610
Anal. Chem. 1980. 52, 610-614
Simultaneous Determination of Vitamins A, D2 or D, E, and K, in Infant Formulas and Dairy Products by Reversed-Phase Liquid Chromatography S. A. Barnett," L. W. Frick, and H. M. Baine Mead
Johnson and Company, 2404 Pennsylvania Avenue, Evansville, Indiana 4772 1
A reversed-phase high performance liquid chromatographic method for the simultaneous determination of vitamins A, D, or D,, E, and K, in milk and soy-based infant formulas and in dalry products has been developed. The method requires enzymatic hydrolysis of the lipid component of the sample, extraction with n-pentane, and addition of cholesterol phenylacetate internal standard. Separation is achieved using a methanokethyl acetate (86:14):acetonitrile gradient elution on two Zorbax ODS 25-cm columns connected in series. Detection of the compounds of interest is accomplished using a microprocessor controlled variable wavelength UV detector with vitamin A forms detected at 325 and 365 nm. Vitamins D2, D,, E forms, K,, and the internal standard are detected at 265 nm. Chromatographic separation requires approximately 50 mln with typical relative standard deviations of 4.6, 4.8, 2.3, 2.9, and 3.8% for vitamins A, D,, D,, E, and K,, respectively.
Compendia1 methodologies currently available for the determination of vitamins A, D, E, and K, in milk and soy-based infant formulas, and dairy products lack specificity, display poor internal precision, are time consuming, and are not amenable t o simultaneous determination. Vitamin A is currently analyzed by the Cur-Price colorimetric method ( I ) . This procedure relies on the formation of a transient blue color complex produced by the interaction of vitamin A with antimony trichloride. T h e method does not correct for inactive isomeric forms of vitamin A which also react with the reagent resulting in analytical bias. Similarly, a compendial vitamin E determination, using the Emmerie-Engel reaction, is based on the reduction of ferric ion to ferrous ion by a-tocopherol ( 2 ) . T h e ferrous ion is reacted with a,a'-dipyridyl resulting in the formation of a red-colored complex. Other reducing substances will also react with this reagent resulting in analytical bias. Recent improvements in the determination of vitamin E include application of thin-layer and gas chromatographic techniques. These methods, while capable of increased specificity, are also time consuming and subject to inaccuracies associated with oxidative loss. T h e compendial vitamin D bioassay is performed by placing weanling rats on a rachitogenic diet for an 18-25 day depletion period (1). After rickets has developed, a seven-day assay follows during which some of the rats receive a reference standard dosage of vitamin D, while others receive vitamin D from the test product. The animals are then sacrificed, and a leg bone is dissected and examined for calcium deposition. T h e bone sections are placed in a silver nitrate solution and exposed to light. The resulting photochemical reaction causes a black deposit of silver on the calcified areas of the rat bone. Evaluation of the extent of calcification is made by comparing t h e healing of the reference group t o the group fed the test product. 0003-2700/80/0352-06 10$01 O O / O
The biological determination of vitamin K1 is achieved by using young chicks which have been maintained on a vitamin K1 depleted diet ( 3 , 4 ) . After vitamin K1 depletion, the hemorrhagic chicks are fed either a reference standard or a test product. They are then sacrificed, whole blood prothrombin time or blood clotting time is measured, plotted, and the potency of the product tested is estimated. Alternately, vitamin K1 can be determined by separation of the lipid fraction of the sample by open column adsorption chromatography with detection and quantitation by thin-layer reflectance densitometry ( 5 ) . T h e literature contains many references to liquid chromatographic separations of the fat soluble vitamins as standards, raw materials, premixes, concentrates, pharmaceutical preparations, and from milk products (6-25). However, search of the literature has revealed no methodology for the simultaneous determination of vitamin A forms, D2 or D,, E forms, and K1 from infant formula and dairy products. This paper outlines a procedure for the simultaneous determination of the fat-soluble vitamins from a single sample preparation using nonaqueous reversed-phase liquid chromatography with internal standard techniques.
