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Liquid Chromatographic Analysis of Vitamin K1in Medical Foods Using Matrix Solid-Phase Dispersion
BALA
Jayashree
GSRS
JFCA*20000935
DATE ......................... 11*7*2000
DISC USED YES ! (2000) 13, 765}771 doi:10.1006/jfca.2000.0935 Available online at http://www.idealibrary.com on
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
ORIGINAL ARTICLE Liquid Chromatographic Analysis of Vitamin K1 in Medical Foods Using Matrix Solid-Phase Dispersion G. William Chase Jr.* , R. R. Eitenmiller-, and A. R. Long* *U.S. Food and Drug Administration, Northwest Regional Laboratory, 22201 23rd Drive, S.E., Bothell, WA 98021-4421, U.S.A.; and -Department of Food Science and Technology, University of Georgia, Athens, GA 30602, U.S.A. Received June 3, 1999, and in revised form January 26, 2000
A liquid chromatographic method for vitamin K in medical foods is described. The vitamins are extracted from medical food by matrix solid-phase dispersion (MSPD) and quantitated by reversed-phase chromatography with #uorescence detection. Vitamin K is converted to the #uorescent hydroquinone with a post-column zinc reductive reactor. The limit of detection is 6.6 pg and the limit of quantitation is 22 pg on column. Linear response ranged from 45 to 908 pg on column (r"0.998). Recoveries were determined on an analyte-forti"ed zero control reference material (ZRM) for medical foods and averaged 97.9% (n"25) for vitamin K . The method provides a rapid, speci"c and easily controlled assay for the analysis of vitamin K in forti"ed medical foods. 2000 Academic Press Keywords: vitamin K; MSPD; liquid chromatography.
INTRODUCTION Recently, Chase et al. (1999a, b) developed methods to quantitate vitamin K in forti"ed milk and soy-based infant formula. The extraction protocol utilized matrix solid-phase dispersion (MSPD) followed by a concentration step. The extract was injected directly onto a C8 LC column connected in series with a zinc post-column reduction column. The zinc reduction column induced formation of the #uorescent hydroquinone for the quanti"cation of total vitamin K by #uorescence detection. This technique and others currently used for vitamin K analysis resulted from the e!orts of the people at the United States Department of Agriculture Vitamin K Laboratory at Tufts University (Ferland and Sadowski, 1992a, b; Ferland et al., 1992; Booth et al., 1993, 1994, 1995, 1996a}c, 1997; Davidson et al., 1996; Booth and Sadowski, 1997). The MSPD technique has been patented and used extensively for isolating drugs from milk and tissue (Barker and Long, United States Patent). Methods reported by Chase et al. (1999a, b) provide speed, low solvent requirements and improved repeatability when compared to the O$cial AOAC International method for vitamin K in milk-based infant formula (Method 992.27, 50.106) (1995). The AOAC International, O.cial Methods of Analysis, 16th edn., does not To whom correspondence and reprint requests should be addressed. Tel.: 425 483 4990. Fax: 425 483 4996. E-mail: [email protected] 0889}1575/00/050765#07 $35.00/0
2000 Academic Press
766
CHASE JR, EITENMILLER AND LONG
provide a method for the analysis of vitamin K in medical foods. Method 992.27 applies to milk-based infant formula products. Due to sample matrix di!erences, the milk-based infant formula method for vitamin K is not collaborated and often not applicable for medical food assays. Problems with the AOAC International method include hard to interpret chromatograms due to lipid interference, inability to measure cis-vitamin K in matrices containing corn oil, and an overall high RSD of 20.9% (Eitenmiller and Landen, 1999). Since infant formula and medical food formulations are diverse and the resulting matrices are dissimilar, issues of solvent polarity, miscibility, partitioning characteristics, density characteristics and other physiochemical parameters become critically important in developing a sound validated method. The purpose of this study, therefore, was to apply the MSPD extraction technique for vitamin K from the earlier work with infant formula to medical foods (Chase et al., 1999a, b). METHOD Apparatus (a) Liquid Chromatograph * LDC Analytical Constametric 4100 pump (Thermo Separation Products, Riviera Beach, FL) and Waters 715 autoinjector (Waters, Inc., Milford, MA). (b) Column * Supelco C18, 5 lm, 4.6;250 mm, part number 58 298 (Supelco, Bellefonte, PA). (c) Integrator * Waters Millenium Data System, Software Revision 2.15 (Waters, Inc.). A stand-alone integrator can also be utilized. (d) Fluorescence detector * Model 1046A programmable #uorescence detector (Hewlett-Packard, Avondale, PA) or equivalent. (e) Reservoirs with frits * Varian 15 mL size, part number 1213}1016 (Varian, Harbor City, CA). (f ) Turboevaporator * Turbo Vap II (Zymark, Hopkinton, MA) or a suitable technique to evaporate the extracts. (g) Vortex mixer * Maxi-Mix I, (Thermolyne, Dubuque, IA). (h) Reduction reactor * 2.5 cm;3.2 mm stainless steel, packed with zinc powder, 100 mesh (Aldrich Chemical Co., Milwaukee, WI). (i) Ultrasonic cleaner * 5.2 gallon ultrasonic cleaner (Thomas Scienti"c, Swedesboro, NJ). Reagents (a) (b) (c) (d) (e) (f ) (g) (h)
