Anal. Chem. 1982, 5 4 , 1287-1291 (4) Doran, T.; McTaggart, N. G. J . Chromafogr. Sci. 1974, 12,715-721. (5) Choudtiury, D. R.; Bush, 8.Anal. Chem. 1981, 53, 1351-1356. (6) Tomkins, B. A.; Griest, W. H.; Caton, J. E.; Reagan, R. R . , Presented on 6th International Symposium on Polynuclear Aromatic Hydrocarbons, Oct 27-29, 1981, Columbus, OH. (7) Yu, M.-,L.;Hites, R . A. Anal. Chem. 1981, 53,951-954. (8) Baifanr, E.; Konig, J.; Funcke, W.; Romanowskl, T. Z . Anal. Chem. 1981, 306, 340-346. (9) Wiik, M.; Rochlitz, J.; Bende, H. J . Chromatogr. 1988, 2 4 , 414-416. (10) Oelert, H. H. 2.Anal. Chem. 1969, 244, 91-101.
1287
(11) Streuli, C. A. J . Chromafogr. 1971, 56, 225-229. (12) Hill, H. H., Jr.; Chan, K. W.; Karasek, F. W . J . Chromafogr. 1977, 131, 245-252. (13) Cautreels, W.; Van Cauwenberghe, K. Wafer, Air, Soil Polluf. 1978, 6 , 103-110.
RECEIVED for review February 8,1982. Accepted March 29,
1982.
Determination of Glyburide in Human Serum by Liquid Chromatography with Fluorescence Detection W. J. Adams," 0. S. Skinner,' P. A. Bombardt, M. Courtney, and J.
E. Brewer
Pharmaceutical Research and Development, The Upjohn Company, Kalamazoo, Michigan 4900 1
A specific ireversed-phase high-performance liquid chromatography method which rvtillzes fluorescence detection for the quantitatlon of glyburide In human serum Is descrlbed. Sample cleanup prlor to chromatographlc analysis was accompllshed by extraction of the drug from acid-buffered rerum wlth toluene. The fluorescence response at 360 nm (308 nm excltatlon) was linear for glyburide concentratlons up to 10 pg/mL and the Sensitivity of the method for quantltatlon was approximately 10 ng/mL (slgnaknoise, 4: 1). Analytical precision, as assessed by analyzing a control standard containing 140 ng/mL glyburide over a 45-day period, was 6.1 % (relative standard devlatlon, m = 18). The analytlcal results were compared wlth those obtained by uslng two different radioimmunoassays.
Glyburide (Micronase, registered trademark, The Upjohn Co.) is an orally active sulfonylurea for the management of maturity-onset diabetes mellitus ( I ) . Because of the potency of glyburide, the classical spectrophotometric, fluorometric, and colorimetric (a) methods that were developed during early phases of its development lack the sensitivity and specificity for monitoring blood levels following administration of therapeutic doses of the drug (maximum serum glyburide concentrations rarely exceed 300 ng/mL in humans following a 5-mg oral dose of the drug). More recently, RIA (3-7);GLC (8)and high-performance liquid chromatography (HPLC) (9, IO) methods have been reported for glyburide in plasma or serum. Although several of the radioimmunoassays are sufficiently sensitive for therapeutic drug monitoring (4, 6, 7), all of these methods suffer to some degree from lack of specificity because of one or more of the following reasons: (1) cross-reactivity with known metabolites of glyburide (3-5); (2) nonspecific binding to plasma proteins (4, 6); (3) crossreactivity with other sulfonylureas (6, 7). The GLC method that was developed for the analysis of glyburide in human plasma requires reaction of the drug with 2,4-dinitrofluorobenzene to form a volatile, thermally stable derivative that is suitable for electron-capture detection (8). This methodology provides high sensitivity and the known metabolites Senior Independent Project Participant, Department of Chemistry, Kalamazoo College, Kalamazoo, MI 49007.
of glyburide do not interfere; however, several other sulfonylureas (e.g., acetohexamide and glipizide) that form identical 2,4-dinitrofluorobenzenederivatives as glyburide can interfere. The HPLC methods that have been reported for glyburide in dog (9) and human (10)serum are reversed-phase procedures that utilize ultraviolet detection. These methods are not suitable for pharmacokinetic and relative bioavailability studies in man following administration of therapeutic doses of glyburide since they do not have sufficient sensitivity to allow glyburide to be determined at serum concentrations less than 50 ng/mL. In the present report we describe a specific and sensitive reversed-phase HPLC method for the quantitation of intact glyburide in human serum which utilizes fluorescence detection. The utility of the methodology was demonstrated by analyzing serum samples from selected subjects participating in a relative bioavailability study. The analytical results were compared with those obtained by using two different radioimmunoassays (4, 7)which have been found convenient for studying the relative bioavailability of glyburide, owing to the speed and ease of sample preparation that these methods afford.
