in blood by differential pulse polarography

---

computer or when applied signals of very high frequency are employed.

proach to ac polarography in the harmonic multiplex mode because it replaces numerous analog circuits, which require tuning, offset adjusts, etc., by a drift-free, highly automated digital program. On the other hand, because it places minimal demands on data acquisition capability, an analog conditioning approach such as the one used in this work is favored whenever digital data acquisition rates are too slow to meet the demands of digital signal conditioning-e.g., whenever one has to operate with a slob dedicated minicomputer system, or with a low-priority time shared terminal to a large

ACKNOWLEDGMENT

The authors are indebted to Barry J. Huebert for helpful discussions and programming assistance. RECEIVED for review January 5 , 1972. Accepted February 9, 1972. D. E. G .is an NASA Fellow 1971-72. Work supported by National Science Foundations Grant GP-16281.

Determination of Glibornuride (a Tolylsulfonyl Urea Hypoglycemic Agent) in Blood by Differential Pulse Polarography J. Arthur F. de Silva and Martin R. Hackman Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche lnc., Nutley, N.J. 07110 A sensitive and specific differential pulse polarographic assay was developed for the determination of glibornuride (a tolylsulfonylurea hypoglycemic a ent) in blood. It involves the selective extraction the compound from whole blood into diethyl ether and Following suitable back-extraction into 1.ON “,OH. “clean-up” of the sample, the compound is nitrated in 10% KNOa/H2S04at 105 O C for 2 hours. The nitro derivative is extracted into ethyl acetate, the residue is dissolved in 0.1N NaOH, deoxygenated, and analyzed by differential pulse polarography. The overall recovery of the assay is 80.7% i 8.0 (Std dev) from blood and the sensitivity limit is 0.05-0.10 pg/ml of blood (using a 2-ml sample per assay). The method was applied to the determination of blood levels of the intact drug in man following single oral doses of 50 and 100 mg of glibornuride.

09

GLIBORNURIDE; 1-[(1R)-d-endo-hydroxy-3-endo-bornyl]-3-(ptolylsulfony1)urea (Ro 6-4563/00) is a new member of the tolylsulfonylurea class of compounds being tested as hypoglycemic agents (1). The chemistry (2), pharmacology (3-6), and toxicology (7) of the drug in several animal species have been reported. The compound has been extensively tested in man as a oral hypoglycemic agent (8-11) and has been reported to be more effective at lower doses than either tolbutamide or chlorpropamide (12,13). (1) “Recent Hypoglycemic Sulfonylureas-Mechanisms of Action and Clinical Indications,” U. C. Dubach and A. Biickert, Ed., Hans Huber, Bern, Switzerland, 1971, pp 1-327. (2) H. Bretschneider, ibid., pp 22-33. (3) 0. Oelz and E. R. Froesch, ibid., pp 36-48. (4) E. Lorch and K. F. Gey, ibid., pp 49-55. (5) J. Beyer, U. Cordes, H. Krall, W. Ewald, and K. Schoffling, ibid., pp 56-63. ( 6 ) U. Cordes, J. Beyer, H. Krall, E. Haupt, C. Rosak, and K . Schoffling, ibid., pp 6 4 8 . (7) K. Scharer and H . Hummler, ihid., pp 163-70. (8) D. Pometta, W. Stauffacher, G. Zahnd, H. Micheli, and A. E. Renold, ibid., pp 182-8. (9) U. C. Dubach, A. Biickert, and I. Forg6, ibid., pp 187-99. (10) E. Haupt, J. Beyer, W. Koberich, K. M. Bartelt, U. Cordes, C . Rosak, and K. Schoffling, ibid., pp 200-205. (11) N. 0. Lunell, B. Persson, and J. Thorell, ibid., pp 234-40. (12) W. Ewald, W. Kunkel, M. D. Saenger, and K . Schoffling, ibid., pp 278-82. (13) A. Biickert and E. Schweda, ibid., pp 283-8.

