(4) P. K. Mueller and E . L. Kothny, Anal. Chem., 45, (5),1R (1973). (5) R. C. Lao, R. S.Thomas, and J. L. Monkman, J. Chromatogr., 112, 681 (1975). (6) R. C. Lao, R. S.Thomas, H. Oja, and L. Dubois, Anal. Chem.,45,906 (1973). (7) D. A. Lane, H. K. Moe, and M. Katz, Anal. Chem., 45, 1776 (1973). (6)K. D. Bartle, M. L. Lee, and M. Novotny, lnt. J. Environ.Anal. Chem., 3,349 (1973). (9) W. Giger and M. Blumer, Anal. Chem., 46, 1663 (1974). (10) I. P. Fisher and A. Johnson, Anal. Chem., 47, 59 (1975). , (11) M. L. Lee, K. D. Bartle, and M. V. Novotny, Anal. Chem., 47, 540 (1975). (12) G. M. Janini, K. Johnvton, and W. L. Zielinskl, Jr., Anal. Chem., 47, 670 (1975). (13) M. A. Fox and S.W. Staley, "Analysis of Polycyclic Organic Compounds in Atmospheric Particulate Matter by HPLC with Fluorescence Detection", 170th National Meeting, Am. Chem. Soc.,Chicago, Ill., August 25-29, 1975. (14) . , J. A. Schmit, R. A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromatogr. Sci., 9, 645 (1971). (15) S.Soedigdo, W. W. Angus, and J. W. Flesher, Anal. Biochem., 67, 664 (197.51 . - . -,. (16) C. G. Vaughan, B. B. Wheals, and M. J. Whitehouse, J. Chromatogr., 78, 203 (1973). ~
(17) J. C. Suatoni and F. R. E. Swab, J. Chromatogr. Sci., 13, 361 (1975). (18) J. C. Suatoni, H. R. Farber, and B. E. Davis, J. Chromatogr. Sci., 13, 367 (1975). (19) M. Novotny, M. L. Lee, and K. D.Bartle, J. Chromatogr. Sci., 12,606 (1974). (20) R. Gladen, Chromatographia, 5, 237 (1972). (21) J. L. Monkman, J. Chromatogr., 26, 456 (1967). (22) M. Dong, D. C. Locke, and E. Ferrand, Anal. Chem., 48, 368 (1976). (23) B. Wheals, C. G. Vaughan, and M. J. Whitehouse, J. Chromatogr, 106, 109 (1975). (24) H. J. Klimisch, J. Chromatogr., 23, 11 (1973): Anal. Chem., 45, 11 (1973). (25) R. E. Jentoft and T. H. Gouw, Anal. Chem., 40, 178 (1968). (26) B. L. Karger, M. Martin, J. Loheac, and G. Guiochon, Anal. Chem., 45,496 (1973). (27) R. Vivilecchia, M. Thiebaud, and R. W. Frei, J. Chromatogr. Sci., 10,411 (1972). (28) C. H. Lochmuller and C. W. Amoss, J. Chromatogr., 108, 85 (1975).
RECEIVEDfor review February 23,1976. Accepted May 19, 1976. Work supported by Grant No. CA 1760301 from the National Cancer Institute.
Gas Chromatographic and Mass Spectral Properties of Sulfonylurea N-MethyI-"-perf Iuoroacyl Derivatives W. E. Braselton, Jr.,* E. D.'Bransome, Jr., and H. C. Ashline Deparfment of Medicine, Medical College of Georgia, Augusta,
J.
Ga.30902
1.Stewart and 1. L. Honigberg
School of Pharmacy, University of Georgia, Athens, Ga. 30602
N-Methyl and N-methyltrlfluoroacetyl, N-methylpentafluoropropionyl, and N-methylheptafluorobutyryl derlvatlves of the sulfonylureas tolbutamide, hydroxyltolbutamide, carboxytolbutamide, chlorpropamide, and tolazamide were prepared. The derlvatlzed compounds were thermally stable as shown by mass spectrometry and exhibited excellent gas chromatographic properties on the liquid phases 3% OV-I, 3% OV-17, and 3% SP-2401.The derivatives showed high sensitivity to electron-capture detection: tolbutamlde-N-methyltrlfluoroacetate sensitivlty = 2.8 X mol s-' at 3:l signal to noise. The mass spectral properties of the methyl-perfluoroacyi derlvatlves of chlorpropamide, tolbutamide, and tolbutamide metabolites were characterized by rearrangemenf involving loss of SO1, In contrast to the toiazamide derivative which did not lose SO2.
