Technical Note: A Simplified Procedure For Vitamin E Determination in Beef Muscle 1,2

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1 Technical Note: A Simplified Procedure For Vitamin E Determination in Beef Muscle 1,2 Qingping Liu, Kristine K. Scheller, and Daniel M. Schaefer Department of Meat and Animal Science, University of Wisconsin-Madison, Madison ABSTRACT: A simplified method for the determination of a-tocopherol concentration in beef muscle was developed and evaluated. The method consists of a saponification step applied to 1-g samples of intact, fresh muscle, followed by a single isooctane extraction of the saponified samples. a-tocopherol in the extract was separated by normal phase chromatography and quantified by fluorescence detection. A single extraction with the simplified method accounted for 95% of the total muscle a-tocopherol concentration obtained by two extractions with the Arnold et al. (J. Food Sci. 58:28, 1993) method. Recovery of added a-tocopherol standard after two extractions of a saponified muscle sample was 91% for the simplified method, which was not different ( a =.78) from recovery using the Arnold method, and the efficiency of the single extraction in the simplified method was 89%. The coefficients of variation for the simplified and Arnold methods were both 3.1%. This method should permit the duplicate analysis of 100 fresh muscle samples within 24 h. Key Words: Beef, Muscle, Vitamin E, a-tocopherol, Normal Phase Chromatography J. Anim. Sci : Introduction Long-term dietary supplementation of feedlot cattle with vitamin E improves the oxidative stability of fresh, frozen, and cooked beef under simulated retail conditions (Liu et al., 1995; Sherbeck et al., 1995), presumably due to accumulation of a-tocopherol in subcellular membranes (Schaefer et al., 1995). Benefits to cattle performance of such supplementation have been more evident early in the confinement feeding period (Hill and Williams, 1993) and less evident over the total finishing period (Arnold et al., 1993; Hill and Williams, 1993). Implementation of this technology in the U.S. beef industry requires compensation of cattle feeders for their additional vitamin E expense. Verification that cattle have been fed a sufficient amount of vitamin E would be 1 Generous support was provided by the Beef Industry Council of the National Live Stock and Meat Board; Hoffmann-La Roche, Nutley, NJ and Basel, Switzerland; the Wisconsin Beef Council, Madison, WI; Oscar Mayer Foods, Madison, WI; Packerland Packing, Green Bay, WI; and the College of Agricultural and Life Sciences, University of Wisconsin-Madison. Research was conducted as described in protocol 406, which was approved by the UW Research Animal Resources Center. The authors appreciate the valuable contributions of Scot Williams, Martin Frigg, and Wendy Clemens. 2 This article is a portion of a paper, Verification of Vitamin E in Beef, presented at the Meat Industry Research Conference, September 20, 1995, Chicago, IL. Received October 17, Accepted June 3, facilitated by development of a rapid procedure for muscle a-tocopherol determination in packing plants. Our goals for such a procedure were to sample an accessible, inexpensive skeletal muscle on the day of slaughter. It was desired that a-tocopherol concentrations be available before carcass fabrication (which may begin by 24 h after slaughter) and that carcass sampling capacity be maximized. On the basis of knowing the a-tocopherol concentration for the sampled muscle, the a-tocopherol concentrations in other valuable muscles could be predicted based on the uniform responsiveness of skeletal muscles observed thus far following vitamin E supplementation (Arnold et al., 1993). The objective was to develop a method for direct determination of muscle a-tocopherol concentration in accordance with the goals stated above. Following initial experimentation, it became apparent that a simplification of the Arnold et al. (1993) procedure would be the optimum solution. Rectus capitis dorsalis major ( RCDM) muscle was chosen as the carcass sampling site. The quantitative relationship involving a-tocopherol concentrations between RCDM and other valuable skeletal muscles will be reported in a subsequent publication. Materials and Methods Reagents. Ethanol ( ETOH), tetrahydrofuran ( THF), methanol, and isooctane were HPLC grade and purchased from Fisher Scientific (Pittsburgh, 2406

