ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
on the classical chromatography of insulin. We also thank Mark Hayes for samples of bovine and porcine insulin, and Ed Logsdon for discussions concerning the HPLC of peptides.
PORCINE INSULIh
BOVINE
INSULIN
LITERATURE CITED
1\
PORCINE 3ESA"lDS
INSULIN
-1.24
ik-yi;
'
'
8 7 i . i iEc'
-6
'
iihr'
1873
'
i3k.l
'
'
i515.
'
;.in.
Figure 1. Sample chromatogram of insulin mixture
and accurate method for the determination of bovine insulin. I t is also apparent from the above data that application of this method to the isolation of various insulin components will be quite facile.
ACKNOWLEDGMENT The authors thank Ronald Chance of these laboratories for a variety of insulin samples, as well as for helpful discussions
M. Gurkin, Am. Lab., 9(1), 29 (1977). A listing of recent articles is found in -1. Chromatogr., 144, 8311, sec. 32 (1977). R. Burgus and J. Rivier, 14th European Peptide Symposium, Wepion, Belgium, April 1976, A. Loffet, Ed., Editions de I'Universite' de Bruxelle. K. Krummen and R. W.Frei, J . Chromatogr., 132, 27 (1977). K. Krumrnen and R. W.Frei, J . Chromatogr., 132, 429 (1977). J. J. Jansen et al., J . Chromatogr.. 135, 155 (1977). W. Monch and W.Dehnen, J . Chromatogr., 140, 260 (1977). I. Molnar and C. Horvath, J . Chromatogr., 142, 623 (1977). K. S. Axelsen and S. H. Vogeisang, J Chromatogr., 140, 174 (1977). W. Monch and W. Dehnen, J . Chromatogr., 147, 415 (1978). W.S. Hancock et al., Science, 200, 1168 (1978) International Liquid Chromatography Symposium 11, 5-6 October, 1978, Boston, Mass. (a) R. Chance, Diabetes, 21 (suppl. 211, 461 (1972); (b) R. Chance, R. M. Ellis. and W.W.Bromer, Science. 161, 165 (1968). A. Savitzky and M. J. E. Golay, Anal. Chem., 36, 1627 (1964). L. F. Smith, Diabetes, 21 (suppl. 2), 457 (1972).
RECEIVED for review February 5 , 1979. Accepted June 4, 1979.
On-Column Chromatographic Extraction of Aflatoxin M, from Milk and Determination by Reversed Phase High Performance Liquid Chromatography Wray Winterlin," Gregory Hall, and Dennis P. H. Hsieh Department of Environmental Toxicology, University of California, Davis, California 956 16
Aflatoxin MI is a hydroxylated animal metabolite of aflatoxin B1, found in milk. Procedures presently available for the determination of aflatoxin MI in milk are relatively long, time consuming, and expensive due mainly to sample extraction and cleanup which require large quantities of solvents, laboratory supplies, and analyst time for an analysis. Beebe ( I ) , along with other investigators, have cited several references (2-9) for determining aflatoxins in foods and agricultural products and also discussed the advantages of reverse phase HPLC and fluorescence detection. None of these procedures are described as suitable for milk with the combined advantages of simplicity, accuracy, sensitivity, rapidity, and low operating cost. Presented here is a procedure that has all the advantages listed for aflatoxin M1 in milk.