EXPERIMENTAL SECTION Reagents. Glyburide (Figure 1, Ia; 1-[[4-[2-(5-chloro-2methoxybenzamido)ethyl]phenyl]sulfonyl]-3-cyclohexylurea)and 4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenylsulfonamide, an intermediate used to synthesize the internal standard, were supplied by the Pharmaceutical Research and Development Laboratories of The Upjohn Co. The internal standard, 1-[[4[ 2-(5-chloro-2-methoxybenzamido)ethyl]phenyl]sulfonyl] -3-(4isopropylphenyl)urea, was synthesized by condensing 4-isopropylphenyl isocyanate and 4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenylsulfonamide using preparative conditions similar to those used t o synthesize glyburide (11). The 4-isopropylphenyl isocyanate was commercially prepared (Jakem Industries, New Haven, CT) and used without further purification. Synthetic samples of the known metabolites of glyburide in man, 4-trans-[ 1-[ [4-[ 2-(5-chloro-2-methoxybenzamido)ethyl] phenyl]sulfonyl]ureido]cyclohexanol(Figure 1, Ib) and 3-cis-[1-[[4-[2(5-chloro-2-methoxybenzamido)ethyl] phenyl]sulfonyl]ureido] cyclohexanol (Figure 1,IC)were kindly provided by Farbewerke Hoechst AG (Frankfurt, West Germany). Distilled-in-glass spectroscopic grade solvents (Burdick and Jackson, Muskegon, MI) were used as received. Inorganic chemicals were AR grade and were prepared in distilled, deionized water. Apparatus. The high-performanceliquid chromatograph used in this study was a modular component system consisting of a
0003-2700/82/0354-1287$01.25/00 1982 American Chemical Society
~
1288
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
1:
R1
l
la: R 1
=
Ib: R 1
= --OH,
R2
IC: R1 = H, R 2
%-OH
H, R 2
Spiked Control
=H
IS
H
FEgure 1. Molecular structures of glyburide and its major metabolites.
dual piston solvent pump (Model 6000A, Waters Associates, Milford, MA), an in-house designed and fabricated autoinjector (12)fitted with a 250-pL sample loop, a commercially prepared 25 sm x 4.6 mm i.d. column packed with 10-pmLiChrosorb RP-8 (E. Merck Laboratories, Elmsford, NY), a dual monochromator spectrofluorometer equipped with a 28-pL flow cell (Model 204B, Perkin-Elmer, Norwalk, CT), and a variable sensitivity recorder (Model 355, Linear Instruments, Irvine CA). The autoinjector was modified to provide for the accurate injection of small sample volumes by replacing the vacuum sample aspirator with a precision motorized buret (Metrohm Model E412, Brinkmann Instruments, Westbury, NY) and using microsample vials (Waters Associates, Milford, MA). A 2.5 cm X 3.9 mm i.d. guard column (Waters Associates, Milford, MA) dry-packed with 3C-40 pm Perisorb RP-8 (E. Merck, Darmstadt, West Germany) was used to protect the analytical column from fouling. A two-speed reciprocating shaker (Eberbach and Sons, Ann Arbor, MI) was used for extraction of the samples. Glassware was rinsed with distilled water and distilled-in-glass acetonitrile prior to use. Standards. Stock solutions of internal standard (10 pg/mL) and glyburide (1pg/mL) were prepared in acetonitrile. A 1 pg/mL working internal standard solution was prepared by diluting an aliquot of the stock solution with acetonitrile. Reference standard glyburide solutions containing 500,400, 300, 200,100,40, 20, 10, and 0 ng/mL glyburide, respectively, were prepared by making appropriate dilutions of the 1pg/mL stock solution of glyburide with acetonitrile. Control serum containing 140 ng/mL glyburide was prepared by transferring an aliquot of the 1pg/mL glyburide solution to a volumetric flask, evaporating the solvent to dryness at 40 "C using a stream of filtered nitrogen, and diluting with blank serum. Sample Preparation and Extraction. Calibration curve standards bere prepared for extraction by adding 1 mL of the appropriate calibration curve standard and 1 mL of internal standard to 16 X 125 mm screw-cappedcentrifuge tubes that were fitted with Teflon-lined caps and evaporating the solvent to dryness at 40 OC using a stream of fitered nitrogen (Organomation Associates, Shrewsburg, MA). One milliliter of control serum was then added to each standard tube and the samples were throughly mixed on a vortex mixer (Lab-line Instruments, Melrose Park, IL). The