2996
Anal. Chern. 1985, 57,2996-2997
total quantity was considerably larger than that of gas evolved from the inlet. The percentage of hydrogen was relatively lower than the first measurement, and there was a corresponding increase in the percentage of m / z 28 component; however, the quantity of gases was significantly less than in the first analysis. This method offers rapid characterization of high-ternperature outgassing from many materials used in high-vacuum furnaces, and furthermore, it can be used to evaluate different
treatments of such materials so as to enhance their quality. Registry No. Hz, 1333-74-0;CH4,74-82-8;CzHz,74-86-2; Nz, 7727-37-9; CO, 630-08-0; C2H6, 74-84-0; C,H,, 74-98-6; BN, 10043-11-5.
LITERATURE CITED (1) Colwell, 8.H. Vacuum 1970, 2 0 , 481-490. (2) Hu, J. C. A. Anal. Chem. 1981, 53, 942-943.
RECEIVED for review May 3, 1985. Accepted July 19, 1985.
Apparatus for Trace Determination of Volatile N-Nitrosamines in Small Samples Harry M. Pylypiw, Jr., Frank Zimmerman, and George W. Harrington*
Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122 Lucy M. Anderson
Laboratory of Comparative Carcinogenesis, National Cancer Institute, Ft. Detrick, Frederick, Maryland 21701 Because of their importance as a potential major human health hazard (1, 2), N-nitrosamines (NAs) have been the subject of many investigations. In all such studies, analytical chemistry plays a key role (3). In fact, guidelines were recently suggested to ensure accuracy in quantitation techniques (4). A significant problem that has existed in various in vivo metabolic experiments concerns difficulties associated with analyzing very small samples for very low levels of NAs. For example, the mouse, which is frequently used as the laboratory animal for metabolism studies, supplies the analyst with about 0.5-1.0 mL of blood per animal and organs that weigh between 0.3 and 1.5 g. When the levels of NAs expected are of the order of a few parts per billion, sample preparation becomes the most important step in the analysis, since the instrumental method, gas chromatography coupled with the thermal energy analyzer (GC-TEA), is well established and has a detection limit of less than 1 ppb (5). The usual method of sample preparation for volatile NAs in a wide variety of samples is the mineral oil distillation (6). While this technique is acceptable for large samples (ca., 5-20 g), it falls short of the demands imposed by small samples (ca., 0.2-1.5 9). The apparatus and procedures described here minimize sample handling and yield recoveries in the 90-100% range for small samples containing a few parts per billion volatile NAs.
EXPERIMENTAL SECTION Chemicals. All chemicals and solvents were ACS reagent grade or better. Water was distilled and purified with a Barnstead NANOpure I1 system. Morpholine (Aldrich Chemical Co. no. 13, 423-6) was double distilled, and the second distillate was collecbd and stored under nitrogen gas. The defoaming agent used was Antifoam B (Fisher Scientific Co., CS-283-4). NAs were received from the NCI Chemical Carcinogen Reference Standard Repository, a function of the Division of Cancer Etiology, NCI, NIH, Bethesda, MD. Instrumentation. A thermal energy analyzer,Model TEA-502, manufactured bv Thermo Electron Corm. interfaced with a Hewlett-Packari Model 5790A series paiked-column gas chromatograph was used for separation and detection of NAs. Specific conditions were as follows: column 6 f t X 2 mm i.d. glass, packed with 10% Carbowax 20M + 2% KOH on Chromosorb W AW, 80/100mesh; programmed from 120 to 190 "C at 5.0 OC/min; final hold time, 1.0 min; total run time, 15.2 min; carrier gas, He; flow rate, 12 mL/min; on-column injection, 250 "C; interface line, l/s in. 0.d. glass-lined stainless steel, 250 OC; TEA furnace, 525 O C ;
-8
lllr- I
.~
I
I
_____-_______
1.50-1.75mmIDcapillary
t _ _ _ _ _ _ .
Flgure 1. Distillation-extraction apparatus. All dimensions are in millimeters except where noted. All tubing is standard wall unless otherwise indicated. All tubing sizes are outer diameters except the capillary. Dimensions marked with an asterisk are critical.
