2000
Anal. Chem. 1981, 53,2000-2003 AMP
i
ATP
Figure 5. Separation of adenosine monophosphSte, dlphosphate, and triphosphate, with tartarate-Mg buffer.
adenosine cyclic 2,3-monophosphate and adenosine cyclic 3,5-monophosphate showed almost same retention time as adenosine phosphate. For the separation of AMP, cyclic AMPs, and orthophosphate, an eluant with weaker ionic strength is necessary. LITERATURE CITED (I) Ohashi, S.;Tsuji, N.; Ueno, Y.; Takeshita, M.; Muto, M. J. Chromatogr. 1970, 50, 349. (2) Ueno, Y.; Yoza, N.; Ohashi, S. J. Chromatogr. 1970, 52, 481. (3) Kura, Genichiro; Ohashi, Shigeru J . Chromatogr. 1971, 56,111. (4) Julin, B. G.; Vandenborn, H. W.; Kirkland. J. J. J. Chromatow. - 1975,
(6) Virkola, P. J. Chromatogr. 1970. 51, 195. (7) Brook, A. J. W. J. Chromatogr. 1970, 47, 100. (8) Murakami, F.; Rokushika, S.; Hatano, H. J . Chromatogr. 1970. 53, 584. (9) Kirkland, J. J. J . Chromatogr. Scl. 1970, 8, 75. (10) Brown, P. J. Chromatogr. 1970, 52,257. (11) Pennington, S. N. Anal. Chem. 1971, 43, 1701. (12) Drobishev, V. I.; Mansurova, S. E.; Kulaev, I. S. J. Chromatogr. 1972, 69,317. (13) Shmukier, H. W. J. Chromatogr. Scl. 1972, IO, 139. (14) Gabrlei, R. F.; Michalewsky, J. J . Chromatogr. 1972, 67, 309. (15) Henry, R. A.; Schmk, J. A.; Williams, R. C. J. Chromatogr. Sci. 1973, 7 1 , 360. (16) Baker, D. R.; Williams, R. C.; Steichen, J. C. J. Chromatogr. Sci. 1974, 12,501. (17) Kreis, W.; Greenspan, A.; Woodcock, T.; Oordon, C. J. Chromatogr. Scl. 1976, 14,331. (18) Anderson, F. S;Murphy, R. C. J. Chrowtogr. 1976, 721,251. (19) Henry, R. A.; Schmit, J. A.; Dieckman, J. F. Anal. Chem. 1971. 43, 1053. (20) Frei, R. W.; Lawrence, J. F. J . Chromatogr. 1973, 83, 321. (21) Seiber, J. N. J. Chmmatogr. 1974, 94,151. (22) Lawrence, F.; Renauk, C.; Frei, R. W. J . Chromatogr. 1976, 127, 345. (23) Erdahl, W. L.; Stolyhwo, A.; Privett, 0. S. J . Am. 011 Chem. SOC. 1973, 50,513. (24) Jungalwala, F. B.; Evans, J. E.; McCluer, R. H. Blochem. J . 1976,
755, 55.
112,443.
(5) Yoza. N.; Kouchiyama, K.; Miyajima, T.; Ohashi, S. Anal. Lett. 1975, 8, 641.
RECEIVED for review March 9,1981. Accepted July 23,1981.
Ion-Exchange Chromatographic Separation of A/-Nitrosodiethanolamine in Cosmetics Yoji Fukuda,* Yoshlhlro Morlkawa, and Isao Matsumoto Shiseido Laboratories, 1050 Nippa-Cho, Kohoku-Ku, Yokohama, Japan 223
A cleanup method is presented for the analysis of N-nitrosodiethanolamine (NDELA) as a contaminant in cosmetic products and their ingredlents. NDELA was found to be speclficaily adsorbed on a strongly bask anion exchange resin (OH-Form) in 80 % ethanol and quantitatlveiy recovered by elution wlth 10% acetic acid/ethanol. Samples were examined in the presence of various ionic and nonlonlc polar compounds used in common cosmetic formulations, and recoverles were found to be satisfactory (80-100%). The detectlon llmit of NDELA obtained wlth a Thermal Energy Analyzer (TEA, registered trademark of Thermo Electron Corp., Waltham, MA) attached to a high-performance liquid chromatograph (HPLC) was 1 X IO-' g (1 ng). Detection limits at the 10 parts-per-blillon (ppb) level were obtained for NDELA in cosmetic products.
