Gas Chromatographic Determination of Nonvolatile Fatty Acids in Cigarette Smoke M. R. Guerin, Geraldine Olerich, and W . T. Rainey Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
The acidic components of cigarette smokes are of interest to tobacco scientists because of the relationship such components have with the quality of the smoking product ( I ) . Acidic components are of interest in experimental tobacco carcinogenesis through the well established tumorpromoting, accelerating, and/or co-carcinogenic activity of the weak acid subfraction of smoke condensate (2-7). Lower molecular weight carboxylic acids have received the greatest attention in terms of smoke “quality,” and phenolics in terms of tumor promotion. Higher molecular weight fatty acids may also be important in these phenomena. It may be particularly important ( 3 ) to expedite a consideration of acidic compounds other than phenols as indicators of the tumor-promoting and/or co-carcinogenic activity of cigarette smoke condensates. Fatty acids in the 16-18 carbon range have been determined in tobacco leaf (8) and tobacco smokes ( I ) by gas chromatography of the free acids and following conversion to methyl esters. Trimethylsilylation has been applied to tobacco leaf acid “fingerprinting” for qualitative analysis (9). The best defined procedure for the quantitative determination of smoke fatty acids involves serial extraction, methyl ester formation, florisil column cleanup, gas chromatography, and isotope dilution (10). This note presents a procedure, based on the trimethylsilylation of whole condensate or particulate matter, which allows the convenient quantitative determination of free fatty acids with minimal sample preparation.
EXPERIMENTAL Quantitative Analyses. The procedure is equally applicable to analyses of smoke condensate and particulate matter. For the analysis of particulate matter, four weight selected ( A 2 0 mg of the average weight of 100 cigarettes) cigarettes are smoked using standard (11) puff characteristics through each of four standard CM-113A “Cambridge” filters (Phipps and Byrd, Richmond Va.). Each filter is placed in a separate 30-ml capacity septum capped vial (Schwarz/Mann, Orangeburg, N.Y.) and 10 ml of distilled pyridine is added to each. The vials are allowed to stand overnight, are then mixed for 5 minutes using a Vortex mixer, and centrifuged to compact the disrupted filter pad. For the analysis of condensate, approximately 500 mg of a 50 wt 70suspension of dry condensate in acetone or 250 mg of dried condensate is weighed into a 15-ml capacity centrifuge tube fitted with a cap, 10 ml of distilled pyridine is added, and the tube is allowed to stand overnight. The sample is mixed using the Vortex mixer and may then be aliquoted. Stedman, D. Burdick, W . J . Chamberlain, and Irwin Schrneltz, TobaccoSci., 8, 79 (1964) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco Smoke,” Academic Press, New York, N . Y . , 1967, pp 226, 206. B. L. Van D u u r e n , T . Blazej, B. M . Goldschmidt. C. Katz, S. Melchionne. and A . Sivak, J . Nat. Cancer lnst., 46, 1039 (1971). 6. L. Van Duuren. A . Sivak, C. Katz, and S. Melchionne, J. Naf. Cancer lnst.. 47, 235 (1971) D. Hoffmann and E. L. Wynder, Cancer, 27, 848 (1971). F. G . Bock, J. Nat. Cancer Inst., 48. 1849 (1972) F. G . Bock, A . P. Swain, and R . L. Stedrnan, J. Mat. Cancer lnst.,
(1 j R. L.
(2) (3) (4) (5) (6) (7)
47,429 (1971). ( 8 ) I . Schmeltz, R. L. Stedman, and R. L. Miller, J . Ass. Offic, Chem., 46, 779 (1963). (9) T. C . Jones and I . Schmeltz, Tobacco Sci., 12, 10 (1968) (10) D. Hoffrnann and H. Woziwodzki, Beitr. Tabak, 44, 167 (1968). ( 11j C. L. Ogg, J. Ass. Offic.Agr. Chem., 47, 356 (1964).
Agr.
A 0.4-ml aliquot of the pyridine extract is transferred to a 3.5ml capacity septum capped vial and 0.2 ml of BSTFA-1% TMCS (Bistrimethylsilyltrifluoroacetamide-Trimethylchlorosilane,Regisil, Regis Chemical Company, Chicago, Ill.) is added. The vial is heated a t 65 “C for 30 minutes, cooled to room temperature, and a 5-pl aliquot is subjected to gas chromatography. Chromatographic conditions employed are as follows: single hydrogen flame ionization detector, column of 6 feet x 0.25-in. 0.d. standard wall Pyrex tubing containing 4% (w/w) OV-101 on 80-100 mesh Chromosorb G(HP);column, inlet, detector, temperatures of 190 “C, 210 “C, and 230 “C, respectively; helium carrier gas flow cate of 70 cm3/minute. A Tracor Model 220 gas chromatograph was used for this work. Pure fatty acids (Analabs, North Haven, Conn.) are dissolved in pyridine and the pyridine solutions are combined t o provide a master mixed standard containing palmitic (0.2 mg/ml) oleic, linoleic, linolenic, (total of 0.3 mg/ml equally divided between the three acids) and stearic acid (0.1 mg/ml). The master standard is diluted with pyridine to provide four standards with the following concentration ranges: palmitic, 0.04 to 0.20 mg/ml; oleic + linolelinolenic, 0.06 to 0.3 mg/ml total; and stearic, 0.02 to 0.10 ic mg/ml. The standard solutions are derivatized as are the samples and a calibration curve of peak height us. quantity of acid is used to quantitate the chromatograms. Combined G a s Chromatography-Mass Spectrometry. The T M S (Trimethylsilyl) derivatives were prepared by placing approximately 1 mg of t h e fatty acid in a 3.5-ml septum-capped vial and adding 50 p1 each of pyridine and BSTFA. The mixture was shaken and placed in a heated block at 50-60 “C for several hours. Mass spectra were obtained using an instrument constructed a t this laboratory. A Varian Model 1200 chromatograph using a single column and flame ionization detector was used for all GC-MS work. A 60-cm X 8-mm 0.d. (4-mm i.d.1 glass column packed with 3% OV-101 on 80/100 mesh Chromosorb G(HP) was followed by a stream splitting which diverted approximately 95% of the carrier gas through heated stainless steel tubing to the carrier separator. The separator was constructed (12) of 1.25-cm 0.d. porous stainless steel tubing of 0.3-cm wall thickness and 0.1-micron mean pore diameter. A 5-cm section of this material was mounted concentrically in a stainless steel jacket which was attached through large bore tubing to a mechanical vacuum pump. In use, a Nupro Bellows Metering Valve a t the entrance to the separator controlled the flow such that the effluent left the chromatograph column a t slight positive pressure, allowing flow also to the flame detector. The effluent passed through the metering valve into the porous tubing, and, during passage down the length of the tubing, a large portion of the helium carrier was removed through the pores and exhausted to the pump. The remaining, enriched gas passed through an isolation valve and heated stainless steel capillary into the ionization box of the mass spectrometer. The single stage mass spectrometer with a 30-cm radius, 90O-sector magnet was operated a t 4-kV accelerating voltage and 25-eV ionizing electron energy. A 750 I./sec oil diffusion pump was attached through a molecular sieve trap to the source region. The analyzer region was differentially pumped by two 80 l./sec ion pumps. During introduction of the samples, the source region was maintained at