Hexane * LC grade (Burdick and Jackson, Muskegon, MI). Isopropyl alcohol * LC grade (EM Science, Gibbstown, NJ). Ethyl acetate * LC grade (Burdick and Jackson). Methylene chloride * LC grade (Burdick and Jackson). Methyl alcohol * LC grade (Burdick and Jackson). Acetic acid * glacial, reagent grade (GR) (EM Science). Zinc chloride * ZnCl , catalog Z-33 (Fisher Scienti"c, Fairlawn, NJ). Sodium acetate * anhydrous, catalog S-8750 (Sigma Chemical Co., St. Louis, MO). (i) Reductive ionic solution * 2.0 M zinc chloride, 1.0 M sodium acetate and 1.0 M acetic acid per liter of methanol. Prepare this solution by weighing 68.1 g of zinc chloride, 20.5 g of sodium acetate into a 250 mL volumetric #ask and adding about
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
767
200 mL of methanol and 15 mL of acetic acid. Dissolve by stirring and then dilute it with methanol to the required volume. ( j) Diluting solution * one liter of methanol containing 5.0 mL of the reductive ionic solution. (k) Mobile phase * the diluting solution containing 10% hexane. Filter the mobile phase through a 0.45 lm "lter prior to use. (l) Vitamin K standard * Accurately weigh c. 25 mg of vitamin K (USP reference standard Phytonadione Lot L) into 50.0 mL volumetric #ask and dilute it to volume with hexane. Determine the exact concentration from E% "419 at 248 nm. Appropriate dilutions were made with hexane so that when the "ve "nal working standards are injected the concentration on column ranged from 45 to 900 pg. Each working standard should contain 10% hexane with the remaining volume being the diluting solution. (m) Bondesil * C preparative grade, part number 1221}3013 (Varian). (n) Isopropyl palmitate * catalogC 29,178-1, tech 90% (Aldrich Chemical Co., Milwaukee, WI). (o) Spiking solutions * The appropriate dilutions were made from the vitamin K stock standard in hexane so that an aliquot not exceeding 100 lL would contain x, 5x, 10x, 20x and 40x levels where x corresponds to 16 lg/100 g of dry powder. Chromatographic Conditions (a) Instrument parameters * Injection volume, 30 lL; #ow rate 1.0 mL/min. Fluorescence detector parameters: excitation wavelength (exj)"248, emission wavelength (emj)"418, gain"9. The zinc reduction column is placed after the analytical column. In addition, a scrubber column was installed after the pump but before the injector. This was comprised of a 15 cm stainless-steel column "lled with zinc metal (catalog no. Z15, Fisher Scienti"c) to aid in reducing background noise. (b) LC con"guration * First, inject the working standard to establish linearity (r"0.998) allowing for a 11 min run time per injection. Upon completion of the standard injections, inject the samples while interspersing with alternate standard injections. Sample Description and Preparation A ZRM originally used for other fat-soluble vitamin method development studies was utilized for vitamin K (Chase et al., 1998). Five pounds of a powdered medical food were obtained from a domestic manufacturer that was formulated without the addition of fat- and water-soluble vitamins. The formulation is milk based with medium-chain triglycerides and mimics commercial medical food matrices. Analytical examination documented that the matrix was devoid of vitamin K . The medical food, therefore, provides a ZRM for this analyte. For analysis, 1 lb was mixed, divided into 30 g airtight containers, stored under nitrogen at 03C until needed. Then, approximately 10 g of the powder was taken from one of the airtight containers and thoroughly mixed with 50 g of hot water (803C). Two commercial medical foods were locally purchased that contained milk, soy and whey protein as well as various oils. Twelve cans of each (12 #. oz.) were combined to provide composite samples. Sampling and preparation were completed as described in AOAC 50.1.01, section A and B (AOAC, 1995).
