Chapter 39
Nonvolatile N-Nitrosamides in Dried Squid Analysis by High-Performance Liquid Chromatography-Photolysis-Chemiluminescence 1
2
S. H. Kim and J. H. Hotchkiss 1
Cheju National University, Cheju-do, Korea Institute of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853
Downloaded by FUDAN UNIV on February 15, 2017 | http://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch039
2
The consumption of dried and salted fish products has been associated with nasopharyngeal and gastric cancers (1). N-nitroso compounds (NOC) have been suggested as one potential etiological factor (2). Relatively high amounts of volatile N-nitrosamines (VNA) are formed when dried squid is broiled (3). It is possible that dried squid also contains nonvolatile N-nitroso compounds (NVNOC) which might play a role in the risk associated with dried fish. However, analytical methods for NVNOC, and particularly N-nitrosamides in foods, have not been developed. Recently developed instrumentation, based on the photolysis of NOC to produce nitric oxide, makes detection of NVNOC possible by interfacing a chemiluminescence detector to an HPLC. We report here an analytical method for detecting nitrosamides in uncooked dried squid. As a model nitrosamide, we used N-nitrosotrimethylurea (NTMU), which is relatively stable, of low toxicity, and not reported to occur in nature. Our goal is to develop a method which we can apply to dried fish after cooking or reaction with nitrite. As a preliminary step, we undertook analysis of squid which was spiked with a model N-nitrosamide. It is likely that an analytical method capable of detecting NTMU would also detect other unknown NVNOC. Experimental Imported Korean dried squid was locally obtained. N T M U was synthesized by nitrosation of trimethylurea (TMU). HPLC-photolysis-chemiluminescence (HPLC-Nitrolite-TEA) conditions were as follows: HPLC, (4.6 χ 250 mm Altex C-18 column with 5 μτη particle size; injection volume of 20 μΐ); mobile phase, H 0 : A C N , 85:15; 1 mL/min. For UV-photolysis and interfacing to chemiluminescence detector, we used the Nitrolite (Thermedics Inc., Woburn, MA) which is based on a published design (4). Sample preparation is outlined in Figure 1. N T M U was extracted with MeOH, and the lipids were removed from the extract by a modification of the method of McLeod (5). This procedure removes lipids and waxes by precipitation at -85 °C while the extract is agitated by bubbling N gas to keep the particles small. The sample was filtered at -85 °C in a cold-jacketed Buchner funnel. The filtrate was rotoevaporated to ca 10ml at 35 °C. The concentrate was then adjusted to pH 3-4 with 3N HjS0 . The extract was mixed with 8g Celite, packed over 15g N a S 0 in a glass column (2 χ 29cm) and eluted 2
2
4
2
4
0097-6156/94A)553-0355$08.00/0 © 1994 American Chemical Society
Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
356
NITROSAMINES AND RELATED JV-NITROSO COMPOUNDS Squid ca. 20g + 1.194pg N T M U I SOml M e O H + 5 ml 1% Sulfamic acid Homogenize Filter Repeat homogenization Solid
Filter (discard) e
ppt fat (ca.,-85 C) Fat (discard)
Filter (ca.,-85*C)
Rotary evaporation to ca. 10ml Adjust p H 3-4 (3N H S 0 )
Downloaded by FUDAN UNIV on February 15, 2017 | http://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch039
2
4
M i x w/8g Celite & pack over 15g N a , S 0 in column 4
Extract w/80ml hexanes Rotary evaporation to ca. 3ml Load on SPE (SiOH) (prewetted w/5ml hexanes) Elutc with 2.5ml A C N
I Concentrate w / N , to 0.5ml & add 0.5ml water
ι H P L C photolysis chemiluminescence
Fig 1. Analytical Scheme for N T M U in Dried Squid with 80ml hexanc. The hexane was rotoevaporated to ca 3ml. The hexane extract was then loaded on a pre-wetted (5 mL hexanes) SPE(SiOH) column. N T M U was eluted from the column with 2.5 ml acetonitrile (ACN). The A C N was concentrated under a stream of N gas to 0.5ml and 0.5ml H 0 added. This solution (20 ml) was analyzed by HPLC-Nitrolite-TEA. 2
2
Results The HPLC-Nitrolite-TEA response to N T M U was linear (r=0.99) over a range of 2 to 20ng injected. The MeOH extract was viscous, cloudy and yellow after rotoevaporation to 10ml. Celite column extraction with hexanes and concentration to 3ml resulted in a fine precipitate which was removed by solid phase extraction (SPE). Moderately polar SPE packings were tested using A C N and dichloromethane (DCM) as eluting solvents to further clean the extract. Silica gel and florisil SPE with A C N as eluent, and cyano and amino SPE with D C M as eluent, produced satisfactory recoveries and baseline. Silica gel was chosen because A C N was compatible with the mobile phase. N T M U was most stable in the pH range of 3 to 5. The temperature of the water bath was important in achieving acceptable recoveries. With MeOH, the highest recovery was obtained of 30 °C to 40 C . With hexanes, maximum recovery was obtained at 25 C and 30 C . The detection limit (3x noise) was 10/jg/kg and recovery of N T M U from 60 μζ/kg spiked squid was 79% (s.d.= 13%; c.v.» 16%; n«12). Figure 2 is a representative chromatogram of a N T M U standard, spiked sample and unspiked sample. N T M U was not found in any unspiked samples but e
e
Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
e
39. KIM AND HOTCHKISS
Nonvolatile N-Nitrosamides in Dried Squid
357
a ο
Downloaded by FUDAN UNIV on February 15, 2017 | http://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch039
ce α
ζ
ζ ο & Ul
M (Γ
8 RT STD
0
4
8
0
4
8
\ΜΙΝ.) NPRO, NTMU + SQUID
UNSPXKED SQUID
Fig 2. Chromatograms of a standard, spiked sample and unspiked sample several samples produced unknown peaks with different retention times than N T M U . This indicates that dried squid contains unknown NOC. Because of the selectivity of the photolysis-chemiluminescence reaction and this analytical procedure, it is likely that these responses are due to unknown nonvolatile N nitroso compounds such as nitrosamides. Literature Cited
1. Yu, M.C.; Ho, J.H.C.; Lai, S-H.; Henderson, B.E. Cancer Res., 1986, 46, 956-961. 2. Mirvish, S. J. Nat. Cancer Inst. 1983, 71(3), 631-647. 3. Matsui,M; Ishibashi, T; Kawabata, T. Bull. Japan. Soc. Fish. 1984,50(1), 151-154. 4. Conboy, J.J.; Hotchkiss, J.H. Analyst 1989, 114, 155-177. 5. McLeod, H. Anal. Chem. 1972, 44, 1328-1330. R E C E I V E D January 26, 1994
Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