TEA cold trap, -151 "C; and TEA detector pressure, 1.4 torr. All data were accumulated and calculated on a Hewlett-Packard 3390A reporting integrator. Apparatus. The apparatus is shown in Figure 1. It is a modification of a commercially available model (6826 distillation-extraction apparatus, Ace Glass, Inc., Vineland, NJ). The dimensions are given in Figure 1 and the caption. Those dimensions marked with an asterisk are critical. The heights of the flask arms are critical to ensure that the liquid-liquid interface occurs just below the capillary tip. If the interface is not properly located, a mixture of liquids will return to the lower flask. The dimension of the capillary tip is important to regulate flow rate. It was found that an optimum flow rate was required to obtain
0003-2700/85/0357-2996$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
2997
Table I, N-Nitrosodimethylamine Recoveries by GC-TEA
NDMA concn, sample
n
size range, g
blood liver kidney brain
6 6 6 6
0.8-1.2 1.2-1.6 0.4-0.7 0.3-0.7
ppb 2.3 3.0 3.6 4.2
& 1.3
f 0.3 f
0.9
f 1.5
I
3
recovery range, 70 94 f 2 95 f 1 93 f 2 97 f 1
a sharp interface without emulsion formation. These dimensions were established by experiment and are not simply a reduction of the original device. Samples. Male strain A/J mice from the Jackson Laboratory, Bar Harbor, ME, or A/JCr mice from the Animal Production Area of the Fredrick Cancer Research Facility were sacrificed and samples of blood, liver, kidney, and brain were placed in small plastic cryotubes, frozen in liquid nitrogen, and shipped in dry ice to the laboratories of the Chemistry Department of Temple University for NA analysis. Sample Preparation Technique. Each sample was weighed and placed in 100-mL round-bottom flask with 1 g of sulfamic acid, 0.5 mL of concentrated H2S04,1g of Antifoam B emulsion, 100 p L of morpholine, and 25 mL of water. N-Nitrosodi-npropylamine (NDPA) was added as an internal standard. The flask was attached to the higher arm of the apparatus. Another 100-mL round-bottom flask containing 50 mL of methylene chloride was attached to the lower distillation arm. Boiling chips were added to each flask, and each was heated via a heating mantle to the boiling point of the respective solvent for 15-16 h. After cooling for 30 min, the contents of the flask with methylene chloride were poured through a pad of anhydrous sodium sulfate into a Kuderna-Danish evaporator. The pad was washed three times with 10-15 mL of methylene chloride. The methylene chloride was concentrated to 2.0-2.5 mL, and 25-30 pL of the concentrate was analyzed for NAs via GC-TEA. R E S U L T S AND DISCUSSION The apparatus involves simultaneous distillation and extraction. The samples, blood or tissue, are digested during the boiling process. The volatile NAs are distilled along with water and methylene chloride into the condenser. The mixed condensate flows into the capillary tubing to the bottom of the apparatus where the methylene chloride, now containing the NAs, returns to the methylene chloride flask and the water, to the sample flask. After the apparatus was run for 15-16 h, the NAs originally in the sample flask have been transferred to the methylene chloride flask. Small samples of blood and tissues were prepared, and sample results with recoveries are given in Table I. Recoveries for N-nitrosodimethylamine (NDMA) added at the start of the procedure by direct spiking and/or injection into organs averaged 93.5%. These recoveries show an improvement over those from previously published methods (3, 6) for similar samples, along with decreased contamination from added water. No N-nitrosomorpholine was found in the extracts. It has been documented that NAs can form in deionizing cartridges (7). The original apparatus requires 250 mL of
I 0
I
I
I
4
8
12
i 16
Minutes Flgure 2. Chromatograms of reagent blanks: curve I, unmodified apparatus showing peaks for NDMA (1) and unidentified compound (2) (see text) from water contamination; curve 11, modified apparatus showing absence of contaminatlon. Peak 3 is the internal standard, NDPA.
water in the sample side. Water containing 0.01 ppb NDMA contributes 2.5 ng of NDMA when 250 mL are used. When a 1-g sample is then analyzed, a significant error from NDMA contamination is introduced that is on the order of 2.5 ppb, based upon sample size. However, the modified extractor requires the addition of only 25 mL of water, one-tenth of the water needed in the commercially available apparatus. Figure 2 shows duplicate blank samples, one processed via the commercially available extractor and the other by the modified extractor. Contamination from the water has been eliminated with the reduced volume apparatus. Curve I shows a peak (1)from NDMA in the water plus a peak (2) due to an unidentified non-N-nitroso compound. The latter was established by the photolysis procedure of Doerr and Fiddler (8). The extractor unit provides the investigator with a better choice over the mineral oil distillation. The latter involves seven separate operations whereas this procedure consists of only three. Minimal handling is, of course, critical in trace analysis. Where sample size is not critical, the larger apparatus can be used with the same minimal sample handling, and the results are superior when compared to other techniques. Registry No. N-Nitrosodimethylamine, 62-75-9. LITERATURE C I T E D (1) Magee, P. N.; Barnes, J. M. Br. J. Cancer 1956, 7 0 , 114-122 (2) Fine, D. H. In “Nitrosoamlnes and Human Cancer”; Magee, P. N., Ed.; Cold Spring Harbor Lab: New York, 1982; Banbury Report Series, No. 12, pp 199-210. (3) Hotchkiss, J. H. J. Assoc. Off. Anal. Chem. 1981, 6 4 , 1037-1054. (4) Fine, D. H. I n “Nitrosamines and Human Cancer”; Magee, P. N., Ed.; Cold Spring Harbor Lab: New York, 1982; Banbury Report Series, No. 12, pp 185-174. (5) Fine, D. H.; Rufeh, F.; Lieb, D.;Rounbehler, D. P. Anal. Chem. 1975, 47, 1188-1191. (6)Havery, D. C.; Fazio, T.; Howard, J. W. J . Assoc. Off. Anal. Chem. 1978, 67, 1374-1378. (7) Kimoto, W. I.; Dooley, J.; Carre, J.; Fiddler, W. Wafer Res. 1980, 74, 869-876. (8) Doerr, R. C.; Fiddler, W. J. Chromatogr. 1977, 740, 284-287.
RECEIVED for review May 24,1985. Accepted August 12,1985. This investigation was supported, in part, by Grant No. CA18618, awarded by the National Cancer Institute, DHEW.
CORRECTION Photoelectroanalytical Chemistry: Electrochemical Detection of a Photochemically Active Species, Tris(2,2’-bipyridine)rut henium( 11)
J. M. Elbicki, D. M. Morgan, and S. G. Weber (Anal. Chem. 1985, 57, 1746-1751).
In Table I, under the heading Q, the last two labels have been reversed. The correct order going from top to bottom [Co(NH3)&1I2+,Co(cysS03)t-, and should be [CO(C,O~)~]~-, Co(gly),. The numbers are correct in their positions as published.