EXPERIMENTAL Reagents and Solvents. Acetonitrile, ethyl acetate, methanol, and n-pentane HPLC grade reagents were obtained from Fisher Scientific Company, Pittsburgh, Pa., and Burdick and Jackson Company, Muskegon, Mich. Retinol, retinol palmitate, d,l-atocopherol, d,l-a-tocopherolacetate, vitamin K,, cholecalciferol, lipase, and cholesterol phenyl acetate were obtained from Sigma Chemical Company, St. Louis, Mo. Ergocalciferol was obtained from the United States Pharmacopeia, Rockville, Md. A mixed vitamin standard solution was prepared containing approximately 0.2 mg/mL vitamin E alcohol, 0.8 mg/mL vitamin E acetate, 20 pg/mL vitamin A alcohol, 20 pg/mL vitamin A palmitate, 10 pg/mL vitamin K1, 4 mg/mL cholesterol phenylacetate, and 0.75 pg/mL vitamin D2 or vitamin D, in a solution of 50:50 acetonitri1e:ethyl acetate. Instrumentation. The liquid chromatograph used was a Hewlett-Packard dual pump microprocessor controlled Model 1084B equipped with a variable wavelength detector. Two Zorbax ODS (Dupont), 4.6 mm x 25 cm analytical columns were used in series. Separations were flow, solvent, and wavelength programmed with an operating back pressure of approximately 11 MPa. All separations were performed a t ambient temperatures. Sample Preparation. An accurately measured sample containing approximately 3.5-4.0 g of fat and 12.5210 units of vitamin A, 2 5 4 0 units of vitamin D2or D,, 0.85 20 mg of vitamin E and/or 3.8-18.5 pg of vitamin K1 is transferred to a I-L round-bottom flask containing 2.5 g of lipase and 200 mL of pH 7.7 buffer. The sample is vigorously stirred at 37 f 2 "C for 1 h. Immediately after incubation, 10 mL of 10 N sodium hydroxide solution and 200 mL of 95% ethanol are added to the sample hydrolysate. The sample is immediately extracted with three 200-mL portions of n-pentane; the extracts are collected and water-washed until the aqueous phase is neutral to phenolphthalein. In subdued light, the combined extracts are filtered through anhydrous sodium sulfate and evaporated to dryness under reduced pressure with 1980 American Chemical Society
50
40
-
-
r
---- - - - - - -1
I
____ -
I .I
SOLVENT PROGRAM FLOW P R O G R A M . . . . . . . . . .W . AVELENGTH
I
'
I
i
..........................
I. . . . . . . . . . . . . . . . . ....I.. . . . . . . . . . . . . . . ..: I I 15 _ _ _ _ _ _ _ _ _ - - - - J
400nm
FLOW (mllminl
1 3
I ...... 2.0
300nm
1.0
200nm
I
I I L
'ill
I
3.0
kI
20
10
-
I
.................................
-
- 4.0
I
100-%B
30
-
__
,
L.-20 30
40
50
60
4.0
3.0
400nm
FLOW /mllmin)
2.0 300 n m
1.0 200nm
10
20
30
40 TIME I m n !
50
60
Figure 2. Profile of solvent system, flow, and wavelength progress used with two Zorbax ODs columns connected in series. Insert illustrates elution of peaks of interest relative to profile. (1) Vitamin A alcohol, (2) vitamin D, (3) vitamin E alcohol, (4)vitamin E acetate, (5)vitamin K,, (6) cholesterol phenylacetate, (7) vitamin A palmitate
the aid of a warm (NMT 40 "C) water bath. The sample is cooled to rmm temperature under vacuum and transferred quantitatively to a 2.0-mL volumetric flask with ethyl ether. The ethyl ether is evaporated to dryness under a stream of nitrogen. After the final ether transfer has been evaporated to dryness, 1 mL of cholesterol phenylacetate internal standard solution dissolved in equal parts of ethyl acetate and acetonitrile is added to the volumetric flask. Final volume is obtained using a diluent composed of equal parts of acetonitrile and ethyl acetate. Chromatography. The diluted sample, 75 /IL, is injected on two 25 cm X 4.6 mm Zorbax-ODS columns connected in series. Figures 1 and 2 depict the microprocessor programs controlling separation of the compounds of interest from samples containing either D2or DB. Both separation systems require a gradient elution using a binary system of 86% methanol149i ethyl acetate against 100% acetonitrile. Solvent flow rates are changed during the chromatographicrun to optimize separations. Wavelength changes are required to selectively detect compounds of interest. All chromatograms are developed at ambient temperatures and analytes are separated in approximately 50 min.
