1205
Anal. Chem. 1981, 53, 1205-1208
Results are shown for 2-nitrotoluene (MNT), 2,4-dinitrotoluene (DNT), and 2,4,6-trinitrotoluene (TNT). The data were obtained from a series of experiments designed to give the precision of determination for the nitrotoluenes using a standard, commercially available detector (TEA Model 502A Analyzer-see Experimental Section), the simple integrator provided with it, and manual injection into an isothermally operated gas chromatograph. This is the simplest practical instrumental arrangement that can be used to obtain quantitative results. The experimental data are tabulated in Table 11. Ten determinations were obtained for each concentration level. The volume injected was 10 pL in all cases. The precision of the results range from 11% relative standard deviation for 2,4-dinitrotoluene at the picomole level (0.0182 mg/L) to 2% for 2-nitrotoluene a t the 10 nmol level (137 mg/L). When the data were plotted as in Figure 4, a linear regression analysis yielded correlation coefficients of greater than 0.999 for each of the three compounds.
e
0
;i
LITERATURE CITED
I
\
8 12 TIME (MINUTES)
4
(1) Fan, T. Y.; Ross, R.; Fine, D. H.; Keith, L. H.; Garrison, A. W. €nviron. Sci. Technoi. 1978, 12,692-695. (2) Lafleur, A. L.; Morriseau, B. D.; Fine, D. H. “Proceedings, New Con-
L 16
Figure 4. Plot of detector response vs. moles injected for GC-TEA detection. Substances tested were 2-nitrotoluene (MNT) 2,4dinitrotoluene (DNT), and 2,4,6-trinitrotoluene (TNT).
determination of molar response is the accuracy of the response for a given quantity of nitric oxide gas. It has been found that calibrated gas samples from different sources may vary in composition and that the choice of materials in handling the gas is critical. Therefore, although it is possible to obtain reproducible results to sometimes better than 1 or 2% relative standard deviation if the conditions are favorable, the absolute accuracy is suspected to be poorer than the precision. With this in mind, it can be seen that the relative response between different nitrotoluenes will be more accurate than their absolute response referred to nitric oxide, although the precision of either result will be approximately the same. Figure 4 shows a plot of detector response vs. quantity injected into the gas chromatograph expressed in moles.
cepts Symposium and Workshop on Detection and Identification of VA. ExDlosives”, National Technical Information Service: Springfield, . 1978;pp 597-598. (3) Lafleur, A. L.; Morriseau, B. D. Anal. Chem. 1960, 52, 1313-1318. (4) Spanggord, R. J.; Keck, R. G. J . Pharm. Sci. 1980, 69, 444-446. (5) Preussman, R.; Castegnaro, M.; Walker, E. A,; Wassermann, A. E. Environmental Carcinogens: Selected Methods of Analysis; Vol. l., Analysis of Volatile Nitrosamines In Food”; International Agency for Research on Cancer: Lvon. 1978. (6) Fan, T. Y.; Vita, R.; Fine, D.’ H. Toxicol. Left. 1978, 2, 5-10. (7) Yinon, J. Crk. Rev. Anal. Chem. 1977, 7(4), 1-35. (8) Fine, D. H.; Lieb, D.; Rufeh, F. J . Chromatogr. 1975, 107. 351. (9) Fine, D. H.; Rufeh, F.; Lieb, D.; Rounbehler, D. P. Anal. Chem. 1975,
47, 1188. (IO) Urbanski, T. “Chemistry and Technology of Explosives”; Pergamon Press: New York, 1965;Vol. 2, Chapter 6. (11) Reference 10,p 52. (12) Kaye, S. M. “Encyclopedia of Explosives and Related Terms”; U.S. Army Armament Research and Development Command: Dover, NJ, 1978;Vol. 8,p P86. (13) Krauss, S . A.; Glattstein, B.; Landau, D.; Almoz, J. “Proceedings, New Concepts Symposium and Workshop on Detection and Identification of Explosives”; National Technical Information Service: Sprlngfieid, VA, 1978;pp 873-676.
RECEIVED for review October 31, 1980. Accepted March 18, 1981.