With regard to the detection and determination of nitrosoamines in our environment, a selective and highly sensitive method has been developed by Fine et al. (1, 2) using a chemiluminescence detector called the Thermal Energy Analyzer (registered trademark of Thermo Electron Corp.). However, to improve the accuracy of nitrosoamine determination in complicated matrices, this detector must be used in conjunction with a procedure to isolate nitrosoamines from samples. The determination of nitrosamine in cosmetic products is difficult, and for this reason cosmetic products were analyzed.
A cleanup method used for the first time for the analysis of NDELA in cosmetic products was an adsorption chromatography using silica gel (3). However, this method is known to be affected by the type of cosmetic products and their components, resulting in poor recovery and reproducibility. Mitchell and Rahn (4), Rosenberg and co-workers (5),and Fellion and co-workers (6)have reported HPLC methods using a UV detector, instead of the TEA. Ion-exchange chromatography has been used for the separation of various ionics, or mixtures of ionic and nonionic compounds such as surface active agents (7), and also applied to cosmetic analysis (8). At the beginning of this study, the authors thought that NDELA might be adsorbed by a cation exchanger because of its structure, but NDELA was found not to be adsorbed. Later, it was found that NDELA was specifically adsorbed on a strongly basic anion exchange resin (OH form) and the NDELA was quantitatively recovered by elution with an acetic acid/ethanol solution. This means that most of ionic and nonionic components in cosmetic products can be removed by this method. This paper describes the conditions of ion-exchange chromatography required for the separation of NDELA from cosmetic products and the ingredients to be analyzed. Recoveries from various compounds commonly used in cosmetic formulations are also discussed. EXPERIMENTAL SECTION Materials. NDELA. Reagent NDELA (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was purified as follows; Approximately 0.5 g of NDELA was dissolved in 10 mL of 1-propanol
0003-2700/81/0353-2000$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
and ca. 5 g of anhydrous sodium sulfate was added to the solution with stirring. After atanding for an hour or two, the solution was filtered and the filtrate passed through a chromatographic column pakced with 10 mL of AG50WX8 (H form, 50-100 mesh), a strongly acidic cation exchange resin (a sulfonated styrene-divinylbenzene copolymer) obtained from Bio-Rad Laboratories (Richmond, CA). The eluent and washings (50 mL of ethanol) were combined and evaporated to dryness at 35 "C. The residue was used as the standard. The standard solution of 1 pg/mL concentration was prepared in ethanol. The working solutions from 10 to 250 ng/mL concentration for calibration were prepared in ethanol:2,2,44rimethylpentane(10:90). Cosmetic Ingredients. Commercial cosmetic ingredients, sodium dodecyl sulfate (Clz;95% by gas chromatography) and dodecanoic acid diethanolamide (Clz;98%) obtained from Toho Chemical Industry Co., Ltd. (Tokyo, Japan), polyoxyethylene tetradecyl ether (15mol of ethylene oxide adduct; Nihon Emulsion Co., Ltd., Tokyo, Japan), and poly(ethy1ene glycol) (PEC:) 400 Sanyo Chemical Industry Co., Ltd., Kyoto, Japan) were extracted with ethanol to remove inorganic salts and used after being dried. Triethanolamine (99%,Dow Chemical Co., Midland, MI) wm used without purification. All other reagents were analytical grade and were used without further purification. It was necessary to confiim that ethanol had no peak at the NDELA retention time as required in the blank test. Preparation of Ion-Exchange Columns. The cation exchange column was prepared by packing with 20 mL of AG50WX8 (H form, 50-100 mesh) into a 20 mm i.d. X 30 cm length ordinary g h s chromatographic tube equipped with a Teflon stopcock. The column was washed with water (ca. 200 mL) followed by 80% ethanol (200 mL) and then degassed. The anion exchange column was prepared by packing with 30 mL of AGlX2 (C1 form, 50-100 mesh), a strongly basic anion exchanger (a styrene-divinylbenzene copolymer containing trimethylammonium groups) obtained from Bio-Rad Laboratories (Richmond, CA). This resin was converted to OH form by the usual way (9). The column was washed with water (ca. 500 mL) and then with 