The recently renewed controversy over the University Group Diabetes Program (UGDP) report concerning increased cardiovascular mortality from treatment with tolbutamide, an antidiabetic sulfonylurea (1-5),highlights the need for a rapid and precise method of measurement of this class of drugs and their metabolites in body fluids. Although sulfonylureas have been used in the treatment of maturityonset diabetes for more than 20 years, inadequate quantitative methods have resulted in a paucity of reliable information on the pharmacokinetics and mechanism of action of this class of drug. Quantitative procedures employing colorimetric and spectrophotometric methods to measure sulfonylureas and their metabolites in body fluids have suffered from lack of sensitivity and specificity unless involved solvent extraction procedures were used (6, 7). High pressure liquid chroma1386
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
tography (HPLC) has been used to analyze pharmaceutical preparations (8), but present HPLC methodology is not sensitive enough for measurements of therapeutic levels in blood. Matin and Knight (9) recently reported a sensitive and specific chemical ionization mass spectrometry method although the requirements of expensive specialized equipment limit its use as a routine clinical procedure. GLC methods of quantitation have been described (6,10-12)but, for the most part, these have been unsuccessful because of the thermal instability of the sulfonylureas and their N-methyl derivatives. We have recently found that the N-methyl-N'-perfluoroacyl derivatives of the sulfonylurea drugs I, IV, V, and some sulfonylurea metabolites 11, I11 (Figure l),are stable to conditions of gas chromatography and thus provide the basis for a simple and accurate quantitative procedure employing flame ionization GLC (13). This paper describes in detail the mass spectral (electron impact) and GLC characteristics of the N-methyl-N'-trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl derivatives. This work has led to development of a more sensitive electron capture GLC method of quantitation. A forthcoming paper will describe methodology for routine extraction, derivatization. and measurement of sulfonylureas in 50-pl serum samples.
EXPERIMENTAL Apparatus. Compounds were analyzed o n a F i n n i g a n 1015D gas chromatograph-mass spectrometer interfaced w i t h a Systems Industries (Sunnyvale, Calif.) System 150 data acquisition a n d control system. T h e injector and separator temperatures were 240 OC; spectra were t a k e n a t 70 eV. Columns were 1.5 m X 2 mm i.d. glass, a n d H e carrier gas f l o w was approximately 30 ml/min. Compounds were chromatographed o n 3% OV-1 (on SO/lOO Supelcoport, Supelco, Inc.),
0
0
.. . O H
H R I = CH3;
R2 = C4Hg
II HYDROXYTOLBUTAMIDE
Rl = CH,OH;
R2 = CH ,,
111, CARBOXYTOLBUTAMIDE
RI = COOH:
R,
IV. CHLORPROPAMIDE
R l = CI
R, = C1H7
R I = CHI;
R, = N
I TOLBUTAMIDE
V TOLAZAMIDE
Figure 1. Kmethyl-Nf-perfluoroacyl derivative of
C&
3
sulfonylurea drugs
and metabolites
o
e
i
b
C
f
P
i
d
n
k
described by Sabih ( 19)for KMe-tolbutamide They are shown here as general structures since they appear in the mass spectra of a number of the sulfonylurea derivatives (TableI). Other ions are shown in Scheme I and Scheme II. Ion fof Sabih ( 19)probably exists as shown above Figure 2. Ions a-k
(22)(see text). I) R' = CH3, R" = CH3; II) R' = ROCHp, R" = CH3; Ill) R' = CHjOOC, R" = CH3; IV) R' = CI, R" = H; TFA) R E COCFs; PFP) R = COCpFs; HFB) R
COC3F7.
3% OV-17 (on 100/120Gas-Chrome Q)and 3% SP-2401 (on 100/120
%. C
%.
9
a
v
L-i-
oal
Supelcoport) at 230 "C. Reference compounds and derivati;es were also analyzed by direct probe insertion into the ionizer region of the mass spectrometer for comparison with spectra obtained by GC-MS. GLC was carried out on a Varian 2100 GC equipped with a 63Ni electron capture detector. Columns were 1.8 m X 2 mm glass, with Ns carrier gas flow at 37 ml/min. Reagents and Chemicals. Tolbutamide, chlorpropamide, and tolazamide were kindly supplied by Francis Henderson of the Upjohn Company. Diazomethane was prepared as an ethereal solution by the action of ethanolic KOH on a suspension of N-methyl-N-nitrosop-toluene-sulfonamide (Diazald,Aldrich) in diethyl ether, and subsequent distillation at 65 "C. N-Butyl-N-(4-~arboxybenzenesulfonyllurea (111)was prepared by methods of Popova (14)and Cardani et al. (15).N-Butyl-N-(4-hydroxymethylbenzenesulfonyl)urea (11) was prepared as follows: Synthesis of 4-Bromomethylbenzenesulfonamide.p-Toluenesulfonamide 17.1 g (0.1mol) and 19.5 g (0.11 mol) of N-bromosuccinimide were suspended in 400 ml of ethylene chloride and refluxed for 20 h in an apparatus fitted with a calcium chloride drying tube. The refluxing solution was illuminated for an initial period of 4 h with a 275-watt sunlamp (General Electric Corporation, New York) during which time the color of the solution changed from red to yellow. After 20 h, the mixture was then cooled to room temperature and the resulting precipitate filtered and washed several times with water. The filtrate was evaporated to dryness and the residue extracted with acetone. Evaporation of the acetone yielded a solid which was combined with the original precipitate and recrystallized from 95%ethanol to yield a white powder, mp 185-189 "C, 52.8% yield. The bromomethyl compound was contaminated with a small amount of starting material and was used as such in the synthesis of acetoxymethylbenzenesulfonamide. ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
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