Gas Chromatographic Determination of Rafoxanide [ 3 ’ - C h l o r o - 4 ‘ - ( 4 - C h l o r o p h e n o x y ) - 3 , 5 - ~ i i ~ d o ~ ~in~ iPlasma cy~~~~~~~~~ by Electron Capture Detection of Its Trimethylsilyl Derivative Charles P. Talley, Nelson R. Trenner, George V. Downing, Jr., and W. J. A. VandenHeuvel Merck Sharp & Dohme Research Laboratories, Rahway, N . J . 07065 Experimental conditions have been established for gas chromatography at the submicrogram level of the anthelmintic rafoxanide, 3‘-chloro-4‘-(4-chlorophenoxy)-3,5-diiodosalicylanilide. The drug is converted to its di-trimethylsilyl derivative (characterized by combined gas chromatography-mass spectrometry) and chromatographed on a 4411. column with electron capture detection. Rafoxanide, but not the derivative, undergoes a photolytic reaction to form the corresponding mono-iodo compound. A procedure has been developed for the isolation and gas chromatographic determination of rafoxanide in plasma with a sensitivity of 0.01 rg/ml.
GASCHROMATOGRAPHY coupled with highly sensitive detectors has proved to be an excellent technique for the separation and quantitation of nanogram amounts of materials of biological origin and of exogenous materials in a biological matrix (1-3). Unfortunately, many of the more interesting substances associated with biological systems contain one or more groups capable of hydrogen bonding, leading to undesirable adsorption phenomena. The polarity imparted by such groups to a molecule, particularly if it possesses a high molecular weight, precludes direct gas chromatography without prior derivative formation. The substitution of trimethylsilyl (TMSi) groups for active hydrogen atoms has permitted gas chromatography of many high molecular weight, polar molecules (4-7). We now report the development of an assay in animal plasma for rafoxanide [3’-chloro4’-(4-chlorophenoxy)-3,5-diiodosalicylanilide], a new anthelmintic agent (8). To detect and quantitate plasma drug levels in the parts per million range soon after oral administration, we have used the technique of gas chromatography with electron capture detection. EXPERIMENTAL
Apparatus and Chromatographic Conditions. Gas chromatography of nanogram and subnanogram quantities of rafoxanide was carried out with a Glowall Model 320 instrument. The column was a 4-in. X 3-mm i.d. glass U-tube packed with 3% OV-17 coated over 1 S Z SE-30 on SOjlOO mesh acid-washed and silanized Gas-Chrom P (9). The (1) B. J. Gudzinowicz, “Gas Chromatographic Analysis of Drugs and Pesticides,” Dekker, New York, N. Y., 1967. (2) J. A. F. de Silva and C. V. Puglisi, ANAL. CHEM., 42, 1725
(1970). (3) E. Townley, I. Perez, and P. Kabasakalian, ibid., p 1759. (4) A. R. Pierce, H. N. Graham, S . Glassner, H. Madlin, and J. G. Gonzales, ibid., 41, 298 (1969). ( 5 ) W. E. Wilson and J. E. Ripley, ibid., p 810. (6) K. Tsuji and J. H. Robertson, ibid., 42, 1661 (1970). (7) M. Katz and Y. Lensky, Experientia, 26, 1043 (1970). (8) H. Mrozik, H. Jones, J. Friedman, G. Schwartzkopf, R. A. Schardt, A. A. Patchett, D. R. Hoff, J. J. Yakstis, R. F. Riek, D. A. Ostlind, G. A. Plishker, R . W. Butler, A. C. Cuckler, and W. C. Campbell, ibid., 25, 883 (1969). (9) E. C. Homing, W. J. A. VandenHeuvel, and B. G. Creech, in “Methods of Biochemical Analysis,” Vol. XI, D. Glick, Ed., Interscience, New York, N. Y., 1963.