unknowns and control serum samples were prepared for extraction by adding 1mL of internal standard to 16 X 125 mm screw-capped culture tubes fitted with Teflon-lined caps and evaporating the solvent to dryness at 40 "C using a stream of filtered nitrogen. One-milliliter aliquots of the unknowns or controls were then added to each tube and the samples were thoroughly mixed on a vortex mixer. The samples were buffered with 1mL of freshly prepared 2.55 M NaH2P04and extracted with 10 mL of toluene for 20 min on a reciprocating shaker (280 cpm). After separation of the phases by centrifugation for 10 min at 2200 rpm, approximately 9.5 mL of the toluene was transferred to a 15-mL conical test tube and evaporated to dryness at 40 "C using a stream of filtered nitrogen. The walls of the test tubes were rinsed with 1mL of acetone and the solvent was evaporated to dryness at 40 "C using a stream of filtered nitrogen. The residues were solubilized in 60 pL of acetone, diluted with 240 ,uL of 0.05 M NH4H2P04,and thoroughly mixed by vortexing. Chromatography. A 250-pL aliquot of each sample was chromatographed at ambient laboratory temperature using a mobile phase containing 0.05 M NH4H,PO4/acetonitrile (52/48, V/V) and a flow rate of 1.4 mL/min. Under these chromatographic
-
0
. -
8
5
10 15 20 RETENTION TIME (Min )
25
Figure 2. Typical chromatograms of extracts of human control serum, and human serum containing approximately 100 ng/mL glyburide (G) and internal standard (IS.). Endogenous components present in the extraction solvent are indicated by an asterisk.
conditions, the capacity factors (k') for glyburide and the internal standard were 4.6 and 7.3, respectively. The column eluate was monitored with a dual monochromator fluorescence detector using an excitation wavelength of 308 nm, an emission wavelength of 360 nm, and 10-nm slit widths. The concentration of glyburide in the unknowns was calculated from peak height ratios using the slope and intercept calculated by linear regression analysis of the calibration curve data. I n Vivo Studies. Apparently healthy nondiabetic adult male volunteers were fasted 10 h prior to treatment and for 6 h following administration of a 5-mg compressed tablet of glyburide. Blood specimens were obtained by venipuncture at predetermined times ranging from 0 to 24 h after drug administration. The blood was allowed to clot and the collected serum was stored frozen at -20 "C until analysis. Statistical Analyses. Statistical comparisons were made by using the SAS statistical analysis programs (13). R E S U L T S A N D DISCUSSION Typical chromatograms of extracts of blank serum and serum containing approximately 100 ng/mL glyburide are shown in Figure 2. The retention times of glyburide and the internal standard were approximately 10 and 15.5 min, respectively. The known metabolites of glyburide in man (14), 4-trans- [ 1-[ [ 4- [ 2-(5-chloro-2-methoxybenzamido)ethyl]phenyl]sulfonyl]ureido]cyclohexanoland 3-cis-[l-[[4-[2-(5chloro-2-methoxybenzamido)ethyl]phenyl]sulfonyl]ureido] cyclohexanol, eluted near the solvent front (approximately 2.7 and 3.2 min, respectively), precluding their quantitation using this methodology. All other sulfonylureas that are currently marketed, acetohexamide, carbutamide, chlor-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
1289
Table I glYburide concn, ng/mL 0 20 40 60 100 200 300 400 500
slope intercept r2 a
peak height ratio
-day 1
day 2
a a
0.00 0.0639
0.1221 0.1869 0.2943 0.6240 0.89!56 1.1912 1.4932
a
0.002973 0.0073 0.9 9 9 6
0.003919 0.0080 0.9966
0.2705 0.4094 0.7334 1.2508 1.6064 1.9215
day 3
day 4
0.00 0.0470 0.1032 0.1519 0.3046 0.5841 0.8812 1.2158 1.4203
0.00 0.0995 0.1362 0.1764 0.2880 0.5382 0.8625 1.1782 1.4171 0.002832 0.012 0.9980
0.002935
--0.0052 0.9978
--
day 5 a
a a
0.1580 0.2917 0.5227 0.7613 1.0644 1.3361 0.002638 0.0030 0.9980
day 6
day 7
0.00 0.0825 0.1379 0.2396 0.3723 0.7900 1.1082 1.6356 2.1012
0.00 0.0814 0.1458 0.1556 0.3223 0.5517 1.2263 1.3068 1.7349
day 8
0.0041 50 --0.032 0.9958
0.003496 -0.015 0.9807
0.00 a
0.1262 0.2016 0.3300 0.6693 1.0244 1.4008 1.8575 0.003650 -0.029 0.9972
Sam& lost because of instrument/technical problems.