768
CHASE JR, EITENMILLER AND LONG
Sample Extraction Weigh 2 g Bondesil C into a mortar. Add 100 lL isopropyl palmitate and gently blend the isopropyl palmitate onto the C with a pestle. Accurately weigh 0.50 g reconstituted sample into the C -isopropyl palmitate mixture, followed by the addition of the spike solution and two drops of the reductive ion solution. Use the pestle to gently blend the reconstituted sample and the C -isopropyl palmitate into a #u!y, slightly sticky powder. Accurately transfer the C -matrix blend into a 15 mL reservoir tube with a frit at the bottom and then insert the top frit on the powdery mix. Tightly compress the reservoir contents with a 10 cm syringe plunger. Pass 9 mL 0.5% isopropyl alcohol in hexane (v/v), followed by 9 mL of ethyl acetate, followed by 9 mL of methylene chloride through the reservoir. Collect all the eluates into a 50 mL Turbo Vap vessel. Evaporate the combined eluates at 453C in the Turbo Vap under 5 psi of nitrogen to near dryness. Dilute the residue to 1 mL with hexane. Vortex the Turbo Vap vessel for 15 s and then sonicate for 15 s. Quantitatively transfer the hexane extract and bring to volume in a 10.0 mL volumetric #ask with the diluting solution. Calculation Inject each of the "ve standards in duplicate and record the peak area. Calculate the concentration (pg) of vitamin K in the sample extract injection volume by linear regression analysis. RESULTS AND DISCUSSION Figure 1a illustrates the chromatogram of the vitamin K standard overlaid with the chromatogram of a vitamin K in a commercial medical food extract (Fig. 1b) at a concentration of 114 pg on column. Fluorescence responses for vitamin K hydro quinone were linear for the standard range from 45 to 900 pg vitamin K on column (r"0.998). Vitamin K hydroquinone was observed to have a limit of detection (LOD) (3p) of 6.6 pg vitamin K on column, corresponding to about 0.4 lg/100 g of dry powder. The limit of quantitation (LOQ) (10p) was 22 pg vitamin K on column corresponding to 1.5 lg/100 g of dry powder. A series of recovery studies were performed on the medical food ZRM. Table 1 shows the recoveries obtained when the blank was spiked at the x, 5x, 10x, 20x, and 40x levels, where x is equivalent to 16 lg/100 g of dry powder. Each spiking level was assayed 5 times, as was the blank. Recoveries of vitamin K were deemed acceptable over the spiking range studied. Since commercial formulations contain overages in forti"cation, the method is more than adequate to assay vitamin K at levels of 200% of label declaration and at levels approaching the LOQ. The peak purity of the vitamin K was established by peak ratioing (Haroon et al., 1986). The emission wavelength was kept constant for the analytes while #uorescence was measured at three excitation wavelengths. The #uorescence emission of vitamin K at 418 nm was determined at excitation wavelengths of 238, 248 and 258 nm. Ratios were calculated for 238/248 and 258/248. The ratios were compared for the standard and the commercial medical food extract (Table 2). Good agreement was obtained for ratios of standard and sample for vitamin K , indicating the purity of the vitamin K peaks. Earlier work by Chase et al. (1999a, b) utilized a C8 liquid chromatograph column. In this study, due to interferences observed in the medical food ZRM as well as the
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
769
FIGURE 1. The chromatogram of the vitamin K standard (a) overlaid with the chromatogram of the extract of a commercial medical food at an on column level of 114 pg (b). Flow rate was 1.0 mL/min, injection volume of 30 lL, excitation wavelength of 248 nm and emission wavelength of 418 nm and a gain of 9.
commercial medical food, a C18 column was utilized to obtain better separation from the interferences. The retention time for vitamin K was observed to shift considerably from the earlier work (Chase et al., 1999a, b) from 4 to 8.5 min; however, the total run time only increased by 1 min. The addition of hexane to the mobile phase, standards and samples as previously discussed (Chase et al., 1999a) is essential in order to maintain low column back pressure and good recoveries. There were no observed di$culties with the miscibility of hexane in methanol at levels of 10% (v/v).
770
CHASE JR, EITENMILLER AND LONG TABLE 1 Recovery of vitamin K from a forti"ed medical food ZRM
Forti"cation level vitamin K
Average recovery (%)
40x 20x 10x 5x x Blank
95.2$1.9 (2.0) 102$3.9 (3.9) 98.9$2.1 (2.2) 96.8$1.7 (1.8) 96.7$3.1 (3.2) Not applicable
Values are mean$S.D. Percent cvs are given in parentheses. Five replicates were assayed at each spiking level and blank. x is equivalent to 16 lg/100 g dry powder. No peaks were observed above the baseline in the ZRM chromatograms.
TABLE 2 Evaluation of peak purity Peak response ratio Nutrient Vitamin K
Peak ratio wavelength (nm)
Standard
Sample
238/248 258/248
0.59 0.12
0.59 0.12
Emission wavelengths were constant for vitamin K (418 nm). Ratios are based on the average of triplicate injections.