80-100% ethanol and subsequently degassed. Apparatus. An HPLC was constructed with a Model LC-3A high-pressure pump (Shimadzu Corp., Kyoto, (Japan)and a ]Model SIL-1A injecter (Shimadzu Corp., Kyoto, Japan) equipped with a 200-pL sample loop, with which a 4 mm i.d. X 3.3 cm length guard column packed with a 10 pm LiChrosorb SI-100 (E. Merck, D m t a d t , Germany) and an adsorption chromatographic column, 4.6 mm i.d. X 25 cm length Zorbax SIL (I. E. duPont de Nemours Co., Wilmiigton, DE), were connected in series). The mobile phase was a mixture of ethanol and 2,2,4-trimethylpentane (2080) and its flow rate was 2 mL/min. A Model TEA-502 Thermal Energy Analyzer (Therm0 Electron Corp., Waltham, MA) was used for NDELA determination The catalytic furnace temperature of the TEA was 550 OC and the reaction chamber vacuum was 1.4-1.5 torr. The cooling materials of the cold traps were dry ice/ethanol(-70 "C)for the first trap and liquid nitrogen/ethanol (-130 "C) for the second trap. Ion-Exchange Chromatographic Isolation of NDELA. Each 2 g of samples, whether spiked with a known quantity of NDELA or not, was weighed into a 100-mL beaker and then dissolved with 80% ethanol (40 mL). Ifnecessary,it was dispersed by ultrasonification and then filtered. This solution or filtrate (test solution) was introduced into an anion exchange coluinn or a pair of columns consisting of a cation and an anion exchange columns in series. The beaker and filter were rinsed with 10 mL of 80% ethanol, and the rinsings were added to the test solution in the column. The solution was passed through the column at a flow rate of 2-3 mL/min. The nonadsorptionable materiakg were washed out with 150 mL of 80% ethanol. NDELA was eluted from the anion exchange column with 10% acetic acid/etlhanol (150 mL). The eluent was evaporated to almost dryness by a rotary evaporator at 35 "C. The remaining acetic acid was removed with a jet of nitrogen. A 2-mL portion of ethanol:2,2,4trimethylpentane (1090) was added to dissolve the residue. A 100-pL portion of this solution was injected into the HPLC-TEA. RESULTS AND DISCUSSION T E A Response. As for the peak response to the comcen-
2001
NDELA W
9L2
0
63 W a a
a
8 W a
Ovi6816
MIN
b
2 4 6 8
16 12 MIN
RETENTION TIME
Flgure 1. HPLC-TEA chromatograms of (A), 1 ng, and (E), 25 ng, of standard NDELA: sample size, 100 pL; TEA attenuatlon, X4 (A) and X 8 (B).
tration of NDELA working solutions (10-250 ng/mL), good linearity was obtained. Relative error of peak height was less than 3% in the range from 50 to 250 ng/mL concentration. The chromatogram (A) in Figure 1 shows the case of a 1 ng injection (100 pL portion of 10 ng/mL), while (B) shows the case of a 25 ng injection. In order to enhance the detectable level, we injected as much as 100 pL of a solution. One nanogram was the detection limit, because the signalto-noise ratio (S/N) was approximately 5 as shown in (A). This indicates that the detection of NDELA at the 10 ppb level can be achieved when 2 g of sample is subject to the present method. Chromatographic Conditions. The adsorption of NDELA occurred especially on OH form resin and did not occur on the other form resin such as C1 form or HCOs form. It was also observed that the adsorption occurred for the nitrosoamines having some hydroxyl groups in these molecules such as NDELA and N-nitrosodiisopropanolamine but did not occur for the nitrosamines having no hydroxyl group such as N-nitrosomorpholine or N-nitrosodiethylamine. This shows that the adsorption is due to a weak interaction between the hydroxide ion of the resin and hydroxyl groups of NDELA. This seems to be the same phenomenon observed as glycerol and sugars are adsorlbed on OH form anion exchanger in nonaqueous solvent (IO). Therefore, in order to obtain the best conditions for the ion-exchange chromatographic isolation of NDELA, various factors which might affect the adsorption of NDELA were discussed with evaluation by NDELA recoveries. First, the quantitative adsorption of NDELA on the resin depended on ethanol concentration. When ethanol concentration was varied stepwise from 100 to 80%, the maximum recovery of 1pg of NDELA introduced into an anion exchange column was 93% at 80% ethanol. The recovery was decreased to 60% according to the increase of ethanol concentrations to 100%. In other words, nearly 40% of the NDELA was eluted without adsorption. Thus, 80% ethanol was selected in the subsequent experiments in order to dissolve the oily components in cosmetic products as much as possible and maintain the sufficient recoveries for practical analysis. There are two reasons for the use of a cation exchange column. One is the removal of metal cations in a sample. Various ionic materials are used in a cosmetic formulation, e.g., anion surface active agents such as fatty acid soap as an emulsifier and alkylsulfuric acid salt as a detergent in shampoo. These anionic surface active agents usually have metal counterions such as sodium or potassium. When the test solution of sodium dodecyl sulfate (LS-Na)