propamide, glibornuride, glipizide, tolazamide, and tolbutamide, elute prior to glyburide and do not interfere in the analysis. The three peaks eluting a t approximately 18.5,22, and 25 min, respectively, correspond to impurities present in the distilled-in-glass solvents. The retention time of the potential interference eluting just after glyburide was sensitive to the composition of the mobile phase and care was necessary in the preparation of the mobile phase to ensure adequate resolution of glyburide from this compound. Linear regression analysis of calibration curve data indicated no significant deviations from linearity (r2= 0.9998) for glyburide concentrations up to 10 pg/mL. Correlation coefficients for standard curves run over a 45-day period and having glyburide concentrations ranging from 0 to 500 ng/mL were better than r2 = 0.9807, with the mean correlation coefficient being r2 = 0.9954 (Table I). Standard curve intercepts were not significantly different, from zero (p 7 0.05) for all curves, indicating negligible interference from endogenous compounds. The day-to-day variation in the slopes of the standard curves is a reflection of both the interassay variability of the method and the fact that not all of the curves were prepared by using the same concentration of internal standard. An estimate of the interassay reproducibility and precision was obtained by comparing the standard curves prepared on assay days 3,4, and 5-all of which were prepared with the same internal standard solution. The ialopes for these three curves ranged from 0.002 64 to 0.002 94 mL/ng with a mean f percent relative standard deviation of 0.002 80 mL/ng f 5.4%. The percent relative standard deviations of the interassay ordinate values for the three curves were f7.970, h3.0%, h5.870, f7.7%, *6.8%, and f3.4% for concentrations (abscissa) of 60, 100, 200, 300, 400, and 500 ng/mL, respectively. The sensitivity of the method for quantitation is approximately 10 ng/mL glyburide, which gives a response equivalent to 4 times the peak-to-peak noise in the detector signal. Analytical precision and accuracy were established by fortifying control serum with 140 ng/mL glyburide (approximately half the maximum serum glyburide concentration achieved in man following a 5-mg oral dose of the drug) and analyzing replicate samples each day unknowns were analyzed. An average recovery of 96.4 f 6.1% (n = 18) was obtained for samples analyzed over a 45-day period, in excellent agreement with the recovery previously reported for the extraction of glyburide from dog serum using identical extraction conditions (9). Serum samples from selected nondiabetic male subjects participating in a glyburbide bioavailability study were analyzed by using the described HPLC procedure and compared with the analytical results obtained by using the radioimmunoassay methods of Royer et al. (4)(RIA1) and Heptner
100 80
-
60
-
A
G
2
5
40
z
P
8
A 0
-
0
-
4 K
z
Ez
20
-
0
u w
P
2
10-
d
8 -
>
-
z
~6 u
A
l o )
0
-
4 t 0
0
2
4
6
8
10
12
TIME AFTER DRUG ADMINISTRATION (HRS)
Flgure 3. Serum glyburide profiles in a nondiabetic subject following oral administration of a 6-mg compressed tablet. Samples were anref 7; and HPLC (0). alyzed by: RIA, (A),ref 4; RIA, (O), Serum glyburide concentrations less than the sensitivity of the RIA,, RIA,, and HPLC methods for qluantitation (5,3, and 10 nglmL, respectively) were set equal to zero. 'The calculated half-lives of elimination for the RIA,, RIA,, and HPLC methods were 1.87, 1.47, and 1.42 h, respectively. The RIA, Serum drug concentration data suggest a biphasic elimlnation profile for tho drug.