Analysis for Nitrosourea Antitumor Agents by Gas Chromatography-Mass Spectrometry Ronald G. Smith,” Silas C. Blackstock, Lily K. Cheung, and Ti Li Loo The University of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
An assay for nltrosoureas applicable to the antitumor agents BCNU, CCNU, and MeCCNU has been developed by using the gas chromatography-mass spectrometric technlque of selected Ion monltorlng. The analysis follows derlvatlration by trlfluoroacetlc anhydride, which converts the 1,3-dialkyl-lnltrosoureas to 1,3-dlacyl-l ,t-dlalkylureas. The lower limits for quantifying these drugs in plasma are 1-3 ng/mL. Appllcatlon Is made to determine the rates of decomposition for the three nltrosoureas In plasma and to determine the plasma clearance of MeCCNU In a dog.
Three established antitumor agents, 1,3-bis(2-chloro-
ethyl)-1-nitrosourea (BCNU), l-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethy1)-3-trans4-methylcyclohexyl)-l-nitrosourea(MeCCNU), Figure 1, are active against certain neoplastic diseases in man (1-3). Their physical and chemical properties allow them to penetrate the blood-brain barrier ( 4 , 5 ) and are thus especially useful in the pretreatment of tumors of the central nervous system (6, 7). The activity of these agents apparently comes from their rapid nonenzymatic decomposition to unstable intermediates capable of DNA alkylation (8-10). Despite the clinical usefulness of nitrosoureas for over 15 years, relatively little is known about their disposition and metabolism in vivo. Methods previously applied to the
0003-2700/81/0353-1205$01.25/0 0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
Scheme I
-0NfiO c I - C H ~ C H ~ - N H - C - N - C H ~ CI H ~ - C
F ) Q
CF3C-0-C CF3
BCNU
R-N-C-N-R'
C=O
9 NfiO
CF3
C)NH-C-N-CH~CH~-C I
lo
lCF3 !-OH
CF&-OH
CCNU
P
?
R-NH- -NH-R'
R-YC-NH-R'
MeCCNU
Flgure 1. Chemical structures of the antitumor drugs BCNU, CCNU, and MeCCNU. analysis of nitrosoureas include colorimetric assays (II,12), differential pulse polarography (131,radiochemical assay of labeled compounds (5,14,15),high-pressure liquid chromatography (16),and chemical ionization mass spectrometry (17). None of these methods presently combines high sensitivity with selectivity and general applicability. As a result, the clinical pharmacokinetics of only BCNU have been studied (18). Metabolites arising from enzymatic hydroxylation and denitrosation have been identified from microsomal incubations (16,19,20),but not directly detected in animal models (21); the significance of these metabolites in the clinical pharmacokinetics of these compounds has not been determined. This report describes a gas chromatography-mass spectrometric (GC-MS) assay for nitrosoureas using selected ion monitoring after derivatization with trifluoroacetic anhydride. This method is selective and sensitive to low nanogram quantities and has been applied to BCNU, CCNU, and MeCCNU.
EXPERIMENTAL SECTION Materials. Samples of BCU, CCNU, and MeCCNU were obtained from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute. Standard solutions of the nitrosoureas were prepared from weighed samples by serial dilutions in methylene chloride. Fresh solutions were prepared for quantitative analysis. The homologue internal standard for BCNU was synthesized by the addition of 3-chloropropylaminehydrochloride (Aldrich Chemical Co., Milwaukee, WI) to 2-chloroethylisocyanate (Eastman Kodak Co., Rochester, NY) followed by nitrosation as previously described (22). Most solvents and reagents were used as received, including acetonitrile and tetrahydrofuran (silylation grade, Pierce Chemical Co., Rockford, IL), methylene chloride (nanograde,Mallinckrodt Inc., St. Louis, MO), acetic, trifluoroacetic, pentafluoropropionic, and heptafluorobutyric anhydrides, trifluoroacetylimidazole, and N-methylbis(trifluoroacetamide)(Pierce Chemical Co.). Ethyl acetate (Fisher Scientific Co., Houston, TX) was stored over molecular sieves (4A). Derivatization. The nitrosoureas were derivatized by adding 40 pL of acetonitrile and 30 pL of trifluoroacetic anhydride to the dried sample in a 0.3-mL conical vial. Derivatives of CCNU and MeCCNU were obtained by heating the reaction mixture at 85 "C for 3 h; BCNU samples were