RESULTS AND DISCUSSION An analytical problem as formidable as the simultaneous separation and quantitation of vitamins A, D, or D,, E, and K1 from milk and soy-based infant formulas and dairy
products requires as a first approach, careful consideration of the chemical interdependencies between the sample matrix and the compounds of interest. Development of a suitable analytical method requires an understanding of the composition of the starting material, particularly the different vitamin forms present and their approximate concentrations. Application of classical methodology for the determination of fat-soluble vitamins generally requires alkaline hydrolysis of the sample. Initial work in our laboratory identified the necessity for the development of a chromatographic system with sufficient resolution and flexibility to quantitate the fat-soluble vitamins from organic extracts of nonsaponifiable sample fractions. The nonpolar nat,ure of this extract suggested the use of some form of reversed-phase chromatography for quantitative separation. Several binary and ternary solvent systems containing combinations of water, methanol, tetrahydrofuran, and acetonitrile were examined for separation suitability on both single and coupled phenyl and phenyl-CI8 columns. All variations of these systems failed to provide the degree of separation required within practical analytical time. In addition, sample solubility in the mobile phase or sample solubility in a solvent compatible with the mobile phase was unsuitable. The nature of the sample also shortened column
612
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980 m 41
I
l 0
k
l
131
I
Figure 3. Chromatograms of mixed vitamins and internal standard solutions containing: ( 1 ) vitamin A alcohol, 20 pg/mL; (2a) vitamin D, 0.75 pg/mL; (2b) vitamin D, 0.75 pglmL; (3)vitamin E alcohol, 0.2 mg/mL; (4) vitamin E acetate, 0.8 mg/mL; (5) vitamin K,, 10 pg/mL; (6) cholesterol phenylacetate, 4.0 mg/mL; (7) vitamin A palmitate, 20 pg/mL
life by irreversible retention of nonpolar species on the column surface resulting in rapidly increasing operating back pressure and occasional peak ghosting. Nonaqueous reversed-phase (NARP) liquid chromatography was examined for application as an alternative reversed-phase mode of separation. NARP is simply an extension of the reversed-phase separation mechanism utilizing a stationary phase of high carbon load and requiring a more polar than normal solvent eluting system. Advantages offered by NARP include increased solubility of low polarity samples in the mobile phase and extended column life (16). Zorbax ODS (Dupont) analytical columns were selected for use owing to controlled particle size distribution, high carbon loads, and flexibility in terms of applicable mobile phases. In order to establish optimum nonaqueous chromatographic conditions, a sample of infant formula was hydrolyzed with potassium hydroxide and prepared as per a modified compendial procedure for the determination of vitamin E. The sample was chromatographed on a single Zorbax ODS column using various mixtures of methanol, acetonitrile, ethyl acetate, and tetrahydrofuran in binary and ternary combinations. Chromatographic systems containing tetrahydrofuran were found to provide acceptable separations but could not be reproduced. Further work revealed that even trace amounts of tetrahydrofuran on Zorbax ODS columns modified the kinetics of the separation mechanism sufficiently to cause a permanent change in column efficiency. Substitution of ethyl acetate for tetrahydrofuran resulted in a reproducible system when used in combination with methanol and acetonitrile; however complete resolution of the compounds of interest from associated interference could not be achieved. Earlier work in our laboratory indicated that improved separation of complex samples could be obtained by utilizing the flexibility offered by simultaneous flow and solvent programming (17). I t is important to note that the objective of this chromatographic system was to optimize the separation of seven compounds of