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
Table I. Influence of Nonionic Compounds on NDELA Recovery compd
polyoxyethylene tetradecyl ether (15 mol ethylene oxide) glyceryl 1-monododecanoate dodecanoic acid diethanolamide
Table 11. Recoveries from Cosmetic Products
recovery, % 107
PEG 400 propylene glycol
93 87, 92 111,102 97
glycerol
40
spiked with 1pg of NDELA was passed through only anion exchange column, the recoveries decreased to 90,70, and 60% according to the increase of LS-Na to 0.5, 1.5, and 2 g, respectively. It was confirmed that sodium hydroxide, formed in the process of ion exchange, interfered with the adsorption of NDELA on the resin. Therefore, to prevent the formation of sodium hydroxide, the test solution was passed through a cation exchange column before an anion exchange column. NDELA recoveries were satisfactory (98-102%) under the presence of LS-Na ranging from 0.5 to 2 g. Also, the recoveries from fatty acid soap were complete. Lauryl sulfuric acid adsorbed on the resin was not eluted with an acetic acid/ethanol. A strong acid such as hydrochloric acid was able to elute the acid. This result indicates that the isolation of NDELA from some cosmetic ingredients such as triethanolamine alkylsulfate is easy. Although soap fatty acid was eluted together with NDELA, with acetic acid/ethanol, it was easily removed by solvent extraction using n-hexane and acetonitrile. NDELA was recovered from the acetonitrile layer. However, this was not a necessary step, because such a small amount of fatty acid in a usual cosmetic product did not interfere with the detection of NDELA by HPLC-TEA. Another reason for the application of a cation exchange column is the removal of ethanolamines, especially of diethanolamine which contaminates some cosmetic ingredients such as triethanolamine and its salts and fatty acid diethanolamide. If diethanolamine is introduced into an anion exchange column without removal, it may form NDELA in the following processes: elution with acetic acid and evaporation of solvent. Influence of Nonionic Compounds. The hydroxyl groups in the NDELA molecule take part in the adsorption on an anion exchanger as mentioned above. Therefore, it was possible that cosmetic ingredients having hydroxyl groups in their molecules were adsorbed on the resin as was NDELA and that they interfered with the adsorption of NDELA because of their large quantities. Thus, six representative nonionic compounds used in common cosmetic formulations were selected to examine their influence (Table I). They have one to three hydroxyl groups in their molecules. Each 2 g of these compounds spiked with 1pg (500 ppb) of NDELA still did not decrease the recoveries, with exception of glycerol which was found to decrease the recovery to 40%. As the amount of glycerol was decreased to 0.2 g, the recovery was increased to 80%. By this it was meant that the amount of glycerol introduced into a column must be less than 0.2 g in order to maintain at least 80% recovery which was satisfactory for practical analysis. In other words, more than 80% recovery is expected when a sample containing less than 10% of glycerol is subject to the present method. Recovery from Cosmetic Products and Ingredients. The levels of 100 and 500 ppb of NDELA spiked to triethanolamine and LS-Na were determined by repeating five times. The recoveries were 90-100% with less than 5% relative standard deviation. The recoveries of NDELA spiked to eight cosmetic products (two creams, two lotions, two foundations, and two shampoos)
NDELA recovery,a spiked, ng/g %
product
500 250 50 500
cream (A)
cream (B) lotion (C)
500
250 50 500 500 500 500 500
lotion (D) foundation (E) foundation (F) shampoo (G) shampoo (H) a
90.3 93 81 80.8 83.9 85 82 78.6 89.3 87.7 89.5 95.2
Mean value ( n = 3).