heated at 85 "C for 6 h. The samples were cooled, evaporated to dryness under a stream of nitrogen, and redissolved in ethyl acetate. Derivatization with other acylating agents and cosolvents was attempted under similar conditions. Plasma Samples. Samples for the standard curve of each agent were prepared by evaporatingportions of a standard solution of the drug, reconstituting with 100 pL of ethyl alcohol, and adding 2 mL of plasma. The internal standard (0.6-1.0 pg) was added followed by extracting twice with 1.0-mL portions of 1:l etherhexane. The extracts were combined, dried over anhydrous magnesium sulfate, and evaporated to dryness under a nitrogen stream before derivatization. The nitrosoureas were readily separated from any denitrosated metabolites by thin-layer chromatography. Plasma extracts were
\
l3-V-j-
N-R'
CF3
CF3
c=o $=o
applied to silica gel plates (E.M. Laboratories Inc., Elmsford, NY) and the plates developed with benzene. The nitrosoureas, RI 0.5-0.7, were scraped from the plate, free from the corresponding ureas that remained at the origin. The rates of decomposition for the three nitrosoureas were determined in plasma at 37 "C. Multiple samples were prepared similar to those used for the standard curve using initial nitrosourea concentrations of 1.0 pg/mL in plasma prewarmed to 37 "C and kept at this temperature in a thermostatically controlled bath. Sampleswere periodically removed, the appropriate internal standard was added, and the samples were extracted and derivatized as described above. The plasma clearance of MeCCNU was measured in a beagle dog which had received a 15 mg/kg intravenous dose over 15 min. Plasma samples were collected at intervals and frozen. For analysis the samples were thawed; after the internal standard was added, they were extracted and derivatized as described above. In a separate experiment MeCCNU was dissolved in plasma, divided into 2-mL aliquots, and frozen. These samples were periodically analyzed for MeCCNU, which was found to be stable up to a month. Instrumentation. Mass spectrometry was performed by using a Finnigan Model 3300F GC-MS system interfaced with an Incos 2300 data system. Gas chromatographic columns, 1.2 m X 2 mm, used in this study were packed with 3% OV-17 on 90/100 Anakrom Q, 10% SE-30 on 100/120 Gas Chrom Q, and chemically bonded Carbowax 20M, 8O/lOO (Ultrabond 20M, RFR Corp., Hope, RI). Mass spectra were obtained by electron impact ionization at 70 eV, scanning m / z 50-550 every 2 s. For selected ion monitoring the ion at m / z 315, common to the CCNU and MeCCNU derivatives, was used. For BCNU, using the chloropropyl homologue as the internal standard, the ion at m/z 202 was monitored. Sampling time was approximately 0.4 s for each ion.
RESULTS AND DISCUSSION Derivatization. The key to this assay is the unusual reaction of 1,3-dialkyl-l-nitrosoureas with trifluoroacetic anhydride. Trifluoroacetylation has become a standard method of derivatization because of its ability to convert polar compounds to volatile derivatives (23)and its applications include assays for urea (24) and several trisubstituted ureas (25). In the present application trifluoroacetic anhydride performs two functions, as proposed in the sequence of Scheme I. Acylation at the N3 position generates trifluoroacetic acid which removes the nitroso group. As the reaction proceeds and acylation at the N' position generates more trifluoroacetic acid some of the nitrosourea probably is denitrosated before acylation. The mass spectra of the resulting 1,3-diacyl-l,3-dialkylureas derived from BCNU, CCNU, and MeCCNU are shown in Figures 2-4. Analogous derivatives were obtained by using pentafluoropropionic and heptafluorobutyric anhydrides, but the corresponding reactions with acetic anhydride, trifluoroacetyl-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981 2c8 I
1
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MW = 376
420
440
r
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280
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320
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mlr
400
380
360
420
440
mlz
Figure 2. Mass spectrum of the principal product derived from the reaction of BCNU with trifluoroacetic anhydride.