EXPERIMENTAL Apparatus. HPLC was performed using a Model 6000A pump (Waters Associates, Milford, Mass. 01757) a U6K injector (Waters), a model 420 fluorescence detector (Waters) operated at excitation at 365 nm and detection at c a 400 nm. A pre-column, 12 cm long by 4.2 mm i.d., was packed with 30/44 p Vydac reversed phase (Applied Science) and was placed ahead of a 30 cm x 3.9 mm i.d. pC18Bondapak high efficiency column. A mobile phase of 28% acetonitrile in water with a flow rate of 2.5 mL m i d was used throughout the study. Injection volumes into the U6K injector were 500 pL or less. If more sensitivity is needed, a preparative pC18 column can be substituted for injections up t o 1500 pL. Materials. Only two solvents were used in this procedure, spectroquality acetonitrile (Burdick and Jackson Laboratories, Muskegon, Mich.) and glass distilled water filtered through a 0.5-pm Gelman Metricel filter (Gelman No. 60173, Type G-A-6, Fisher Scientific, Pittsburgh, Pa.), then sonication under vacuum 5 min. Sep Pak (Waters Associates) consisting of Bondapak CIS (Waters) was used for separating aflatoxins from milk constituents. Procedure. Ten milliliters of milk are withdrawn from a milk container and diluted to 25 mL with water. The CISSep Pak is prewashed with 5 mL of water followed by 5 mL of acetonitrile 0003-2700/79/0351-1873$01 .OO/O
Table I. Recovery of Aflatoxin M,- Fortified Milk ( 0 . 5 ppb) Residue Found Ohours
sample 1 2 3 4
amount found
6 0 hours ~~-
%recovered
;amount found
%recovered
0.438
87.6
0.476
95.2 98.8
0.535 0.486 0.452 0.504
107.0 97.2 90.4
0.494 0.503
100.6
100.8
using a 5-mL Luer-Lok syringe (Becton, Dickenson & Co., Rutherford, N.J.). Using the same syringe, the sample of milk is transferred to the Sep Pak followed by a 5-mL wash with water. Twenty milliliters of 10% acetonitrile in water i3 then added to the Sep Pak column and discarded. Four milliliters of 30% acetonitrile in water is then added to the Sep Pak column and collected in a graduated centrifuge tube or a 5-mL volumetric flask. The sample is made to volume with distilled water followed by mixing and injection directly onto the HPLC.
RESULTS AND DISCUSSION The method as described here is capable of detecting 0.1 ppb or less. Figure 1 shows chromatograms of M1-free milk and fortified milk at 0.5- and 0.1-ppb levels. Figure 2 shows a chromatogram of MI-contaminated milk containing 0.38 ppb. Background interferences are sufficiently removed to permit detection a t levels less than 0.1 ppb; however, for practical purposes, the 0.1-ppb level seems sufficient. Eight samples fortified a t 0.5 ppb resulted in a standard deviation of 11.89 with an average recovery of 95.0%. Recovery studies a t 0.1 ppb range between 80 and 100%. It should be mentioned, the results from contaminated M1 milk sample compared very well using the AOAC method and its modifications (IO). Table I shows the recovery of MI-fortified milk immediately after fortification and 60 h later. During this 60-h period, the milk was stored in a 7 "C refrigerator to see what losses of 0 1979 American Chemical Society
1874
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
I
ti
Yn r
f.
-
T
T
-
3
TIME,min
A
~
4
T
~
TIME, min
Figure 1. Chromatograms of aflatoxin Ml-free milk (A), aflatoxin Ml-fortified milk
1
1
20
4
6
TlME,min
at 0.5 ppb (B), and
aflatoxin M,-fortified milk
at 0.1 ppb (C)
Table 11. Residues of Contaminated Aflatoxin M , Milk sample 1 2 3 4 5
AOAC procedure 0.46 0.16 0.17
0.38 0.11
0.81
1.10 1.14 f 0.565
1.07
F 0.534
O
h
4
6
TIME.min
Figure 2. Chromatograms of aflatoxin M,-contaminated milk containing 0.38 ppb
MI might occur during this period. It appears that there were no detectable losses, and that MI stored under these conditions is very stable. Table I1 shows residues of MI-contaminated milk using the AOAC procedure with TLC/densitometric detection (9, 10) and the procedure as reported in this paper. Background levels from the TLC plate make it very difficult to analyze at low levels and may account for the greater differences between the two methods when residues of less than 0.20 ppb were found. Overall, there was good agreement between the two methods with an average residue of 0.534 ppb for the AOAC procedure and 0.565 ppb for the procedure as described in this paper. The time required for a complete analysis including HPLC is less than 20 min in contrast to other procedures which require in excess of 3 h or 4-6 samples per day when detecting levels of