The medical food matrix is highly complex as compared to milk- and soy-based infant formulas and may contain di!erent protein and fat combinations and amounts. This matrix di!erence often results in an extremely slow, limited #ow of the eluting solvents through the reservoir. The protein utilized in the medical food formulation tends to clump once it comes in contact with the eluting solvents and, thus, restricts free #ow of such solvents through the reservoir. The application of air pressure to the reservoir tube in order to increase the #ow rate of the solvent causes a white precipitate to occur in the eluting solvent, which results in poor recoveries. This study di!ered from the earlier work by incorporating the addition of two drops of the reductive ion solution to the sample once it is weighed onto the C18/isopropyl palmitate mix. Addition of the reductive ion solution precipitated the protein, allowing the eluting solvents to #ow freely through the reservoir. Two di!erent commercial medical foods were also evaluated by this study. The "rst medical food was labeled to contain 25.4 g fat/100 g of dry powder, 15.3 g protein/ 100 g of dry powder and 19.0 lg vitamin K /100 g of dry powder. Ten replicate analyses for vitamin K gave 26.0 lg/100 g of dry powder$0.3 (cv."6.1%). Since commercial medical foods are often over forti"ed, this value would be in line with levels that have previously been observed in this laboratory using methods of Landen et al. (1989) for medical foods. The second medical food was labeled to contain higher fat and protein with 32.8 g fat/100 g dry powder, 22.0 g protein/100 g dry powder and 29.7 lg vitamin K /100 g dry powder. Ten replicate analyses for vitamin K gave 29.5 lg/100 g dry powder#0.5 (cv."6.6%). This method provides a simple and rapid technique to assay vitamin K in medical foods using only 27 mL of solvent per sample. The small sample size of 0.5 g is not
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
771
detrimental because properly prepared liquid samples are homogenous and the small sample size does not signi"cantly in#uence the data. In one day, an analyst familiar with the technique can easily extract 20 samples with analysis being completed overnight.
REFERENCES AOAC. (1995). O.cial Methods of Analysis, 16th edn. AOAC International, Arlington, VA. Barker, S. A. and Long, A. R. ;nited State Patent, No. 5,272,094. Booth, S. L., Charnley, J. M., Sadowski, J. A., Saltzman, E., Bovill, E. G., and Cushman, M. (1997). Dietary vitamin K and stability of oral anticoagulation: proposal of a diet with constant vitamin K content. ¹hromb. Haemo. 77, 504}509. Booth, S. L., Davidson, K. W., Lichtenstein, A. H., and Sadowski, J. A. (1996a). Plasma concentrations of dihydro-vitamin K following dietary intake of a hydrogenated vitamin K -rich vegetable oil. ¸ipids 31, 709}713. Booth, S. L., Davidson, K. W., and Sadowski, J. A. (1994). Evaluation of an HPLC method for the determination of phylloquinone (vitamin K ) in various food matrices. J. Agric. Food Chem. 42, 295}300. Booth, S. L., Madabushi, H. T., Davidson, K. W., and Sadowski, J. A. (1995). Tea and co!ee brews are not dietary sources of vitamin K (phylloquinone). J. Am. Diet. Assoc. 95, 82}83. Booth, S. L., Pennington, J. A. T., and Sadowski, J. A. (1996b). Dihydro-vitamin K : primary food sources and estimated dietary intakes in the American diet. ¸ipids 31, 715}720. Booth, S. L., Pennington, J. A. T., and Sadowski, J. A. (1996c). Food sources and dietary intakes of vitamin K (phylloquinone) in the American diet: data from the FDA Total Diet Study. J. Am. Diet. Assoc. 96, 149}154. Booth, S. L. and Sadowski, J. A. (1997). Determination of phylloquinone in foods by high performance liquid chromatography. Meth. Enzymol. 282, 446}456. Booth, S. L., Sadowski, J. A., Weihrauch, J. L., and Ferland, G. (1993). Vitamin K (phylloquinone) content of foods: a provisional table. J. Food Comp. Anal. 6, 109}120. Chase, G. W., Eitenmiller, R. R., and Long, A. R. (1999a). Liquid chromatographic analysis of vitamin K in milk based infant formula using matrix solid phase dispersion. J. AOAC Int. 82, 1140}1145. Chase, G. W., Eitenmiller, R. R., and Long, A. R. (1999b). Liquid chromatographic analysis of vitamin K in soy based infant formula using matrix solid phase dispersion. J. ¸iq. Chrom. 23, 423}432. Chase, G. W., Reid, A. P., Eitenmiller, R. R., and Long, A. R. (1998). Zero control reference materials for infant formula methods development. J. AOAC Int. 81, 453}456. Davidson, K. W., Booth, S. L., Dolnikowski, G. G., and Sadowski, J. A. (1996). Conversion of vitamin K to 2 ,3 -dihydrovitamin K during the hydrogenation of vegetable oils. J. Agric. Food Chem. 44, 980}983. Eitenmiller, R. R. and Landen Jr., W. O. (1999).