Figure 4. Mass spectrum of the principal product derived from reaction of MeCCNU with trifluoroacetic anhydride.
x1 I
30
'
1
1
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'
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'
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120
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'
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'
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Figure 3. Mass spectrum of the principal product derived from the reaction of CCNU with trifluoroacetic anhydride. imidazole, and N-methylbis(trifluoroacetamide)were unsuccessful. As Scheme I implies, trifluoroacetic anhydride converts both the nitrosoureas and their denitrosated metabolites to the same derivative. This ambiguity is removed by the addition of a thin-layer chromatographic (TLC) step before derivatization. This not only increases the specificity but, when combined with a parallel determination that excludes this step, can be used to quantify the denitrosated metabolite. Several reaction parameters were briefly investigated to optimize the yields. Acetonitrile is a better cosolvent than dichloromethane, benzene, tetrahydrofuran, ethyl acetate, or neat anhydride. A temperature range of 55-85 "C and reaction times of 1-6 h are optimal; temperatures higher than 100 "C and reaction times longer than 24 h produced excessive decomposition and side products. Even optimal conditions for derivatization produced several minor side products that were observed by gas chromatography. Three side products have been tentatively identified by mass spectrometry as the isocyanate and trifluoroacetylated amine, both derived from the N3 group, and a triacylated urea in which the chlorine is substituted by a trifluoroacetoxy group. The diacylureas are suitable for gas chromatographic analysis, producing sharp, symmetrical peaks. They are, however, labile to certain conditions, including exposure to hydroxylic solvents, silica gel, and alumina. The derivatives also appear to be very sensitive to active sites on the gas chromatographic column packing; detection limits improved
Figure 5. Selected ion current profile of mlz315 for a plasma sample containing 300 nglmL of CCNU (A) and 10 nglmL of MeCCNU (B). This analysis is complete in 3 min by using a column packed with Ultrabond 20M and heated isothermally at 155 "C. 10-50-fold when the packing was changed from a standard 3% OV-17 to a 10% SE-30 or chemically bonded Carbowax 20M. The latter column performs better because of its lower column bleed and its excellent separation of the CCNU and MeCCNU derivatives (Figure 5). Selected-Ion Monitoring. The selected ion monitoring technique provides a sensitive and specific method for quantifying nitrosoureas that have been derivatized with trifluoroacetic anhydride. The spectra of the derivatives (Figures 2-4) exhibit several abundant ions useful for quantitation. CCNU and MeCCNU are structural homologues and are conveniently used as internal standards for each other. Separation of the two derivatives on a column of chemically bonded Carbowax 20M (Figure 5) allows the common ion a t m / z 315 to be monitored. The chlorine isotope ion at m / z 317 can be added for greater specificity. These ions are presumably derived from McLafferty rearrangements with transfer of two hydrogen atoms ("McLafferty + 1" rearrangement) (24),resulting in the loss of the cyclohexyl and methylcyclohexyl groups. Ions at m / z 194 and m / z 208 have been monitored when using columns that do not separate these derivatives. BCNU concentrations were assayed with high sensitivity using the chloropropyl homologue and monitoring the ion at m / z 202. Under these conditions the lower limits of detection are 1oct500 pg for the three nitrosoureas. When assaying extracts from 2 mL of plasma, the limits useful for quantitation are 1-3 ng/mL. The response curve shows a linear correlation of detector response to MeCCNU extracted from plasma over the range of 3.0 ng/mL to 10 kg/mL.
1208
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981 BCNU
m -.
CCNU
*-*
mg/kg. The plasma clearance curves, with and without the added chromatographic (TLC) step, are shown in Figure 7. The MeCCNU/CCNU values are slightly higher when the TLC step is incorporated, which was found to be due to an impurity (denitrosated CCNU) in the internal standard. The plasma levels of the denitrosated metabolite are considered negligible because the two curves are parallel. Pharmacokinetic parameters calculated from the “before TLC” data include half-lives of 10.7 min for the distribution phase and 86.6 min for the elimination phase. Definitive analyses of biological samples for nitrosourea antitumor agents have previously been elusive due to the low concentrations and relative instabilities of these drugs and the nonspecific nature of some assays. The methods described here are specific for the unchanged drug and sensitive to the low ng/mL range. Work is under way to apply this assay to determine the pharmacokinetic parameters of these agents in laboratory animals and man.
-
ACKNOWLEDGMENT
Minutes
Decomposition of nitrosoureas in plasma at 37 O C . The lines were obtained by exponential curve fitting.
Flgure 6.
The authors with to acknowledge useful discussions of chromatographic conditions with E. C. Homing, Baylor College of Medicine, and technical assistance by Katherine Lu in obtaining samples of dog plasma. This work was supported by National Cancer Institute Contract N01-CM-87185 and Grant CA-14528.