vaporizer wds nldintdiried ar jUii ’cdnd the column at 250 “C. The carrier gas was high purity, dry N? at a flow rate of 75 mlimin. The electron capture detector used was of the Lovelock (10) design containing 22.5 $3 of 226Ra coated on the inner surface of a cylindrical p!atinum foil. The detector was operated at 8 V dc and maintained at 300 “C. Gas chromatography of microgram quantities of rafoxanide was carried out with a Barber-Colman Model 5000 instrument. The colunin was a 2-ft X 3-mm i.d. glass U-tube containing the same packing as above. The vaporizer was maintained at 300 “C and the column at 265 “C. The carrier gas was argon at a flow rate of 50 ml/min. Detection was by hydrogen flame ionization. Combined gas chromatography-mass spectrometry (GCMS) (11) was carried out with an LKB Model 9000 instrument. The column was a 3-ft X 3-mm i.d. glass spiral packed with 1.8% OV-17 on SOjlOO mesh acid-washed ~2 silanized Gas-Chrom P (9). The vaporizer was maintained at 300 “C and the column at 260 “C. The carrier gas was helium at a flow rate of 30 mi/rnin.. The spectrometer was operated with a source tmperaturi: of 270 “C, electron energy of 70 eV, an acce1eratir.y voltage of 3,5 kV, and B trap current of 60 PA. Reagents. Ethanol (9573 was purchased frc U.S. Industrial Chemical Company. Hydrochloric acid (concc) and sodium hydroxidc- (50% soiuiionj were Mrrck :eagent grade. Spectroquality 2,2,4-ii.iri!eChylpentane (isooc; me) and reagent grade bis-;rin,2trnyisil;;la(:~ta~ide (RS/.\ L . . i!rchased from niatheson Colemrn and Bell and Pirrce Chemical Company, iespectivclj.. Sample Preparation. EXTHPCTI~N. A 1.0-ml aliquot of plasma was treated with 5 in1 of 45 % ethanol and centrifuged to remove the dena:urcd proteins. The clear supernatant was decanted. ecidified with 5 nil of 4 % HCI, and extracted twice with 5-ml portions cf isooctane. The isooctane layer was extracted twice with 5 r d portions of 1% NaOH. The basic extract (protected from direct ligrding) was heated in a steam bath for ? hr, tmid, acidified with 15 in1 of 4z HCl, and extracted twice with 5 mi pcrrioiis of isooctane. The isooctane extract was evaporated to dryness under a stream of nitrogen. DERIVATIZATION. The residue from above was taken up in 50 p1 of BSA, which servid as both reagent and solvent. The mixture was capped tightly, heated in an oil bath at 110 “C for 7 min, coo!ea tc rooin temperature, and centrifuged. One microliter was injected into the gas chromatograph. QUANTiTATiON. Peak areas werc measured by height and width at half-height. Quantitation was accomplished by comparison of sample Wdk area to a standard plot detertnined each day from a series of standard solutions of derivatlzed rafoxanide in BSA. ti-.
I_
(10) J, E Lovelock and S. T?. Lipski, J . Anier. G e m . Soc., 82,
431 (1960). (11) J. A. McCloske) iii “hletnods in Enzymology,” XIV, Lipids, J. M. Lowenstein, Ed.. Academic Press, New York, N. Y., 1969.
ANALYTICAL CHEMISTRY, VOL. 43, N3. 11, SEPTEMBER I971
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A
B
4" OV-17 250.C
OV-17 265.C
1 no Std
Sheep Plasma
w I 0
I 2
I
I 0
2
MIN.
Figure 1. (A) Rafoxanide standard (1 ng as the di-TMSiderivative). (B) Plasma of a sheep dosed with rafoxanide. This analysis represents l/m of the extract from 1 ml of plasma. Peak 1results from desiodo-rafoxanide produced during sample workup. Details of the chromatographic conditions are given in the Experimental Part, first paragraph RESULTS AND DISCUSSION The structure of rafoxanide (I) suggests that this drug might not be particularly well suited to direct gas chromatographic analysis.
MINUTES
Figure 2. Gas chromatogram (upper) of trimethylsilylation reaction mixture of an aliquot of an ethyl acetate solution (0.1%) of rafoxanide. The ethyl acetate solution had been exposed to ordinary laboratory fluorescent light for three weeks. Gas chromatogram (lower) of a mixture of rafoxanide (B) and authentic 3-desiodorafoxanide (A) as trimethylsilyl derivatives. Details of the chromatographic conditions are given in the Experimental Part, second paragraph
I Indeed, the compound was not eluted from nonpolar stationary phases such as OV-1 and SE-30 even at high temperatures (300 "C). Trimethylsilylation of rafoxanide with BSA gave a derivative which could be chromatographed at 265 "C on a moderately polar stationary phase composed of OV-17 coated over SE-30. Characterization of the derivative was accomplished by combined GC-MS. The mass spectrum of this compound, clearly di-TMSi-rafoxanide, exhibited prominent ions at m/e 769 (M, molecular ion), m/e 754 (M - 15, loss of a methyl radical), m/e 680 (M - 89, loss of an OTMSi radical), and m/e 642 (M - 127, loss of an iodine radical). Each of these ions displayed the characteristic dichloro isotope cluster. The ion at m / e 445 probably possessed the acylium structure 11. Trimethylsilylation of rafoxanide results in derivatization of both the phenolic group and the secondary amide group (12). Quantitative conversion to this di-TMSi (12) W. J. A. VandenHeuvel, J. L. Smith, and J. L. Beck, Anal. Lett., 4, 131 (1971). 1380
10
0
