1872
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
(2) Jeffery, P. G. "Chemical Methods of Rock Analysis"; Pergamon Press: New York, 1970; pp 415-416. (3) Murthy, A. R. V.; Narayan, V. A.; Rao, M. R. A. Analyst(London) 1956, 81. 373-375. (4) Murthy, A. R. V.; Sharada, K. Anaksf (London) 1960, 8 5 , 288-300. ( 5 ) Morie, G. P. Tob. Sci. 1971, 107, 34.
(6) Orion Research Incorporated, "Determination of Total Sulfide Content in Water", Applications Bulletin No. 12, Orion Research Inc., Cambridge, Mass., 2 pp.
RECEIVED for review April 2, 1979. Accepted May 29, 1979.
High Performance Liquid Chromatographic Determination of Bovine Insulin Alan Dinner" and Leslie Lorenz Eli Lilly and Company, P.O. Box 678, Indianapolis, Indiana 46206
During the past four years, the use of reversed-phase high performance liquid chromatography (HPLC) for the purification and/or quantitation of biologically active molecules has mushroomed ( 1 , 2 ) . Reports of the application of this technique to the separation and analysis of underivatized peptides have recently started to appear in the literature. Since an early report by Burgus (3),the HPLC characteristics of several small (less than 10 amino acids) peptides have been discussed (4-8), and Vogelsang has reported (9) on the HPLC analysis of the polypeptide (15 amino acids) antibiotic, gramicidin. In more recent publications and symposia, the use of reversed-phase HPLC for the analysis of higher molecular weight peptides and proteins (10-12) has been discussed. Work in our laboratory required a rapid and accurate method for the measurement of the polypeptide bovine insulin (51 amino acids, mol wt 5734) in the presence of the byproducts most commonly encountered during its purification (see Table I). The development of the desired method would relieve some of the need for the slow and tedious classical insulin analyses involving DEAE-cellulose chromatography or polyacrylamide disc-gel electrophoresis (13). Alternatively, the method should be amenable to modification to allow collection of minor insulin components which are difficult to obtain in high purity by classical methods.
EXPERIMENTAL A Waters Associates model 6000A solvent delivery pump, an autoinjector with a Rheodyne sampling valve, and a Varian Associates Vari-Chrom absorbance detector are used in all determinations. The column is a Lichrosorb RP-8,lO km, 250 mm x 4.6 mm (id.) from E. Merck and Company. A temperature bath set at 30 "C which circulates water through a jacket surrounding the HPLC column is also employed. Chromatographic Conditions. Reagent grade ammonium sulfate was obtained from J. T. Baker Chemical Company, and the pH of the solution is adjusted to 3.5 with dilute sulfuric acid. Sterile water is used for the aqueous solution. The 24.5/75.5 acetonitrile (Burdick and Jackson, glass distilled) - 0.2 M pH 3.5 ammonium sulfate solution for isocratic elution is prepared by mixing appropriate volumes of each solvent after which the mixture is degassed under vacuum. The flow rate is 6.0 mL/min, the injection volume is controlled at 20 kL, and the detection is at 215 nm. All insulin samples used in this work are obtained from these laboratories. The analysis of data is performed on a digital data system using the approach of Savitzky and Golay (14) to determine appropriate peak parameters.
RESULTS AND DISCUSSION Reversed-phase HPLC proved to be a satisfactory method for the determination of bovine insulin. The higher molecular weight proinsulin-like impurities, porcine insulin dimer, and bovine and porcine proinsulin (the latter contains 84 amino acids with a molecular weight of 9082 (13a,b))were all retained on the RP-8 column under the conditions utilized to elute bovine insulin; an increase in the amount of acetonitrile in 0003-_2700/79/035 l-lm;OO/O
Table I. Potential By-products in Bovine Insulin bovine monodesamido insulin porcine insulin porcine monoarginine insulin porcine monodesamido insulin bovine monoarginine and diarginine insulin bovine and porcine insulin dimer bovine and porcine proinsulin bovine and porcine proinsulin-like components Table 11. Elution Time of Insulin-Like Proteins component bovine diarginine insulin bovine monoarginine insulin bovine insulin bovine monodesamido insulin porcine insulin porcine monodesamido insulin porcine monoarginine insulin
elution time, s
k
357
13.9
441
17.4
590
23.6
770
31.1
910
36.9
1170 720
47.8
29.0
the mobile phase did result in the elution of these components. The insulin-like proteins, such as porcine insulin, porcine monodesamido insulin, bovine monodesamido insulin, and porcine monoarginine insulin, all separated from the bovine insulin under the isocratic conditions employed (see Table I1 and Figure 1). The response curve from bovine insulin was found to be linear from 2.5 to 12.5 mg/mL (50 b g to 250 pg of insulin on the column). A study to ascertain the precision associated with this method was performed on a set of 30 individually prepared samples from a common lot of insulin. The resulting relative standard deviation was 0.9970 for peak area and 1.22% for peak height. I t is apparent that seemingly minor structural changes among the insulin-like proteins result in significant differences in their retention characteristics. Although porcine insulin and porcine monodesamido insulin (13) differ only by the replacement of an aspartic acid in the latter for an asparagine in the former, these two 51 amino acid-containing proteins are resolved from one another. We have also observed that minor alterations in the mobile phase cause drastic changes in elution time. Whereas the proinsulin-like proteins do not elute within 30 min using 24.5% acetonitrile, the elution time drops to 1@15 min with 26-2770 acetonitrile. Finally, we have observed that insulins from other species, (porcine, rabbit, ovine, and human) although structurally similar (15) to one another, also have characteristic retention volumes under the conditions we have employed for the bovine insulin quantitation. We are in the process of correlating the structural features of the various insulins with their retention characteristics, and hope to report on this a t a later date. The different sensitivity of various insulin proteins to binding on the RP-8 column has enabled us to develop a rapid 63 1979 American Chemical Society
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