Studies on the absorption, metabolism, and elimination of the drug in man, dogs, and rats by. Bigler et al. (14-16) indicated that it was well absorbed, extensively metabolized (Figure l), and eliminated in man with a mean half-life of 8 hours (range 4.7 to 11.5 hours). Following oral administration, intact drug was the major component in the blood but only trace amounts of the intact drug could be found in the urine. The compound undergoes oxidation at the methyl group in the phenylsulfonamide portion of the molecule to produce the alcohol (M- l), which in man is further oxidized to the carboxylic acid (M-2). Oxidation and hydroxylation also took place in the borneol moiety of the compound resulting in the introduction of hydroxyl groups in several positions producing the metabolites (M - 3 to M -6.) The chemical structures of the metabolites (M-1 to M-6) were verified by thin layer chromatography (TLC) of their respective dinitrophenyl (DNP)aminoborneol derivatives ( 1 9 , by pyrolysis gas chromatography, and mass spectrometry of the respective trimethylsilyl (TMS)- aminoborneol derivatives (16). The chemical synthesis of the respective authentic reference compounds has also been reported (17). The enzymatic hydrolysis of glibornuride to 3-endo-aminoborneol and p-tolylsulfonamide has not been established although chemical hydrolysis of the compound can be effected (Figure 1). Spectrophotometric methods for the determination of a similar compound, tolbutamide, in plasma based on the formation of a 2,4-dinitrophenyl (DNP) derivative (18) and a p-N-dimethylaminobenzaldehyde derivative (Ehrlich’s Reagent) (19)have been reported. The strong UV absorption of tolbutamide at 228 nm was also used in the determination of the drug in plasma (20-22). (14) F. Bigler, G. Rentsch, and J. Rieder, ibid., pp 171-80. (15) F. Bigler, J. Rieder, and G. Rentsch, F. Hoffmann-La Roche & Co., Ltd., Basle, Switzerland, unpublished data, 1969. (16) F. Bigler, P. Quitt, M. Vecchi, and W. Vetter, Arz/teim.-Forsc/i., in press. (17) P. Quitt, Cliirnia, 24,452 (1970). (18) H. Spingler, K/in. Woc/ie/isc/ir.,35, 533 (1965). (19) T. Chulski, J . Lab. C/i/i.Med., 53,490 (1959). (20) H . Spingler and F. Kaiser, Armeim.-Forsc/i, 6 , 700 (1956). (21) A. A. Forist, Proc. Soc. Ex/J.Biol. Med., 96, 180, (1957). (22) E. Rladh and A. Norden, Acta Pharmacol Toxicol., 14, 188 (1958).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972

0

1145

.

Glibornuride (Ro 6-4563)

CH EM IC AL

-.

6

CH,~SO,NHCO HNH

M

M-3

\

H Y DR 0 LYSIS

M-4

M-1

\\

OX IDATION

M

p-tolylrrulfonomide

3-endo-omino

M-5

- borneol U-6

M-2

Figure 1. Postulated metabolic pathways of glibornuride (Ro 6-4563) in man (M), rat (R) and in the dog (D) according to Bigler et al. (14-16)

These methods were sufficiently sensitive for the quantitation of blood levels of tolbutamide in man ranging from 30 to 200 pg/ml resulting from oral doses ranging from 1000 to 2000 mg of the drug. However, these methods were not sufficiently sensitive for the determination of blood levels of glibornuride in man resulting from oral doses ranging from 25 to 100 mg. A gas chromatographic (GLC) assay for tolbutamide and chlorpropamide (23) was investigated for the analysis of glibornuride but was unsuitable since the compound underwent pyrolysis on the chromatographic column to the p tolylsulfonamide and 3-endo-aminoborneol moieties (Id). A spectrophotometric method for the determination of tolylsulfonylurea derivatives in blood (24) involving ring nitration, reduction to the aromatic amine, and quantitation as the Bratton-Marshall chromophore (25) was explored as a sensitive assay. The principle of controlled nitration was used in this study for the determination of glibornuride in blood. The nitro derivative formed (Figure 2) was extracted and analyzed by differential pulse polarography employing electrochemical reduction of the NOzgroup. EXPERIMENTAL