derivative is possible only in a large excess of BSA. This is to be expected since silylated secondary amides are powerful silyl donors (e.g., BSA) (13). OTMSi
II A low intensity (-2%) signal at mje 697 is found in the mass spectrum of di-TMSI-rafoxanide. This ion could (13) A. E. Pierce, "Silylation of Organic Compounds," Pierce Chemical Company, Rockford, Ill. 1968.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
100-
392
90 -
80
-
70
-
LIGHT
5 60-
6
v)
M 643
Figure 3. Mass spectrum (m/e 300650) of the di-TMSi derivative of the product resulting from photolysis of rafoxanide result from a minor fragmentation path wherein the diTMSi-rafoxanide loses a (CHa)lSiCH? radical (M - 72). A more likely explanation is that the di-TMSi-rafoxanide reacts with “active sites” in the gas chromatographic packing, exchanging one of its TMSi groups for a hydrogen atom to form mono-TMSi-rafoxanide. The effect is so small with a carefully deactivated gas chromatographic support as to be chromatographically undetectable. A typical gas chromatogram obtained from a plasma sample (rafoxanide-treated sheep) using the procedure discussed in this paper is seen in Figure lb. The small peak at 0.7-minute retention time corresponds to approximately 2 % of the area of the major peak. Control plasma samples spiked with pure rafoxanide and carried through the assay also exhibit this small peak found in samples of biological origin. Rafoxanide loses one iodine atom in a photolytic reaction. Thyroxine has been shown recently to undergo loss of an iodine atom ortho to a phenolic group in the plasma of normal human subjects (14). While this is obviously not a photolytic reaction, the interesting aspect is that a second ortho iodine atom is not lost. We have found no evidence for metabolic loss of the one ortho iodine atom or any other type of animal metabolism of rafoxanide in the body fluids examined in the course of this work. Our efforts to identify this minor component led us to a closer examination of the photolytic behavior of rafoxanide. Gas chromatography of the TMSi derivative of the material resulting from exposure of rafoxanide dissolved in ethyl acetate to ordinary laboratory fluorescent light for three weeks gave the result seen in Figure 2. Characterization of the earlier eluted component as the di-TMSi derivative of “mono-iodo-rafoxanide” (mol wt 643) was accomplished by combined GC-MS. Comparison of its partial mass spectrum (Figure 3) with that of di-TMSi-rafoxanide established the similarity of their fragmentation patterns, ions in the former being shifted 126 amu downfield from those
in the latter. This shift corresponded to the replacement of one of the iodine atoms by a hydrogen atom. Several experimental observations indicated that it may have been the iodine atom ortho to the phenolic group (3-position) which was photolytically labile. Identical gas chromatographic retention times were found for the di-TMSi derivative of “mono-iodo-rafoxanide” and authentic 3-desiodo-rafoxanide (lower chromatogram, Figure 2, peak A ) . Spiking of the plasma extract of Figure l b with authentic 3-desiodorafoxanide enhanced peak 1 and gave no new peaks. Further, the mass spectra of the di-TMSi photolysis product and of authentic di-TMSi-3-desiodo-rafoxanide were indistinguishable. The 60 MHz NMR spectrum of the photolysis product, indicative of a 1,2,5- rather than a 1,2,3-trisubstituted benzene, was entirely compatible with that of authentic 3desiodo-rafoxanide. Whereas free rafoxanide in ethyl acetate solution was unstable toward light, di-TMSi-rafoxanide (in BSA) and rafoxanide in 0.5 NaOH solution remained intact even after overnight UV irradiation in a quartz vessel at room temperature. Protection against irradiation was apparently afforded by derivatization of the phenolic group., either as the TMSi ether or as the phenoxide ion. Under the conditions where rafoxanide was photolytically unstable, 3-desiodo-rafoxanide in ethyl acetate solution did not lose iodine. The unavailability of 5-desiodo-rafoxanide precluded a direct comparison with the photolysis product. The sensitivity requirements of our proposed assay necessitated chromatographic detection at the subnanogram level. On a 2-ft X 3-mm i.d. glass spiral column containing the 3 OV-17/1.5 SE-30 packing, our lower practical detection limit for di-TMSi-rafoxanide was approximately 1 ng per injection. [For comparison, Jaakonmaki and Stouffer (15) found that with a 56 pCi 22BRadetector they were able to detect less than 0.5 ng of triiodothyronine methyl ester as the N,O-dipivalyl derivative.] Increasing the flow rate at constant column temperature or increasing the temperature
(14) K. Sterling, M. A. Brenner, and E. S. Newrnan, Science, 169, 1099 (1970).
(15) P. I. Jaakonmaki and J. E. Stouffer, J. Gas Chromatogr., 5, 303 (1967).
x
x
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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Table I. Recovery of Rafoxanide from Spiked Control Plasma Recovery, % Rafoxanide added, ppm GC Radioactivity 0.19 0.39 0.49 0.80
79 77 76 74
74 77 76 80
Table 11. Typical Experimental Data Animals Rats (4) Mice (4) Rats (2) Mice (4)
Rafoxanide Dose, mg/kg/day Plasma level, pg/ml 0.2 0.2 10 10