W
m
z
Bm W
U
a W
0
a
8 W
a
0 2 4
6 8 1012
TIME,
MIN
Flgure 2. Chromatograms of (a) nonspiked lotlon (B) and (b) spiked lotion (B) with 500 ng/g NDELA, TEA attenuation X 8 (a) and X 1 6 (b).
at the 500 ppb level were confirmed to be more than 79% (Table II), with an overall average recovery of 87% for these products. In addition, good recoveries were obtained at the 50 and 250 ppb levels. Figure 2 shows the HPLC-TEA chromatograms from (a) nonspiked lotion (B), and (b) spiked lotion (B) with 500 ng/g. As mentioned above, the detection at the 10 ppb level has been achieved for almost all of cosmetic products and ingredients except for ethanolamines. For tri-, di-, and monoethanolamine, the detection limits were 20,40, and 40 ppb, respectively, because of the limit of exchange capacity of the cation exchange resin. These results indicate that the present method is widely applicable to many types of cosmetic products and ingredients to be analyzed for NDELA, and together with good precision, more than 80% recovery is expected. ACKNOWLEDGMENT We wish to thank S. Ohta for technical advices and T. Ozawa and T. Takamatsu for helpful discussions. LITERATURE CITED ( I ) Flne, D. H.; Rounbehler, D. P. J. Chromatogr. 1975, 109, 271-279. (2) Flne, D. H.; Huffman, F.; Rounbehler, D. P.; Belcher, N. M. IARC Scl. PUbl. 1976, 14, 43-50. (3) Fan, T. Y.; Goff, U.; Song, L.; Flne, D. H.; Arsenault, G. P.; Bleman, K. Food. Cosmet. Toxicol. 1977, 15, 423-430. (4) Mltchell, W.; Rahn, P. Drug Cosmet. Ind. 1978, 123, 56-66. (5) Rosenberg, I. E.; Gross, J.; Spears, J.; Caterbone, U. J . SOC. Cosmet. Chem. 1979, 30. 127-135. (6) Felllon, Y.; de Smedt, J.; Brudney, N. “An HPLC Method for the Direct Evaluation of NDELA In Some Cosmetic Products and Raw Materials”;
2003
Anal. Ghem. 1981, 53, 2003-2006
presented at the Slxth Internatlonal Meeting on Analysls and Formatlon of N-Nitroso Compounds (IARC); Budapest, Hungary, 16-19 Oct
(10)
Samuelson, 0. Nord. KemikermDde, 9th Chem. Absb. 1958, 52, 5086b.
1956,
part 2, 105-116;
1979
Ginn,.M. E.; Church, C. L. Anal. Chem. 1950, 31, 551-555. Newburger, S. H. "Manual of Cosmetic Analysls", 2nd ed.; Association of Officlal Analytical Chemists, Inc.: Washington, DC, 1977; pp 18 and 55. (9) Samuelson, 0. "Ion Exchangers In Analytical Chemlstry";Wlltry: New
(7) (8)
York, 1954; pp 93-97.
RECEIVED for review
h'hch 30, 1981. Accepted July 20,1981. Portions of this paper were presented at the 179th National Meeting Of the American Society, TX, March 23-28,1980, ANAL 79.
Negative Ion Chemical Ioni,zation Mass Spectrometry of Aflatoxins and Related Mycotoxins William C. Brumley," Stanley Nesheim, Mary W,, Trucksess, Eric W. Trucksess,' Peter A. Drelfuss, John A. G. Roach, Denis Andrzejewski, Robert IM. Eppley, Albert E. Pohland, Charles W. Thorpe, and James A. Sphon Division of Chemistry and Physics, Food and Drug Administration, Washington, D.C. 20204
Negatlve Ion chemlcal lonlratlon mass spectra of afliatoxlns and other mycotoxlns were obtained under resonance electron capture conditions using methane. The negattve Ion spectra of aflatoxins consisted of three major Ions: #-e, (M He)-, and (M CHB*)-. Other mycotoxins related to aflatoxlns also exhlblted relatively simple spectra. The ttsmperature dependence of the spectra is presented In detall for aflatoxin B1. The negatlve Ion spectra of aflatoxlns were used to demonstrate the feasibility of confirming the presence of aflatoxin B1In peanut butter, corn, and other commodMes and aflatoxln M1In mllk after approprlate extractlon-cleanup. The aflatoxlns were confirmed at levels as low as 10.5 ppb in food matrices.