Jayashree
GSRS
JFCA*20000935
DATE ......................... 11*7*2000
DISC USED YES ! (2000) 13, 765}771 doi:10.1006/jfca.2000.0935 Available online at http://www.idealibrary.com on
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
ORIGINAL ARTICLE Liquid Chromatographic Analysis of Vitamin K1 in Medical Foods Using Matrix Solid-Phase Dispersion G. William Chase Jr.* , R. R. Eitenmiller-, and A. R. Long* *U.S. Food and Drug Administration, Northwest Regional Laboratory, 22201 23rd Drive, S.E., Bothell, WA 98021-4421, U.S.A.; and -Department of Food Science and Technology, University of Georgia, Athens, GA 30602, U.S.A. Received June 3, 1999, and in revised form January 26, 2000
A liquid chromatographic method for vitamin K in medical foods is described. The vitamins are extracted from medical food by matrix solid-phase dispersion (MSPD) and quantitated by reversed-phase chromatography with #uorescence detection. Vitamin K is converted to the #uorescent hydroquinone with a post-column zinc reductive reactor. The limit of detection is 6.6 pg and the limit of quantitation is 22 pg on column. Linear response ranged from 45 to 908 pg on column (r"0.998). Recoveries were determined on an analyte-forti"ed zero control reference material (ZRM) for medical foods and averaged 97.9% (n"25) for vitamin K . The method provides a rapid, speci"c and easily controlled assay for the analysis of vitamin K in forti"ed medical foods. 2000 Academic Press Keywords: vitamin K; MSPD; liquid chromatography.
INTRODUCTION Recently, Chase et al. (1999a, b) developed methods to quantitate vitamin K in forti"ed milk and soy-based infant formula. The extraction protocol utilized matrix solid-phase dispersion (MSPD) followed by a concentration step. The extract was injected directly onto a C8 LC column connected in series with a zinc post-column reduction column. The zinc reduction column induced formation of the #uorescent hydroquinone for the quanti"cation of total vitamin K by #uorescence detection. This technique and others currently used for vitamin K analysis resulted from the e!orts of the people at the United States Department of Agriculture Vitamin K Laboratory at Tufts University (Ferland and Sadowski, 1992a, b; Ferland et al., 1992; Booth et al., 1993, 1994, 1995, 1996a}c, 1997; Davidson et al., 1996; Booth and Sadowski, 1997). The MSPD technique has been patented and used extensively for isolating drugs from milk and tissue (Barker and Long, United States Patent). Methods reported by Chase et al. (1999a, b) provide speed, low solvent requirements and improved repeatability when compared to the O$cial AOAC International method for vitamin K in milk-based infant formula (Method 992.27, 50.106) (1995). The AOAC International, O.cial Methods of Analysis, 16th edn., does not To whom correspondence and reprint requests should be addressed. Tel.: 425 483 4990. Fax: 425 483 4996. E-mail: [email protected] 0889}1575/00/050765#07 $35.00/0
2000 Academic Press
766
CHASE JR, EITENMILLER AND LONG
provide a method for the analysis of vitamin K in medical foods. Method 992.27 applies to milk-based infant formula products. Due to sample matrix di!erences, the milk-based infant formula method for vitamin K is not collaborated and often not applicable for medical food assays. Problems with the AOAC International method include hard to interpret chromatograms due to lipid interference, inability to measure cis-vitamin K in matrices containing corn oil, and an overall high RSD of 20.9% (Eitenmiller and Landen, 1999). Since infant formula and medical food formulations are diverse and the resulting matrices are dissimilar, issues of solvent polarity, miscibility, partitioning characteristics, density characteristics and other physiochemical parameters become critically important in developing a sound validated method. The purpose of this study, therefore, was to apply the MSPD extraction technique for vitamin K from the earlier work with infant formula to medical foods (Chase et al., 1999a, b). METHOD Apparatus (a) Liquid Chromatograph * LDC Analytical Constametric 4100 pump (Thermo Separation Products, Riviera Beach, FL) and Waters 715 autoinjector (Waters, Inc., Milford, MA). (b) Column * Supelco C18, 5 lm, 4.6;250 mm, part number 58 298 (Supelco, Bellefonte, PA). (c) Integrator * Waters Millenium Data System, Software Revision 2.15 (Waters, Inc.). A stand-alone integrator can also be utilized. (d) Fluorescence detector * Model 1046A programmable #uorescence detector (Hewlett-Packard, Avondale, PA) or equivalent. (e) Reservoirs with frits * Varian 15 mL size, part number 1213}1016 (Varian, Harbor City, CA). (f ) Turboevaporator * Turbo Vap II (Zymark, Hopkinton, MA) or a suitable technique to evaporate the extracts. (g) Vortex mixer * Maxi-Mix I, (Thermolyne, Dubuque, IA). (h) Reduction reactor * 2.5 cm;3.2 mm stainless steel, packed with zinc powder, 100 mesh (Aldrich Chemical Co., Milwaukee, WI). (i) Ultrasonic cleaner * 5.2 gallon ultrasonic cleaner (Thomas Scienti"c, Swedesboro, NJ). Reagents (a) (b) (c) (d) (e) (f ) (g) (h)