Assay in Whole Blood. Reagents. All reagents used were of analytical grade purity (>99W) and were used without any further purification. All aqueous reagents were made in double distilled water. Reagents used included 1.ON ammonium hydroxide (aqueous); 6N hydrochloric acid (aqueous) ; benzene (reagent grade) ; diethyl ether (absolute anhydrous) Mallinckrodt ; ethyl acetate; and 0.1N sodium hydroxide (aqueous) as supporting electrolyte. The nitration mixture (10% KN03/HzS04)is prepared as follows: Dissolve 5 grams of KNOI into 50 ml of concentrated HzS04. Store in refrigerator. Prepare fresh weekly because of limited shelf life. (23) K. Sabih and K. Sabih, J. Pharm. Sci., 59,782 (1970). (24) W. Kern, ANAL.CHEM., 35, 50(1963). ( 2 5 ) A. C. Bratton and E. K. Marshall, J. Biol. Clrern., 128, 537 (1939). 1146

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972

Standard Solutions. Glibornuride (Ro 6-4563), C18H26Nz04S, mol wt = 366.48, mp = 186.8-187.4 "C (decomp.). STOCKSOLUTION.Disolve 10 mg of glibornuride in 100 ml of absolute ethanol to give a stock solution (A) containing 100 /Jg/ml. WORKINGSOLUTION.Dilute 1 ml of (A) to 10 ml with ethanol to give a working solution (B) containing 10 pg/ml. Suitable aliquots of (B) are used as internal standards added to blood for the determination of % recovery. Parameters for Polarographic Analysis. Instrument. Princeton Applied Research Instruments: P.A.R. Model 171 Polarographic Analyzer equipped with a P.A.R. Model 172 Drop Timer and stand assembly. Electrode Assembly. A three-electrode polarographic cell (modified Melabs cell No. 22820-1) containing a dropping mercury electrode, DME (capillary tube of inside diameter = 0.05-0.08 mm from E. H. Sargent No. S-29417) as the analyzer electrode, a saturated calomel electrode (SCE) (Beckman No. 39178-fiber junction calomel) as the reference and platinum wire as the auxiliary electrode, is assembled as shown in Figure 3. The DME had a drop rate of 2.32 mg/sec., [mz'at1'6 = 1.5611, and a drop time of 0.5 second. Instrumental Parameters (26). OPERATING MODE. Derivative pulse (Le., constant amplitude pulse superimposed on linear d.c. ramp. PULSEAMPLITUDE.(-) 50-mV peak to peak (applied for a 56-msec interval before drop dislodgement). CURRENT READOUT.Differential (i.e., signal displayed is due to difference in current sampled for a 16-msec interval prior to pulse application and for a 16-msec interval before drop dislodgement). Other parameters include scan, from (-) 0.300 V to (-) 0.600 V os. SCE; scan rate, 5 mV/second; scan range, 1.5 volts; and current range, 1 pA full scale deflection. Procedure. Into a 50-ml glass stoppered centrifuge tube, add 2 ml of whole blood specimen and 10 ml of diethyl ether. Stopper the tube (seal stopper with a drop of distilled water), and shake on a reciprocating shaker for 20 minutes at a moderate speed; then centrifuge for 5 minutes at 2000 rpm. Along with the samples, process a 2-ml specimen of control (26) Princeton Applied Research Corporation Instruction Manual for PAR Model 171 (IV-31), Princeton, N.J., 1970.

"8

(NIT RAT ION)

3-endo-amino -borneol

GI i bot-nuride (RO -6-4563)

< HO-2

0

vs S.C.E.

"

Figure 2. Postulated nitration and polarographic reduction reactions of glibornuride

