same manner as the standards. Two sets of triplicate samples were prepared in plasma at four levels from 5.5 to 22.5 ng/mL plus a blank. One set of samples were analyzed on preparation; the other set was analyzed after a minimum of 4 days of storage at -4 "C. Likewise, two sets of triplicate samples were prepared in 1to 10 diluted urine at five levels from 4.7 to 57 ng/mL plus a blank. One set was analyzed upon preparation; the other set was stored frozen for a minimum of 4 days before analysis. Extraction and Analysis. A 1OO-fiLaliquot of the internal standard stock solution (500 ng of II/mL in methanol) was added to each sample to give a final concentration of 50 ng/mL. After the sample was mixed, 1 mL of saturated ammonium chloride solution and 12 mL of chloroform were added; the tube was shaken and centrifuged for 10 min. The aqueous layer was discarded and 10 mL of the chloroform was transferred to a clean tube. To the tube was added 5 mL of 0.1 mol/L HC1; the tube was stoppered, shaken, and centrifuged. Four milliliters of the HC1 layer was transferred to a clean tube and neutralized with 0.4 mL of 1 mol/L NaOH, followed by the addition of 2 mL of saturated ammonium chloride solution, 1.8 g of granular ammonium chloride, and 11.5 mL of chloroform. The tube was stoppered, shaken, and centrifuged. The aqueous layer was discarded, and 10 mL of the chloroform layer was transferred to a clean, silanized centrifuge tube. The chloroform was evaporated at 40 "C with the aid of a stream of dry nitrogen. Immediately prior to chromatographic analysis, 100 pL of the mobile phase was added to the residue in the centrifuge tube, which was then placed in an ultrasonic bath for 30 s and vortexed for 10 s. Fifty microliters of the resulting solution 0 1981 Arnerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981 socti3
"0 .-X-
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A
805
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.HCI
CH2NH-Y-CH2
-
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-Y-
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CH(CH3)CHz
CHOH
CH2
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III co C H (CH3)C H2 H Flgure 1. Structures of sulfinalol hydrochloride (I) and the internal standards (I1 and 111). was injected into the HPLC for analysis. The flow rate was 1.75 mL/min resulting in an inlet pressure of ca. 600 psi. The electrode potential was +0.73 V vs. Ag/AgC1(3 mol/L NaCl), and the chromatograph was operated at ambient temperature. Plasma and urine sample analysis followed the same procedure. Calculations. A regression analysis of the peak height ratios (sulfiialol/internal standard) obtained for the standards was performed; the slope and y intercept and the standard errors of the slope and y intercept were calculated (5). The concentrations of sulfinalol in the samples were determined by inverse prediction. The minimum quantifiable level (MQL) of the assay, defined as the concentration whose lower 80% confidence limit just encompasses zero, was estimated from the least-squares regression analysis (6). The assayed levels for the prepared samples were expressed as percent differences from the nominal values and analyzed by a two-way analysis of variance with replication to test for a concentration effect, a time effect, and a concentrationtimes-time effect. The accuracy of the assay was estimated from the mean percent differences at each concentration. The precision of the assay was estimated from the derived standard deviation of the percent differences. RESULTS AND DISCUSSION To minimize the IR effects observed with mobile phases of high organic content, the internal diameter of the detector cell outlet was increased, and the residual current was kept below 10 nA, typically 5-7 nA, by fresh-distilling the acetonitrile used for the mobile phase. Also, the mobile phase was used on the day it was prepared. The response of the detector was linear over the range from 2.5 to, at least, 60 ng/mL, and appeared to be linear to somewhat higher levels. Typically, the regression line for a plot of the peak height ratios (sulfinalol/internal standard) for the standards vs. the sulfinalol HCI concentration was defined by a slope (A standard error) of 0.033 (A 0.002) and a y intercept (istandard error) of -0.007 (i0.023). The electrochemistry of sulfinalol has not been studied; however, preliminary cyclic voltammetry experiments were run in aqueous and acetonitrile solutions. The poor peak shape in the resulting voltammograms indicated slow electron transfer. The absence of a cathodic peak when the potential scan was reversed indicated that the oxidation product reacted chemically before it could be reduced back to sulfinalol. The site of oxidation and the number of electrons involved were not determined. A hydrodynamic voltammogram of sulfinalol, in the chromatographic mobile phase, gave a half-wave potential of +0.70 V vs. Ag/AgCl for the oxidation of sulfinalol and indicated that the maximum sensitivity for sulfinalol would be obtained a t potential of approximately 0.80 V. Potentials higher than 0.73 V, however, compromised the selectivity of the detector, and significant interference peaks, eluting at the retention time of sulfinalol, were observed.
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Figure 2. Typlcal chromatograms for sulfinalol in extracted human plasma: (A) 0 ng/mL plasma standard; (B) 30 ng/mL plasma standard (see text for chromatographic condltlons).
Table I. Results of the Analysis of Prepared Plasma Samples concn concn added, found, no. of % ng/mL ng/mL determns SEM 6 0