analytical interest simulaneously. The separation of very similar molecular species can often be accomplished by simply optimizing sample on-column residence time with mobile phase polarity and velocity. In addition, coupling of appropriate chromatographic columns offers the researcher a simple, rapid scheme from which significant improvements in band resolution can be ultimately achieved. Nonaqueous conditions used with the single Zorbax column were modified with reference t o the ratio of mobile phase for application to Zorbax columns connected in series. Mobile phase flow rates were also adjusted during the course of the separation. Selection of a suitable internal standard for use with this separation system remained an obstacle to the completion of the methodology. Criteria for the suitability of a n internal standard included: (1) elution near the end of the chroma-
togram, (2) stability in the solvent system, (3) similarity in molecular structure to compounds of interest. Retention characteristics of cholesterol phenylacetate met these criteria. Figure 3 is an example of the separation of the mixed vitamin standards from the internal standard using Zorbax ODS columns connected in series and the microprocessor programs depicted in Figures 1 and 2. A sample of soy-based infant formulas containing vitamin D2 was hydrolyzed with potassium hydroxide, extracted as per the AOAC compendial methodology and chromatographed on Zorbax columns in series as discussed above. Figure 4 is a typical chromatogram resulting from this combination of sample preparation and chromatography. Information obtained from this procedure indicated the method would provide adequate quantitation of vitamin A only. a-TOCOpherol is so extremely sensitive to degradation that stability of the sample preparations could not be maintained. Vitamins D, or D, are subject to a variety of isomerization reactions under conditions of alkaline hydrolysis and vitamin K1 is destroyed. Chromatographic separation is sufficiently flexible to separate the compounds of interest but rigorous conditions of sample preparation (alkaline hydrolysis) obviate simultaneous vitamin determination. T o circumvent problems associated with alkaline hydrolysis, alternate approaches to lipid removal from the sample matrices were examined. Our attention was turned to mechanisms involved with lipid utilization in biological systems. The remarkable efficiency, specificity, and speed of enzymatically catalyzed biological reactions were investigated for application to our problems of sample preparation (18). Enzymes are exceptional biocatalysts demonstrating controllable specificity while operating at p H values near neutrality and a t ambient temperatures. Unlike vigorous alkaline saponification, enzymatic hydrolysis is a more manageable reaction; consequently, the vitamins are less subject to degradation. Several lipid hydrolases were evaluated a t a variety of pH levels and buffer compositions in an effort to enzymatically control the hydrolysis of the lipid fraction of the sample. Characteristics of the yeast organism Candida cyclindraccae were found acceptable for sample hydrolysis. The enzyme effectively catalyses the degradation of lipid triglycerides into long chain fatty acids and glycerol (19). By adjusting the p H of the resulting hydrolysate, fatty acids can be precipitated from the solution as metal salts. Solvent extraction of the remaining reaction mixture followed by adequate water-wash yields an organic phase containing primarily the fat-soluble vitamins, trienols, and other sterols. A mixed standard containing vitamins A palmitate, D2,E acetate, and K1 was prepared in an oil mixture approximating the concentration of the vitamins in a lipid fraction of infant formula product. After enzymatic hydrolysis, vitamins A palmitate and E acetate were partially converted to their
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
613
Figure 4. Chromatogram of a soy base infant formula product after alkaline hydrolysis containing: (1) vitamin A alcohol, (2) vitamin D2, (3)vitamin E alcohol, (4) vitamin E acetate, (5) vitamin K, (degraded),(6) vitamin A palmitate