blood (taken prior to medication or from a pooled control source), and separate 2-ml specimens of control blood containing 1.0, 1.5, and 2.0 Mg of glibornuride [prepared by adding 0.1, 0.15, and 0.2 ml of working standard (B), respectively, to 50-ml centrifuge tubes, evaporating the ethanolic solution to dryness in a 50 "C water bath under a stream of nitrogen, and then adding 2 ml of control blood to the residue]. These internal standards are used to construct a calibration curve for the quantitation of the drug in the unknowns and also for the determination of per cent recovery. Transfer an 8-ml aliquot of the supernatant ether layer to a 15-ml conical glass stoppered centrifuge tube. To each tube, add 2.5 ml of 1.ON N H 4 0 H , stopper and seal in the usual manner, and extract on the reciprocating shaker for 10 minutes. Centrifuge the samples for 5 minutes at 2000 rpm, and aspirate off the supernatant ether layer. Add another 5 ml of ether to each tube, stopper and backwash the sample on a vortex action super mixer (Lab-Line) for 1 minute. Centrifuge and aspirate off the ether layer. Remove all traces of residual ether by warming the tubes in a 50 "C water bath under a stream of nitrogen for approximately 2 minutes. Add 0.5 ml of 6N HC1 to neutralize the ",OH, mix well on the vortex super mixer and add 5 ml of benzene. Stopper and shake the sample on a reciprocating shaker for 10 minutes, centrifuge for 5 minutes, and transfer a 4.5-ml aliquot of the benzene layer into a 15-ml conical glass stoppered centrifuge tube. Evaporate the benzene to dryness in a 75 "C water bath under a stream of nitrogen and vacuum dry in a desiccator for 10 minutes to remove all traces of benzene prior to nitration. To the vacuum dried residue, add 0.3 ml of nitration mixture (at room temperature). Mix on the vortex super mixer for 15 seconds and stopper. Seal the stopper by moistening with a drop of the nitration mixture. At this point in the analysis, include a set of external standards of glibornuride of 1.0, 1.5, and 2.0 pg of compound (prepared as described for the internal standards, dissolving the residue of the ethanolic solutions in 0.3 ml of the nitration mixture) as a check on the nitration reaction, Place in an oven at 105-107 "C for 2 hours to complete the nitration reaction. Remove the tubes from the oven and cool to room temperature. Carefully evacuate the nitration mixture in each tube under vacuum (e50p) for 15 min to remove any residual "02 that is present. This step is performed in a vacuum desiccator containing Ca(OH)* as the desiccant, with a dry ice-acetone solvent trap connected between the desiccator and the vacuum pump to trap the HN02/H2S04vapor. Carefully add 2.7 ml of distilled water to each tube, mix well on the super mixer, and evacuate from a water line aspirator for 10 min. Add 5 ml of ethyl acetate to each tube, stopper, and shake on the reciprocating shaker for 10 minutes to extract the nitro derivative of the compound. Centrifuge the samples for 5 minutes at 2000 rpm and transfer the ethyl acetate supernatant into a 15-ml conical centrifuge tube. Repeat the extraction using another 5 ml of ethyl acetate, shaking for 10

9

>.ME.

n

N; DE-GASSING

Figure 3. Modified Melabs polarographic cell (No. 22820-1) containing the three operational electrodes for analysis = dropping mercury electrode Pt Aux. = platinum auxiliary electrode SCE = saturated calomel electrode

DME

minutes and centrifuging for 5 minutes, and combine the extracts. Evaporate the combined ethyl acetate extract to dryness in a 75 "Cwater bath under a stream of nitrogen. To the residue, add 5 ml of 0.1 N NaOH. Mix well on the super mixer and deoxygenate the samples for 5 minutes with nitrogen (prepurified-Matheson) introduced through a sintered glass (fritted) tube. Transfer the degassed sample into the polarographic cell containing the.3 operational electrodes, and blanket the sample with a stream of N2 flowing over the surface of the sample. Obtain a polarogram by scanning the sample from (-) 0.300 volt (us SCE) to (-) 0.600 volt (us. SCE) using the differential pulse mode of operation, and the polarographic parameters previously described. ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972

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B

T

y CURRENT

T

I 1

4500 -hmO POTENTIAL (VOLTS vs.S.C.E.1

I

-0.300 -04Cn

d POTENTIAL (VOLTS vs. S.C.E.) m

Figure 4. Polarograms of ( A ) nitrated derivative of glibornuride and ( B ) nitrated p-tolylsulfonamide and p-tolylsulfonic acid