et al. (7) (RIA,)--met,hods that have been found convenient for studying the relative bioavailability of glyburide owing to the speed and ease of sample preparation that they afford. Typically, higher serum glyburide concentrations were found by RIAl than by RIAz or HPLC (Figure 3). Regression analysis of the concentration data confirmed that substantially higher serum glyburide concentrations were found by RIAl than by either RIA, or HPLC ( p < 0.05); furthermore, significantly higher serum glyburide concentrations were found by RIAz than by HP'LC ( p < 0.05). Comparison of serum glyburide concentration ratios calculated for each pair of analytical methods indicated that the concentration ratio (y) was a linear function of the time after drug administration ( t )that the sample was collected for RIA1/HPLC [ y = (0.084 f 0.014 h-l)t + (1.214 f 0.084); rz = 0.56821 and RIAl/RIAz, [y = (0.77 f 0.014 h-l)t + (1.18 f 0.085); 1.2 = 0.52021, whereas, the concentration ratio was independent of the sample collection time for RIA2/HPLC [ y = (1.078 f 0.025); rz = 0.01. The observed differences in the RIA, and RIA, concentration
1290
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
Table 11. Selected Pharmacokinetic Parameters: Glyburide in Nondiabetic Subjects subject no.
treatment day
1
1 8 1 8 1 1 8 1 8 1 8
2 3 5 7 9
mean ESD
tu?
RIA,d
t,, ,a h RIAZe
HPLCf
6 6 3 3 4 3 6 4 5 4 3 4.3 1.3
6 6 2 3 3 2 6 4 5 4 3 4.0 1.5
6 6 3 3 3 2 6 4 5 4 3 4.1 1.4
RIA, 2.56 2.50 3.36 2.44 2.30 2.13 2.45 1.87 1.70 2.30 5.57 2.65 1.06
j b
h
RIA, 2.02 2.70 2.73 2.02 2.56 1.79 1.70 1.47 1.27 1.93 2.01 2.02 0.48
AUC (ng-h/mL)C HPLC
RIA,
RIA,
2.17 2.20 2.57 2.00 2.23 2.19 2.64 1.42 1.70 1.99 2.24 2.12 0.35
1151 1601 671 851 780 661 457 412 520 1353 615 825 385
778 1012 411 594 397 418 310 286 344 825 257 512 254
HPLC 590 874 232 504 408 394 170 267 266 619 204 41 2 218
a Time after drug administration that the maximum serum glyburide concentration was observed. Estimated half-life of elimination assuming first-order kinetics. Elimination rate constants were calculated by linear regression analysis of the data from the terminal portion of the serum concentration-time curve. For a given subject and treatment day, the rate constants for each method were calculated over the same time interval. Integrated area (trapezoidal rule) under the serum concentration-time curve during the 0-24 h time interval expressed as ng.h/mL. Serum concentrations less than the sensiSerum glyburide concentrations detertivity of the respective methods for quantitation were assigned a value of zero. mined by using the radioimmunoassay of Royer et al., ref 4. e Serum glyburide concentrations determined by using the radioimmunoassay of Heptner et al., ref 7. f Serum glyburide concentrations determined by using the high-performance liquid chromatographic method described in the present report.