Hexane * LC grade (Burdick and Jackson, Muskegon, MI). Isopropyl alcohol * LC grade (EM Science, Gibbstown, NJ). Ethyl acetate * LC grade (Burdick and Jackson). Methylene chloride * LC grade (Burdick and Jackson). Methyl alcohol * LC grade (Burdick and Jackson). Acetic acid * glacial, reagent grade (GR) (EM Science). Zinc chloride * ZnCl , catalog Z-33 (Fisher Scienti"c, Fairlawn, NJ). Sodium acetate * anhydrous, catalog S-8750 (Sigma Chemical Co., St. Louis, MO). (i) Reductive ionic solution * 2.0 M zinc chloride, 1.0 M sodium acetate and 1.0 M acetic acid per liter of methanol. Prepare this solution by weighing 68.1 g of zinc chloride, 20.5 g of sodium acetate into a 250 mL volumetric #ask and adding about
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
767
200 mL of methanol and 15 mL of acetic acid. Dissolve by stirring and then dilute it with methanol to the required volume. ( j) Diluting solution * one liter of methanol containing 5.0 mL of the reductive ionic solution. (k) Mobile phase * the diluting solution containing 10% hexane. Filter the mobile phase through a 0.45 lm "lter prior to use. (l) Vitamin K standard * Accurately weigh c. 25 mg of vitamin K (USP reference standard Phytonadione Lot L) into 50.0 mL volumetric #ask and dilute it to volume with hexane. Determine the exact concentration from E% "419 at 248 nm. Appropriate dilutions were made with hexane so that when the "ve "nal working standards are injected the concentration on column ranged from 45 to 900 pg. Each working standard should contain 10% hexane with the remaining volume being the diluting solution. (m) Bondesil * C preparative grade, part number 1221}3013 (Varian). (n) Isopropyl palmitate * catalogC 29,178-1, tech 90% (Aldrich Chemical Co., Milwaukee, WI). (o) Spiking solutions * The appropriate dilutions were made from the vitamin K stock standard in hexane so that an aliquot not exceeding 100 lL would contain x, 5x, 10x, 20x and 40x levels where x corresponds to 16 lg/100 g of dry powder. Chromatographic Conditions (a) Instrument parameters * Injection volume, 30 lL; #ow rate 1.0 mL/min. Fluorescence detector parameters: excitation wavelength (exj)"248, emission wavelength (emj)"418, gain"9. The zinc reduction column is placed after the analytical column. In addition, a scrubber column was installed after the pump but before the injector. This was comprised of a 15 cm stainless-steel column "lled with zinc metal (catalog no. Z15, Fisher Scienti"c) to aid in reducing background noise. (b) LC con"guration * First, inject the working standard to establish linearity (r"0.998) allowing for a 11 min run time per injection. Upon completion of the standard injections, inject the samples while interspersing with alternate standard injections. Sample Description and Preparation A ZRM originally used for other fat-soluble vitamin method development studies was utilized for vitamin K (Chase et al., 1998). Five pounds of a powdered medical food were obtained from a domestic manufacturer that was formulated without the addition of fat- and water-soluble vitamins. The formulation is milk based with medium-chain triglycerides and mimics commercial medical food matrices. Analytical examination documented that the matrix was devoid of vitamin K . The medical food, therefore, provides a ZRM for this analyte. For analysis, 1 lb was mixed, divided into 30 g airtight containers, stored under nitrogen at 03C until needed. Then, approximately 10 g of the powder was taken from one of the airtight containers and thoroughly mixed with 50 g of hot water (803C). Two commercial medical foods were locally purchased that contained milk, soy and whey protein as well as various oils. Twelve cans of each (12 #. oz.) were combined to provide composite samples. Sampling and preparation were completed as described in AOAC 50.1.01, section A and B (AOAC, 1995).