Table I. Recovery of Glibornuride from Blood Determined Following Nitration by Differential Pulse Polarography Diffusion current measured in PA Concentration Blood glibornuride recovered added : internal External Recovery, pg/ml of blood standard standard 1 .o 1.o 1 .o 1.5 1.5 2.0 2.0 2.0 2.5 2.5

z

0.0393 0.0437 0,0547 0.0737 0,0793 0,0985 0.0793 0.1067 0.1340 0.1367

Overall recovery 80.7

0.0531 0.0492 0,0591 0.0965 0.0933 0.1268 0.1240 0.1280 0.1622 0.1654

74.0 88.8 92.5 76.4 85.0 77.7 64.0 83.4 82.6 82.6

8.0 (std dev).

of the diffusion current (produced by the reduction of the electroactive species) to concentration. The equation can be simplified as follows : Ai,,, = k . C

where Ai,,, = peak current (PA) at E, (peak potential), C = concentration of electroactive species in moles/liter, and k = constant for the polarographic parameters used. The concentration of the nitrated drug in the external standards, the recovered internal standards, and in the unknowns is determined by measuring the peak height as shown in Figure 4A. Calibration curves of the external and internal standards are made by plotting peak current (Aima,) in pA us. concentration (pg/ml) as shown in Figure 5. The amount of drug in the unknowns is determined either by interpolation from the internal standard curve, or by direct comparison to a given internal standard; thus: Concn of Int. standard (pg) (Aimax)pA unknown ml of Sample (AimaI) pA Int. std X =

The polarograms are recorded on an X-Yrecorder and show a peak at ED = -0.500 V us. SCE characteristic of the reduction of the -NOz group (Figure 4A). Calculations. The quantitation of the nitrated compound is based on the direct proportionality of the diffusion current produced (id) us. amount of the nitrated drug present as defined by the Ilkovic equation (27). Parry and Osteryoung (28) have derived an equation for the processes involved in pulse polarography (29) which relates the faradaic component (27) D. R. Crow and J. V. Westwood, “Polarography,” Methuen & Co., Ltd., London, 1968, p 164. (28) E. P. Parry and R. A. Osteryoung, ANAL. CHEM.,31, 1634 (1965). (29) H. Schmidt and M. von Stackelberg, “Modern Polarographic Methods,” Academic Press, New York, N.Y., 1963, pp 63-70. 1148

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972

(1)

pg glibornuride/ml of blood (2)

The overall recovery is determined by direct comparison of the slope [pA/pg/ml of 0.1N NaOH] of the internal standard curve to that of the external standard curve, thus : [pA/pg/ml] internal std X 1.389 X 100 = [pA/pg/ml] external std

Recovery (3)

where 1.389 is the correction factor for the aliquots used (10/8 X 5/4.5). The assay has an overall recovery of 80.7 rt 8.0 (std dev) (Table I) and a sensitivity limit of 0.05 to 0.10 pg glibornuride/ ml of blood using a 2-ml sample per assay and a 2 :1 sample to blank diffusion current (id) value as the limit of detectability. Calibration curves of external standards of pure compound and internal standards of recovered (spiked) compounds should be prepared daily for the routine determination of the

CONC. GLIBORMJRIDE @g/mDin0.1N NaOH Figure 5. Polarographic calibration curves of glibornuride

p-tolyl sulfonamide

3-endo -amino

borneol

+

,dinitrof luoro benzene (DNFB)

* *

. D N P . A M I N O BORNEOL (yellow d e r i v a t i v e )

v

DETERMINED BY:

a) SPECTROPHOTOMETRY AT 380 nm.

b) POLAROGRAPHY OF NITRO GROUP REDUCTION. c) RADIOMETRY USING H : - D N F B REAGENT.

Figure 6. 2,4-Dinitrofluorobenzene(DNFB) reaction with glibornuride per cent recovery and as a check on the precision and reproducibility of the assay.

RESULTS AND DISCUSSION The phenyl and tolylsulfonylurea have achieved widespread clinical use as oral hypoglycemic agents in the management of diabetes mellitus ( I ) . In contrast to the precursor of this series, carbutamide, which has a reactive aromatic amine group, the phenyl and tolyl analogs lack

reactive functional groups amenable to direct chemical reactions. Hence they have to be derivatized either at the aryl (24) or the aliphatic portion of the molecule (18, 30). Unlike other tolylsulfonylureas, glibornuride has very poor W absorption characteristics, probably due to the electron (30) F.

c. McIntire, L. M. Clements, and

Muriel Sproull, ANAL.