Y)
'0 121
Flgure 5. Chromatograms of soy base infant formula products after enzymatic hydrolysis containing vitamins D, or D,. (1) Vitamin A alcohol, (2)&tocopherol,(3) y-tocopherol, (4a)vitamin D, (4b) vitamin D, (5)vitamin E alcohol, (6)vitamin E acetate, (7) vitamin K,, (8)cholesterol phenyhcetate, (9) vitamin A palmitate
alcohol forms while vitamins D2 and K1 were unaffected. A sample of the soy-based infant formula product containing vitamin D2 previously analyzed (see Figure 4) as well as a D3 containing soy-formula were analyzed using the combination of enzymatic hydrolysis and NARP liquid chromatography. Figure 5 provides an example of the resulting chromatograms. Scrutiny of these chromatograms indicates that enzymatic hydrolysis as a means of sample cleanup, permits simultaneous quantitative separation of vitamins A alcohol, Dz or D,, E alcohol, E acetate, Kl and A palmitate. Relative standard deviations of 0.0593, 0.0278, 0.0425 and 0.0404 for vitamins A, D2 or D,, E, and K1, respectively, are typical for milk or soy-based infant formulas. Recovery of vitamin spikes in these samples was greater than 93%. The combination of enzymatic hydrolysis and NARP liquid chromatography previously discussed has been applied to a variety of samples. These include aged infant formula, unprocessed milks, ice cream, evaporated milks, margarines, human milk, laboratory rodent chows, cattle supplement, and a variety of liquid and powdered nutritional specialty products. Experience indicates that some samples may be hydrolyzed as is while others require extraction of the lipid material prior to hydrolysis. Studies are underway to further understand and utilize the hydrolysis mechanism. T o date, application
of this method has resulted in the identification of gamma and delta tocopherol both in finished products (Figure 5 ) and in refined oils. Unavailability of isomeric forms of vitamins A and E is obstructing analytical work designed t o further characterize unidentified peaks on the chromatograms. In the absence of authentic materials, identification of unknown compounds will proceed using a variety of analytical techniques including mass spectrometry. The successful simultaneous determination of vitamins A, Dz or DS, E, and K1 from natural products requires a sample pretreatment designed to protect the compounds of interest during liberation from the sample matrix. Alkaline hydrolysis, while effective in matrix removal, is a n unnecessarily harsh treatment with regard to vitamin stability. Data supporting our work suggests that the combination of enzymatic hydrolysis and nonaqueous reversed phase liquid chromatography provides both efficient sample preparation and simultaneous quantitation of the fat soluble vitamins.
LITERATURE CITED (1) "Official Methods of Analysis". 12th ed.;Association of Official Analytical
Chemists: Washington, D.C., 1975;Chapter 43 (2) "National Formulary", 13th e d ;American Pharmaceutical Association: Washington, D.C., 1975;p 759. (3) DeLuca, Hector F. "Handbook of Lipid Research - T h e Fat-soluble Vitamins", Vol. 2; Plenum Press: New York, 1978;Chapter 4.
614
Anal. Chem. 1980, 52, 614-617
(4) Sebrell, W. H., Jr.; Harris, Robert S. "The Vitamins", Vol. 2; Academic Press: New York. 1954: Chapter 9. (5) Manes, J. D.; Fluckiger, H. B.; Schneider, D. L. J . Agric. Food Chem. 1972, 20, 1130-1132. (6) Williams, R. C.; Schmit, J. A.; Henry, R. A. J . Chromafogr. Sci. 1972, 10, 494-501. (7) Tomkins, D. F.; Tscherne, R. J. Anal. Chem. 1974, 4 6 , 1602-1604. (8) Ray, A. C.; Dwyer, J. N.; Reagor, J. C. J . Assoc. Off. Anal. Chem. 1977, 60, 1296-1301. (9) Hofsass, H.; Grant, A.; Alicino, N. J.; Greenbaum, S. 8. J . Assoc. Off. Anal. Chem. 1978, 59, 251-260. (10) Dolan, J. W.; Grant, J. R.; Tanaka, N.; Giese, R. W.; Karger. B. L. J . Chromatoar. Sci. 1970. 16. 616-622. (11) Thompson: J. N.; Maxwell, W.B.: L;Abb6. M. J . Assoc. Off. Anal. Chem. 1977, 60,998-1009. (12) Thompson, J. N.; Hatina, G. J . Llq. Chromatogr. 1979, 2 , 327-344.