768
CHASE JR, EITENMILLER AND LONG
Sample Extraction Weigh 2 g Bondesil C into a mortar. Add 100 lL isopropyl palmitate and gently blend the isopropyl palmitate onto the C with a pestle. Accurately weigh 0.50 g reconstituted sample into the C -isopropyl palmitate mixture, followed by the addition of the spike solution and two drops of the reductive ion solution. Use the pestle to gently blend the reconstituted sample and the C -isopropyl palmitate into a #u!y, slightly sticky powder. Accurately transfer the C -matrix blend into a 15 mL reservoir tube with a frit at the bottom and then insert the top frit on the powdery mix. Tightly compress the reservoir contents with a 10 cm syringe plunger. Pass 9 mL 0.5% isopropyl alcohol in hexane (v/v), followed by 9 mL of ethyl acetate, followed by 9 mL of methylene chloride through the reservoir. Collect all the eluates into a 50 mL Turbo Vap vessel. Evaporate the combined eluates at 453C in the Turbo Vap under 5 psi of nitrogen to near dryness. Dilute the residue to 1 mL with hexane. Vortex the Turbo Vap vessel for 15 s and then sonicate for 15 s. Quantitatively transfer the hexane extract and bring to volume in a 10.0 mL volumetric #ask with the diluting solution. Calculation Inject each of the "ve standards in duplicate and record the peak area. Calculate the concentration (pg) of vitamin K in the sample extract injection volume by linear regression analysis. RESULTS AND DISCUSSION Figure 1a illustrates the chromatogram of the vitamin K standard overlaid with the chromatogram of a vitamin K in a commercial medical food extract (Fig. 1b) at a concentration of 114 pg on column. Fluorescence responses for vitamin K hydro quinone were linear for the standard range from 45 to 900 pg vitamin K on column (r"0.998). Vitamin K hydroquinone was observed to have a limit of detection (LOD) (3p) of 6.6 pg vitamin K on column, corresponding to about 0.4 lg/100 g of dry powder. The limit of quantitation (LOQ) (10p) was 22 pg vitamin K on column corresponding to 1.5 lg/100 g of dry powder. A series of recovery studies were performed on the medical food ZRM. Table 1 shows the recoveries obtained when the blank was spiked at the x, 5x, 10x, 20x, and 40x levels, where x is equivalent to 16 lg/100 g of dry powder. Each spiking level was assayed 5 times, as was the blank. Recoveries of vitamin K were deemed acceptable over the spiking range studied. Since commercial formulations contain overages in forti"cation, the method is more than adequate to assay vitamin K at levels of 200% of label declaration and at levels approaching the LOQ. The peak purity of the vitamin K was established by peak ratioing (Haroon et al., 1986). The emission wavelength was kept constant for the analytes while #uorescence was measured at three excitation wavelengths. The #uorescence emission of vitamin K at 418 nm was determined at excitation wavelengths of 238, 248 and 258 nm. Ratios were calculated for 238/248 and 258/248. The ratios were compared for the standard and the commercial medical food extract (Table 2). Good agreement was obtained for ratios of standard and sample for vitamin K , indicating the purity of the vitamin K peaks. Earlier work by Chase et al. (1999a, b) utilized a C8 liquid chromatograph column. In this study, due to interferences observed in the medical food ZRM as well as the
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
769
FIGURE 1. The chromatogram of the vitamin K standard (a) overlaid with the chromatogram of the extract of a commercial medical food at an on column level of 114 pg (b). Flow rate was 1.0 mL/min, injection volume of 30 lL, excitation wavelength of 248 nm and emission wavelength of 418 nm and a gain of 9.
commercial medical food, a C18 column was utilized to obtain better separation from the interferences. The retention time for vitamin K was observed to shift considerably from the earlier work (Chase et al., 1999a, b) from 4 to 8.5 min; however, the total run time only increased by 1 min. The addition of hexane to the mobile phase, standards and samples as previously discussed (Chase et al., 1999a) is essential in order to maintain low column back pressure and good recoveries. There were no observed di$culties with the miscibility of hexane in methanol at levels of 10% (v/v).
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CHASE JR, EITENMILLER AND LONG TABLE 1 Recovery of vitamin K from a forti"ed medical food ZRM
Forti"cation level vitamin K
Average recovery (%)
40x 20x 10x 5x x Blank
95.2$1.9 (2.0) 102$3.9 (3.9) 98.9$2.1 (2.2) 96.8$1.7 (1.8) 96.7$3.1 (3.2) Not applicable
Values are mean$S.D. Percent cvs are given in parentheses. Five replicates were assayed at each spiking level and blank. x is equivalent to 16 lg/100 g dry powder. No peaks were observed above the baseline in the ZRM chromatograms.
TABLE 2 Evaluation of peak purity Peak response ratio Nutrient Vitamin K
Peak ratio wavelength (nm)
Standard
Sample
238/248 258/248
0.59 0.12
0.59 0.12
Emission wavelengths were constant for vitamin K (418 nm). Ratios are based on the average of triplicate injections.