CHEM., 25, 1757 (1953).

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972

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12 HCLRS POST DoslNG

Figure 7. Blood levels of glibornuride in man following single oral 50 mg [0--0, G.J. (m)]and 100 mg [m-n, P.F. (m)] doses

withdrawing effects of the 3-endo-aminoborneol moiety. Consequently, it also has no useful luminescence properties. Spectrophotometric methods (based on derivatization at the alkyl portion of the molecule) for the determination of glibornuride as the DNP-aminoborneol derivative (Figure 6) developed by Bigler et a!. (IS) and by Koechlin ef ul. (31) were used to study the pharmacokinetics and the biotransformation of 3H and 35S-labeledglibornuride in the rat and in the dog. However, the method was not sufficiently sensitive to determine blood levels of glibornuride in man following therapeutic doses of 25-50 mg. Gas chromatographic analysis of glibornuride based on a procedure published for tolbutamide (23) was also not feasible because glibornuride undergoes pyrolysis to the p-tolylsulfonamide and 3-endo-aminoborneol moieties (16). Polarography of glibornuride was attempted in a variety of nonaqueous solvents (DMF, ETOH, acetonitrile, DMSO) using 0.1M tetraethylammonium bromide as the supporting electrolyte. The compound was examined at a concentration of approximately 1 x 10-4M and no polarographic activity was noted in the range of 0.000 V to -2.20 V US. a Ag wire reference electrode. Considerable problems were encountered in performing the nonaqueous polarography because of large amounts of electroactive impurities in the solvent and the recrystalized supporting electrolyte. The possibility that these impurities completely masked the polarographic wave cannot be overruled. Since the required sensitivity limits of the assay for glibornuride had to be of the order of 1 X to 1 X IO-ZM to be of use in monitoring clinical blood levels, polarography in nonaqueous solvents was not practicable. Therefore, it was necessary to employ chemical derivatization and the use of aqueous solvents to develop a suitable assay for glibornuride. The principle of the spectrophotometric method was initially used for the polarographic analysis of glibornuride as the DNP-aminoborneol derivative (Figure 6). The polarograms of the derivative showed two peaks at -0.645 volt and -0.825 volt us. SCE. The peak at -0.645 (31) B. A. Koechlin, F. Rubio, and S. A. Kaplan, HoffmannLa Roche Inc., Nutley, N.J., unpublished data, 1971. 1150

ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972

-

~

~~

volt was sufficiently sensitive for the determination of 1 to 2 pg of glibornuride/ml of blood. However, this assay procedure in addition to being time consuming was also neither sufficiently sensitive nor reproducible to be usable. Derivatization of glibornuride at the aryl portion of the molecule by ring nitration was investigated and showed promise of a sensitive assay. Conditions for establishing the optimal and most reproducible yields of the nitro derivative (Figure 2) were obtained by running time-course nitration reactions with glibornuride in parallel with authentic tolylsulfonamide and tolylsulfonic acid as model compounds. Nitration at 105-107 "C (in an oven) for 2 hours gave the most satisfactory results. N o attempt was made per se to isolate and characterize the reaction product/s formed during the nitration of glibornuride; however, the similarity of the polarograms of nitrated glibornuride, tolylsulfonamide, and tolylsulfonic acid (Figure 4B) would suggest that the nitrated product is either a mixture of or one of these two compounds. The strongly acidic conditions of the nitration reaction would probably tend to form the 3-nitrotolylsulfonic acid in preference to the 3-nitro-tolylsulfonamide. It is also estimated that the conversion efficiency of the nitration reaction to the nitro derivative/s is quantitative irrespective of the actual composition of the final products of the reaction. This estimation is based on a direct comparison of the peak height obtained with equivalent (kg) amounts of nitrated tolylsulfonic acid and tolylsulfonamide determined at equivalent sensitivity compared with nitrated glibornuride determined under similar instrumental conditions. Specificity of the Assay. The phenyl and tolylsulfonylurea compounds are weakly acidic compounds (due to the -SOsNH-CGNH- structure) with pK, values usually between 4 and 6. They can be extracted fairly selectively from blood buffered at pH 4 to 8 by extraction with a suitable organic solvent (24). Glibornuride has a pK, of 4.7 and is extractable from aqueous solution between pH 5.5 to 7.5. Quantitative extraction of 35S-labeled glibornuride into diethyl ether was obtained from unbuffered whole-blood (pH 2 7.0). The ether extracts were also relatively uncontaminated with extracted blood impurities at this pH. The compound in the ether extract was selectively back-extracted into 1N N H 4 0 H (31) which was further washed with ether as a clean-up step. The NHIOH was then acidified with HC1 and the compound re-extracted into benzene for subsequent polarographic analysis. Sulfonic acids and other strong acids which are possible metabolites would not be extracted at pH 7.0. The conditions of controlled nitration selected can also influence markedly the degree of nitration of phenyl U S . tolylsulfonylureas (24) and thus also impart an added degree of specificity. Furthermore, metabolic studies in man and in the dog using 3H- and 35S-labeledglibornuride have shown that the intact drug is the major component present in blood (14-16). Minor amounts of metabolites present in blood are either not extracted into diethyl ether at pH 7.0 or are not recovered in the back extraction and clean-up steps used prior to the nitration of the compound. Studies on the nitration of the metabolites M-1 (alcohol) and M - 2 (acid), Figure 1 , have shown that they do not interfere with the specificity of the assay since no measurable yields of nitro derivative were obtained under the conditions used. Thin-layer chromatographic (TLC) analysis of blood extracts from patients treated with glibornuride at therapeutic doses has also revealed the presence of only the intact drug.