data are not unexpected since the Royer et al. radioimmunoassay cross-reacts with the 4-trans-and 3-cis-hydroxymetabolites of glyburide (84% and 75%, respectively) to a much greater extent than does the Heptner et al. radioimmunoassay (0.1% and 1.5%, respectively). Furthermore, the gradual increase in the RIA,/HPLC and RIA1/RIA2 concentration ratios with time after drug administration is symptomatic of cross-reactivity of RIAl with major metabolites of glyburide. The observed differences in the RIA, and HPLC data may be due, at least in part, to the low recovery of the HPLC method (96.4f 6.1%) and/or to nonspecific protein binding by the Heptner et al. radioimmunoassay. Selected pharmacokinetic parameters for glyburide are listed in Table I1 in order to allow further comparison of the analytical methods and to determine the suitability of the RIA methods for bioavailability/bioequivalence studies of glyburide formulations. Sophisticated pharmacokinetic modeling (15, 16) was not attempted since this protocol was not designed as a pharmacokinetic study. Experimentally observed maximum serum glyburide concentrations were achieved at times (tmm)ranging from 2 to 6 h after drug administration, with the mean t,, being approximately 4 h for all three methods. The mean t , of RIAl was slightly longer than the mean t,, values for RIA2 and HPLC; however, the values were not statistically different (p > 0.05). Half-lives of elimination (tllz) were calculated assuming first-order elimination kinetics. For a given subject and treatment day, the elimination rate constants were calculated by regression analysis of the data over the same time interval for all three analytical methods-with the initial time taken as t,, of the analytical method having the longest t,, and the final time taken as the time immediately preceding the time at which the serum glyburide concentration determined by the HPLC method was less than 10 ng/mL (approximately 10-12 h). Although the elimination half-lives calculated for each method were not statistically different 0, > 0.05),the RIAl mean half-life of elimination was somewhat longer than the RIA, and HPLC mean halflives. The observed half-lives are comparable to those reported in previous studies in nondiabetic humans ( I 7,18) and in dogs (9) in which the data were analyzed over a comparable time interval using the procedures of this report. Examination of the RIA, serum drug concentration data, at times greater than approximately 8 h after drug administration and at drug
concentrations 110 ng/mL suggests a biphasic elimination profile for the drug as previously reported (17). As expected, the areas under the serum concentration-time curves (AUC) from 0 to 24 h (trapezoidal rule) increased in the order HPLC < RIAz < RIA1. Regression analysis of the AUC data indicated that the areas of all three methods were significantly different ( p C 0.05) but were highly correlated ( r2 2 0.9897). Because of the high correlation of the AUC data, valid conclusions concerning the relative bioavailability/bioequivalence of glyburide formulations can be reached using any one of the three analytical methods, so long as the same method is used to analyze all the samples from a particular study. In conclusion, the high-performance liquid chromatographic method described in this report is suitable for monitoring the bioavailability/ bioequivalence of pharmaceutical formulations, for therapeutic drug monitoring, and for forensic toxicology. Furthermore, the radioimmunoassay methods developed by Royer et al. and Heptner et al. have been shown to be suitable for monitoring the bioavailability/bioequivalenceof glyburide formulations, with the Heptner et al. RIA being the method of choice for the expeditious analysis of samples because of its specificity and sensitivity.
ACKNOWLEDGMENT The synthesis of 1-[[4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl]sulfonyl-3-(4-isopropylphenyl)ureaby D. Rector and the analysis of serum samples using the Royer et al. radioimmunoassay by J. M. Wozniak are gratefully acknowledged. We thank W. Heptner, Farbewerke Hoechst AG, Frankfurt, West Germany, for sharing with us the analytical results obtained by using the radioimmunoassay developed in his laboratory. Helpful discussions with H. KO, D. G. Kaiser, R. M. Norris, and W. E. Dulin are also acknowledged, as is the preparation of this manuscript by S. E. Yoder.
LITERATURE CITED (1) Drugs 1977, 1 , 116. (2) Hajdu, P.; Kohler, K. F.; Schmidt, F. H.; Springler, H. Arzneim.-Forsch. 1989, 19, 1361. (3) Glogner, P.; Burmeister. P.; Heni, N. Klin. Wochenschr. 1973, 5 1 ,
352.
(4) Royer, M. E.; KO, H.; Evans, J. S.; Johnston, K. T. Anal. Lett. 1976, 9 ,
629.
(5) Glogner, P.; Heni, N.; Nissen, L. Arzneim. Forsch ./Drug Res. 1977, 27, 1703.
Anal. Chem. 1982, 5 4 , 1291-1296 (6) Kawashima, K.; Kuzuya, T.; Matsuda, A. Diabetes 1979, 28, 221.
(7) Heptner, W.; et al., Farbewerke Hoechst AG, Frankfurt, West Germany, unpublished work. (8) Castoldi, D.; Tofanetti, 0.Clin. Chlm. Acta 1979, 93, 195. (9) Adams, W. J.; Krueger, D. S. J . Pharm. Scl. 1970, 68, 44‘15. (10) Reinauer, H.; Lindner, CL; Oldendorp, J. Ffesenius’ 2. Anal. Chem 1960, 301, 110. (11) Hsi, R S. P. J , LabelledCompd. 1972, 9 , 91. (12) Beyer, W. F.; Gleason, D. D. J . Pharm. Scl. 1975, 6 4 , 3420. (13) Helnig, J. T.; Council, I