The medical food matrix is highly complex as compared to milk- and soy-based infant formulas and may contain di!erent protein and fat combinations and amounts. This matrix di!erence often results in an extremely slow, limited #ow of the eluting solvents through the reservoir. The protein utilized in the medical food formulation tends to clump once it comes in contact with the eluting solvents and, thus, restricts free #ow of such solvents through the reservoir. The application of air pressure to the reservoir tube in order to increase the #ow rate of the solvent causes a white precipitate to occur in the eluting solvent, which results in poor recoveries. This study di!ered from the earlier work by incorporating the addition of two drops of the reductive ion solution to the sample once it is weighed onto the C18/isopropyl palmitate mix. Addition of the reductive ion solution precipitated the protein, allowing the eluting solvents to #ow freely through the reservoir. Two di!erent commercial medical foods were also evaluated by this study. The "rst medical food was labeled to contain 25.4 g fat/100 g of dry powder, 15.3 g protein/ 100 g of dry powder and 19.0 lg vitamin K /100 g of dry powder. Ten replicate analyses for vitamin K gave 26.0 lg/100 g of dry powder$0.3 (cv."6.1%). Since commercial medical foods are often over forti"ed, this value would be in line with levels that have previously been observed in this laboratory using methods of Landen et al. (1989) for medical foods. The second medical food was labeled to contain higher fat and protein with 32.8 g fat/100 g dry powder, 22.0 g protein/100 g dry powder and 29.7 lg vitamin K /100 g dry powder. Ten replicate analyses for vitamin K gave 29.5 lg/100 g dry powder#0.5 (cv."6.6%). This method provides a simple and rapid technique to assay vitamin K in medical foods using only 27 mL of solvent per sample. The small sample size of 0.5 g is not
LC ANALYSIS OF VITAMIN K IN MEDICAL FOODS
771
detrimental because properly prepared liquid samples are homogenous and the small sample size does not signi"cantly in#uence the data. In one day, an analyst familiar with the technique can easily extract 20 samples with analysis being completed overnight.
REFERENCES AOAC. (1995). O.cial Methods of Analysis, 16th edn. AOAC International, Arlington, VA. Barker, S. A. and Long, A. R. ;nited State Patent, No. 5,272,094. Booth, S. L., Charnley, J. M., Sadowski, J. A., Saltzman, E., Bovill, E. G., and Cushman, M. (1997). Dietary vitamin K and stability of oral anticoagulation: proposal of a diet with constant vitamin K content. ¹hromb. Haemo. 77, 504}509. Booth, S. L., Davidson, K. W., Lichtenstein, A. H., and Sadowski, J. A. (1996a). Plasma concentrations of dihydro-vitamin K following dietary intake of a hydrogenated vitamin K -rich vegetable oil. ¸ipids 31, 709}713. Booth, S. L., Davidson, K. W., and Sadowski, J. A. (1994). Evaluation of an HPLC method for the determination of phylloquinone (vitamin K ) in various food matrices. J. Agric. Food Chem. 42, 295}300. Booth, S. L., Madabushi, H. T., Davidson, K. W., and Sadowski, J. A. (1995). Tea and co!ee brews are not dietary sources of vitamin K (phylloquinone). J. Am. Diet. Assoc. 95, 82}83. Booth, S. L., Pennington, J. A. T., and Sadowski, J. A. (1996b). Dihydro-vitamin K : primary food sources and estimated dietary intakes in the American diet. ¸ipids 31, 715}720. Booth, S. L., Pennington, J. A. T., and Sadowski, J. A. (1996c). Food sources and dietary intakes of vitamin K (phylloquinone) in the American diet: data from the FDA Total Diet Study. J. Am. Diet. Assoc. 96, 149}154. Booth, S. L. and Sadowski, J. A. (1997). Determination of phylloquinone in foods by high performance liquid chromatography. Meth. Enzymol. 282, 446}456. Booth, S. L., Sadowski, J. A., Weihrauch, J. L., and Ferland, G. (1993). Vitamin K (phylloquinone) content of foods: a provisional table. J. Food Comp. Anal. 6, 109}120. Chase, G. W., Eitenmiller, R. R., and Long, A. R. (1999a). Liquid chromatographic analysis of vitamin K in milk based infant formula using matrix solid phase dispersion. J. AOAC Int. 82, 1140}1145. Chase, G. W., Eitenmiller, R. R., and Long, A. R. (1999b). Liquid chromatographic analysis of vitamin K in soy based infant formula using matrix solid phase dispersion. J. ¸iq. Chrom. 23, 423}432. Chase, G. W., Reid, A. P., Eitenmiller, R. R., and Long, A. R. (1998). Zero control reference materials for infant formula methods development. J. AOAC Int. 81, 453}456. Davidson, K. W., Booth, S. L., Dolnikowski, G. G., and Sadowski, J. A. (1996). Conversion of vitamin K to 2 ,3 -dihydrovitamin K during the hydrogenation of vegetable oils. J. Agric. Food Chem. 44, 980}983. Eitenmiller, R. R. and Landen Jr., W. O. (1999).