1

1

--

I

1

1

I

Figure 8. Blood levels of glibornuride in man [C.F. (m)] after a 100-mg single oral dose followed by 4 consecutive 50-mg oral doses administered 24 hours apart

again showed rapid absorption of the drug with blood level maxima of 1.55 pg glibornuride/ml at 2 hours after the 50-mg dose and a peak level of 6.50 pg glibornuride/ml at 2 hours after the 100-mg dose. The half-life of elimination following the 50-mg dose was 5.7 hours while that following the 100mg dose was 13.8 hours. Blood levels of glibornuride in a fourth subject given a priming dose of 100 mg followed 24 hours later by 4 consecutive 50-mg doses each given 24 hours apart are shown in Figure 8. A peak level of 3.20 pg/ml was seen 1 hour after the 100-mg dose, and measurable levels of drug ranging from 0.60 to 0.85 pg/ml were seen one hour after each consecutive 50-mg dose. The method is therefore applicable for monitoring blood levels following therapeutic single and multiple oral doses of the drug.

Therefore, the assay is deemed to be specific for the parent drug. It can be modified using differential extraction techniques and thin-layer chromatography (TLC) for the analysis of urinary metabolites. It must be noted, however, that the specificity of the assay will be interfered with by the concomitant administration of other drugs containing either a nitratable phenyl ring such as phenobarbital, glutethimide, diphenylhydantoin, benzodiazepines, phenothiazines, or others such as the nitroimidazoles (32) which contain a reducible nitro group in the molecule. Application of the Method to Biological Specimen. A pilot blood level study was conducted in a subject receiving a single daily oral 25-mg dose over a period of time. Blood samples were collected just prior to the administration of a 25-mg dose (to determine a base-line level) and at 1, 2, 4, 6, and 8 hours after dosing. A “Total” blood level peak ranging from 0.780.80 pg glibornuride/ml was seen at 1 to 2 hours post dosing indicating rapid absorption of the drug. Measurable blood levels were seen up to 8 hours after dosing demonstrating the feasibility of the method. Blood level fall-off curves in two other subjects given a single oral 50-mg and 100-mg dose of the drug (Figure 7)

ACKNOWLEDGMEW

The authors wish to extend thanks to Arthur S . Leon of the Department of Clinical Pharmacology for conducting the clinical studies at the Newark Beth Israel Hospital, Newark, N. J., and to Marvin A. Brooks for his critical review of this manuscript.

(32) J. A. F. de Silva, N. Munno, and N. Strojny, J. Pharm. Sci., 59, 201 (1970).

RECEIVED for review December 13, 1971. Accepted March 1 1972.

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