LITERATURE CITED loo-
(1) Proceedings of the Seventh New Drug Seminar on the Nitrosoureas Cancer Treat. Rep. 1976, 60. (2) Wasserman. T. H.: Slavik. M.: Carter. S. K. Cancer (Amsterdam) 1975, 36. 1258. (3) Pratt, W. B.; RuckJon, R. W. “The Anticancer Drugs”; Oxford University Press: New York, 1979;pp 47, 74. (4) Loo, T. L.; Dion, R. L.; Dixon, R. L.; Rall, D. P. J. fharm. Sci. 1986, 55 - - , 492 .- -. (5) Oliverio, V. T.; Vietcke, W. M.; Williams, M. K.; Adamson, R. H. Cancer Res. 1970. 30. 1330. (6) EORTC Brain Tumor Group Eur. J. Cancer 1076, 12, 41. (7) Walder, M. D.; Hilton, J. Cancer Treat. Rep. 1978, 60, 725. (8) Montgomery, J. A.; James, R.; McCaleb, G. S.;Kirk, M. C.; Johnston, T. P. J. Med. Chem. 1975, 18, 568. (9) Brundrett, R. B.; Cowens, J. W.; Colvin, M.; Jardine, I. J. Med. Chem. 1078, 19, 958. (10) Gombar, C. T.; Tong, W. P.; Ludlum, D. 8. Biochem. Biophys. Res. Commun 1079, 90 676. (11) Loo, T. L.; Dion, R. L. J. fharm. Sci. 1965, 54, 809. (12) . . Mirvish, S.S.:Sams, J. P.: Arnold, S.D. Fresenins’ Z.Anal. Chem. 1979, 298, 408. (13) Bartosek, I.; Daniel, S.;Sykora, S. J. fharm. Sci. 1976, 67, 1160. (14) DeVita, V. T.; Denham, C.; Davidson, J. D.; Oliverio, V. T. Ciin. fharmacol. Ther. 1987, 8, 566. (15) Sponzo, T. W.; DeVita, V. T.; Oliverio, V. T. Cancer 1973, 31, 1154. (16) May, H. E.; Boose, R.; Reed, D. J. Biochemistry1975, 14, 4723. (17) Weinkam, R. J.; Wen, J.; Furst, D. E.; Levin, V. A. Clin. Chem. 1978, 24,45. (18) Levin, V. A.; Hoffman, W.; Weinkam, R. J. Cancer Treat. Rep. 1976, 62, 1305. (19) Hill, D. L.; Kirk, M. C.; Struck, R. F. Cancer Res. 1975, 35, 295. (20) May, H. E.; Kohlhepp, S. J.: Boose, R. B.; Reed, D. J. Cancer Res. 1979, 39,762. (21) Hilton, J.; Walder, M. D. Biochem. fharmacoi. 1975, 24, 2153. (22) Johnston, T. P.; McCaleb, G. S.; Opliger, P. S.;Montgomery, J. A. J. Med. Chem. I S M , 9 , 892. (23) Blau, K.; Klng, G. S. “Handbook of Derivatives for Chromatography”; Heyden: Philadelphia, PA, 1978;Chapter 3. (24) Miller, P. W. J. Agric. Food Chem. 1971, 19, 941. (25) Saunders, D. G.;Vanatta, L. E. Anal. Chem. 1974, 46, 1319. (26) McLafferty, F. W. ”Interpretation of Mass Spectra”, 2nd ed.; W. A. Benjamin: Reading, MA, 1973;pp 68-89. .
c .B e f o r e t l c After t l c
t-4
I
.
0
I
I
I
1
I
I
1
2
3
4
5
6
Hours Plasma Clearance of MeCCNU from a dog after an intravenous dose of 15 mg/kg. Part of each sample was derivatized after chromatographing on thin-layer plates; the remainder was derivatized directly after extraction. Figure 7.
The methods described have been used to determine the in vitro rates of decomposition of the three nitrosourea agents in plasma. The results, which are consistent with the rapid first-order kinetics of these drugs, are shown in Figure 6. The decay lines, resulting from exponential curve fitting, give half-lives of 11, 33.5, and 24 min for BCNU, CCNU, and MeCCNU, respectively (correlation coefficients >0.993). Plasma concentrations of MeCCNU were determined in a dog after it had received an intravenous administration of 15
RECEIVED for review June 17, 1980. Resubmitted February 5, 1981. Accepted March 20, 1981.