-
-
The aflatoxins are a group of related compounds that are included under the general term mycotoxins. They have been shown to be potent carcinogens in certain laboratory animals (1). Aflatoxins are produced by the fungi Aspergillus flaws and A. parasiticus and are found in grains, peanuts, and tree nuts throughout the world where climatic conditions are favorable (2). Methods for the determination of aflatoxins in fo0d.s have utilized fluorescence detection upon exposure to ultraviolet light. Most often, thin-layer chromatography (TLC) has been used for the determinative step but more recently highpressure liquid chromatography (HPLC) has also been used (3). Procedures for the identification of the aflatoxins have employed multiple TLC of the sample extract with dijFferent solvent systems, spray reagents to alter fluorescence on the TLC plate, derivatization and additional chromatography by TLC or HPLC, or demonstration of typical biological activity using the fertile chicken egg as the test organism (3). Mass spectrometry (MS), especially electron impact (EI) MS, has also been a useful tool in the analysis of samples for mycotoxins (4). A more systematic approach to the potential application of MS to mycotoxin structure determination has been reported (5), and a computer-based compilation of E1 MS data of mycotoxins has been published (6). E1 ionization has been the most frequent MS technique applied to the structural characterization of mycotoxins and to the confirmation of identity of suspected compounds asNational Science Foundation-American University Summer Research Participation Program, 1980. Thls
article not subject to US. Copyright.
sayed by relatively nonspecific techniques such as gas chromatography (GC), TLC, and HPLC. Field desorption MS has been used to obtain the molecular weights of certain mycotoxins and to screen extracts for mycotoxins (7). Haddon and co-workers have used high-resolution selected ion monitoring E1 MS for the confirmation of aflatoxins (8). In spite of the successes of E1 MS, no one MS technique can be expected to be optimal for all of these compounds. Optimization may include providing an unambiguous molecular weight, complementary structural information, or lower limits of detection. The versatility of chemical ionization (CI) MS (9-12) suggested that it could be of use in complementing E1 MS in the confirmation of mycotoxins. In this paper we discuss the negative ion (NI) CI mass spectra of aflatoxins and related mycotoxins under resonance electron capture (EC) conditions using methane. Then we give applications indicative of the potential of NI CI MS for the confirmation of aflatoxins in certain foods. EXPERIMENTAL SECTION Instrumentation. A modified Finnigan 3300F CI mass spectrometer operated in the negative ion mode (13) was used. Samples were introduced with a direct insertion probe heated independently of the source. Methane was 99.97% pure (Matheson Gas Products, East Rutherford, NJ). The reagent gas was ionized by a 140 eV beam of electrons generated from a heated rhenium filament. Source temperatures were in the range of 50-250 "C and are given when data are reported. Source pressures measured about 1.0 torr CHI on the standard source pressure gauge (about 0.22 torr using a McLeod gauge). Both the source pressures and temperatures measured by the standard Finnigan configurations are subject to error as observed by Bruins (14). Source temperatures are reported uncorrected. Extraction and Two-DimensionalTLC Cleanup. Precoated silica gel 60 TLC plates, 20 X 20 cm, 0.25 mm thick (E. Merck 5763) were used. A Camag Eluchrome apparatus was used for elution. Acetone, chloroform, methanol, diethyl ether, and 2propanol were ACS reagent grade distilled in glass. Aflatoxin M2 and aflatoxicol were obtained from Makor Chemicals Ltd., Jerusalem, Israel, and were used as obtained. Other mycotoxins were reference standarch purchased from commercial sources. The purity of the standards was determined by TLC. Ginger root ( E ) milk , (16),and peanut butter (3) were e x t r a d following published procedures. For ginger, the mobile phases for the fnst- and second-dimensional TLC developments to isolate B1 were ether-methanol-water (9541) and chloroform-acetone (91), respectively. The mobile phases for milk to isolate M1were ether-methanol-water (95:4:1) and chloroform-acetone-2propanol (85:105) and for peanut butter, melon seeds, and corn
Publlshed 1981 by the American Chemical Society