(13) Thompson, J. N.; Maxwell, W. J . Assoc. Off. Anal. Chem. 1977, 60, 766-77 1. (14) Egberg. D. C.; Heroff, J. C.; Potter, R. H. J . Agric. FoodChem. 1977, 25, 1127-1 132. (15) Pickston, L. N . 2. J . Sci. 1978, 21, 338-385. (16) "L. C. Column Report Nonaqueous Reversed Phase Chromatography", Dupont Instruments: Wilmington, Dei., Dec. 1977. (17) Barnett, Stephen A.; Frick, Leroy W. Anal. Chem. 1979, 51, 641-645. (18) Mahler, Henry R.; Cordes, Eugene H. "Basic Biological Chemistry"; Harper and Row: New York, 1968; Chapter 7. (19) "Worthington Enzymes and Related Biochemicals"; Worthington Biochemical Corporation: Freehold, N.J., 1978; p 125.
RECEIVED for review September 4, 1979. Accepted January 7 , 1980.
Determination of Trace Levels of Iron(I1I) by Homogeneous Catalysis and Gas Chromatography Mauri A. Ditzler Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 0 16 10
W. F. Gutknecht" Systems and Measurements Division, Research Triangle Institute, Research Triangle Park, North Carolina 27709
A procedure Is described for the determination of trace levels of Fe3+. The procedure Is based on the gas chromatographic measurement of o-hydroxyanisole, which is a product of the Fe3+-catalyred reaction between anisole and H,O,. Under controlled conditions, the amount of o-hydroxyanisole measured is proportional to the concentration of Fe3+ present in the reaction mixture. The procedure is found to have a detection limit of 0.25 ppb and is shown to be linear up to 1000 ppb. A chemical amplification factor of 125 Is realized with a Tornin reaction time. 01 several metal ions tested, oniy Cu2+ was found to slgnificantly interfere. The procedure has been successfully applied to several real-world samples.
Despite many advances in the area of trace analysis, there exists a continuing need for new analytical techniques that are more selective, accurate, and precise than techniques developed previously. It is desirable, too, that such techniques be relatively simple and inexpensive so that they can be used by a majority of the laboratories involved in trace analysis work. These goals have been met, in part, with a new trace analysis technique using a combination of homogeneous catalysis and gas chromatography. The technique involves first carrying out a chemical reaction catalyzed by the substance of interest and then measuring a product of that reaction using gas chromatography. Under controlled conditions, the amount of product measured will be proportional to the concentration of t h e catalyst. T h e technique was first introduced through a new procedure for the determination of Cu+ involving the Cu+-catalyzed reaction of p-tolyldiazonium and chloride ions to form p-chlorotoluene, with subsequent gas chromatographic measurement of this product ( I ) . It is true that gas chromatographic methods have been used previously for the direct determination of trace metals. These procedures are based on the formation and gas chromato0003-2700/80/0352-0614$01 .OO/O
graphic analysis of volatile, metal ion-ligand complexes (2-5). Indeed, Fe3+ levels in several real-world samples have been determined by gas chromatographic analysis of neutral Fe3+ complexes, though analysis has been limited to samples containing microgram levels of iron (6-8). These procedures have not been extensively applied to routine analyses, however, because of problems associated with the low stability of many of the complexes. Such problems are avoided in the new indirect Fe3+ analysis procedure described herewith. The basis of the new procedure for determination of Fe3+ is the gas chromatographic measurement of o-hydroxyanisole, where this species is the product of an Fe3+-catalyzed reaction between H 2 0 2and anisole in weakly acidic solutions. Hydroquinone must also be present for the Fe3+to be catalytically active. Equation 1 summarizes this reaction. OCH3
2
(9
fH202
-
Hydroquinone
(1)
This reaction was selected for use in the measurement of Fe3+ partially on the basis of studies by Hamilton et al. ( 9 , I O ) . The authors found that the role of Fe3+was that of a catalyst, with over 20 molecules of product produced per Fe3+ ion present. Furthermore, Fe3+ was the only ion found to substantially accelerate the reaction. Another consideration in the choice of this reaction was that the product, o-hydroxyanisole, is amenable to isolation and detection by gas chromatography.
EXPERIMENTAL Equipment. All gas chromatographic studies were carried out using a Perkin-Elmer Sigma 4 gas chromatograph equipped with
a flame ionization detector. This instrument was equipped with a glass-lined injection port. A 6 f t X in stainless steel column packed with 18% DC-550 on 60/80 mesh Chromosorb W HP was
C 1980 American Chemical Society