2 VITAMIN E DETERMINATION IN BEEF MUSCLE 2407 Table 1. Comparison of two HPLC methods for vitamin E determination in muscle Step Arnold et al. (1993) Step Simplified 1 Freeze tissue in liquid N 2 then pulverize 1 Cut and weigh 1 g of fresh muscle into 25-mL test tube 2 Weigh 1 g into 25-mL test tube 2 Add 250 mg ascorbic acid and 7.3 ml 11% KOH digestion solution 3 Add 100 mg of 50:50 of EDTA:ascorbic acid mixture and 2.0 ml of.1 N HCl then vortex 3 Incubate test tube for 15 min at 80 C with shaking in water bath 4 Add 4 ml ethanol; vortex; heat 10 min at 4 Cool tube in ice; add 4 ml isooctane 70 C; vortex every 5 min 5 Add 600 mg KOH; vortex; heat 20 min at 5 Vortex for 2 min with multi-tube vortexer 78 C; vortex every 10 min 6 Cool tube in ice; vortex; add 1 ml water 6 After phases separate, transfer aliquot of upper layer to autosampler vial 7 Add 4 ml isooctane; stopper; vortex 1 min 7 Inject 30 ml into chromatograph with automatic injector 8 Remove 3.4 ml isooctane to second tube; discard 8 Allow 7 min between injections remaining isooctane 9 Repeat steps 7 & 8; 6.8 ml total of extracts 9 Duplicate determinations on each carcass require 14 min of chromatograph time 10 Hold tube at 65 C and evaporate 6.8 ml to dryness under N 2 10 Accumulate data via computer with appropriate software 11 Dissolve residue in suitable volume of isooctane and inject 30 ml into chromatograph with automatic injector 12 Allow 8 min between injections 13 Duplicate determinations on each carcass requires 16 min of chromatograph time 14 Accumulate data via computer with appropriate software 15 Summarize data for previous 24 h of chromatograph operation 11 Summarize data for previous 24 h of chromatograph operation PA). Ascorbic acid, potassium hydroxide (KOH), and dl-a-tocopherol were analytical grade and bought from Sigma Chemical (St. Louis, MO). High-purity nitrogen gas was purchased from Liquid Carbonic (Oak Brook, IL). Saponification Solution. This solution was prepared fresh each day and comprised 11% KOH (wt/vol), 55% ETOH (vol/vol), and 45% deionized distilled water(vol/vol). Potassium hydroxide was dissolved in the distilled water before adding ETOH. Aliquots (7.3 ml) of the saponification solution were dispensed from a 10-mL Repipet dispenser (Fisher Scientific). Muscle Samples. Muscle samples from 51 Holstein steers were collected at a commercial packing plant (Packerland Packing, Green Bay, WI). After steer decapitation, the RCDM was exposed as lying on both sides of the first cervical vertebra (Getty, 1975). A small piece (200 g) of the RCDM muscle was collected, vacuum-packaged, and transported on ice to the authors laboratory. Saponification and Extraction. Immediately after the muscle samples arrived, a 1-g sample was weighed into a mm test tube in duplicate for each muscle sample. Each tube received ascorbic acid (.25 g) and 7.3 ml of saponification solution and was briefly vortexed until the ascorbic acid dissolved. Ascorbic acid addition before saponification was necessary to prevent a-tocopherol destruction by an alkaline solution (Bourgeois, 1992). Tubes were then placed into a test tube rack and incubated in an 80 C shaking water bath for 15 min at 200 rpm. After the incubation, test tubes were cooled in ice and 4 ml of isooctane was delivered to each tube with a 5-mL Repipet dispenser. Each tube was then tightly capped with a Teflon-lined cap and vortexed for 2 min with a multi-tube vortexer (model VB4, Glas-Col, Terre Haute, IN). Tubes were allowed to stand briefly to allow separation of aqueous and isooctane phases. The concentrations and volumes of the saponification solution and isooctane were designed to provide the final proportions of 4 ml H 2 O:4 ml ETOH:4 ml isooctane; the muscle sample was assumed to contribute.7 ml of H 2 O. These are nearly the same proportions used by Arnold et al. (1993, Table 1). Distinct separation of the aqueous and isooctane phases occurred spontaneously in 1 min. Thirty microliters of the upper isooctane phase was injected into an HPLC composed of a model M-45 pump, 712 WISP autosampler, and 470 fluorescence detector (Waters, Milford, MA). Data were collected with software (Dynamax MacIntegrator 2, Rainen Instrument, Woburn, MA) and a Macintosh computer. HPLC Conditions. Precolumn: Waters Resolve Guard-Pak Silica. Stationary phase: Waters Resolve 5 mm spherical silica column ( mm). Column temperature: 15 C. Mobile phase: 96% isooctane (vol/ vol):4% (vol/vol) THF prepared fresh daily. Flow rate:

3 ml/min. Injection volume: 30 ml. Detection: Fluorescence (excitation wavelength of 296 nm and emission wavelength of 325 nm). Calculation: External standard method based on peak area. Standards:.5 to 4 mg a-tocopherol/ml isooctane. Retention time: 5.2 min. Run time: 7 min. Extraction Efficiency. Numerous trials were conducted to determine efficiency of the first extraction. Extraction efficiency was determined by randomly selecting, weighing, and saponifying muscle ( n = 22) in four replications or by adding 3 mg of a-tocopherol standard ( n = 11) into four replicate tubes and saponifying the standard in the absence of meat samples. Saponification of muscle samples or standards was done with the simplified procedure described above or the method of Arnold et al. (1993), which has been routinely used in our laboratory (Table 1). After saponification, two extractions were done. In the first extraction, 4 ml of isooctane was added and vortexed as described above. For each tube, a portion of the first extract was transferred to an autosampler vial (Fisher Scientific) and 30 ml was injected into the HPLC for a-tocopherol determination. Three milliliters of isooctane from the first extract was also removed and transferred into a test tube. Residual isooctane was discarded. For the second extraction, another 4 ml of isooctane was added into the tube, tubes were vortexed for 1 min, and 3.0 ml of isooctane was removed from the tube and combined with the 3.0 ml of isooctane from the first extraction. With a stream of nitrogen gas, the combination of 6.0 ml isooctane from both extractions was evaporated to dryness in a 65 C water bath. The concentration of total a-tocopherol from the two extractions was then determined by redissolving the dried residue in 1 ml of isooctane and injecting an aliquot of each redissolved sample into the HPLC. Calculation of first extraction efficiency was as follows: Extraction efficiency, % = mg/ml in first extract 4 ml mg/ml in first plus second extracts 1 ml (8 ml/6 ml) LIU ET AL. 100 α-tocopherol Recovery. Recovery was determined by adding 1 ml of a known amount ( 3 mg/ml) of a- tocopherol into test tubes, with or without meat samples, and digesting the contents of those test tubes with the simplified procedure described above or the Arnold et al. (1993) procedure. Each determination included four replicate tubes with muscle sample alone, four replicate tubes with 3 mg a-tocopherol standard alone, and four replicate tubes with muscle sample and a-tocopherol standard. For the tubes with added a-tocopherol, 1 ml of the 3 mg/ml standard was added to each of four tubes and evaporated to dryness under a stream of nitrogen gas before making any other additions. If required, muscle samples were then added into those tubes containing added a-tocopherol. [1] All of the tubes were processed with the saponification, extraction, evaporation, and dilution steps described above for extraction efficiency. Two extractions were conducted and the extracts were combined. An aliquot of the redissolved extracts from each tube plus six replicate aliquots of the external standard were injected into the HPLC for a-tocopherol quantification. a-tocopherol recovery was calculated with the following equations: Recovery, % = [(muscle + external standard) (muscle)] external standard 100 [2] Recovery, % = (extracted external standard/external standard) 100 [3] Comparison of the Procedures. Muscle samples from 51 finished Holstein steers were analyzed for a- tocopherol concentration by both simplified and Arnold et al. (1993, Table 1) procedures. The relationship between a-tocopherol concentration from the first extraction of the simplified procedure and a-tocopherol concentration resulting from two extractions with the Arnold et al. (1993) procedure was established. Statistical Analysis. For a-tocopherol recovery and first extraction efficiency, analysis of variance was done by using the GLM procedure (SAS, 1990). Mean comparisons were conducted with the F-protected ( P <.05) least squares mean procedure. Regression analyses were determined by the GLM procedure. Results There was no difference ( a =.78) between the simplified and Arnold et al. (1993) procedures for a- tocopherol recovery regardless of whether a muscle sample was absent or present for the saponification step (Table 2). First-extraction efficiencies were lower ( P <.01) for both procedures for a-tocopherol standard alone vs muscle samples (Table 2). Presumably the presence of muscle enhances the partitioning of a-tocopherol into isooctane. Repeatability of the determination was not different ( a =.5) between procedures for saponifications conducted with or without a muscle sample or following a single or double extraction (data not shown). For saponification conducted in the presence of muscle, the CV for the simplified procedure following a single extraction and for the Arnold et al. (1993) procedure following its typical two extractions were both 3.1%. The KOH concentration in the saponification solution was optimized at 11% based on complete muscle digestion within a 15-min incubation, minimum KOH and maximum a-tocopherol recovery (data not shown). Figure 1 shows the regression relationship of muscle a-tocopherol concentrations determined by the two procedures. The relationship is linear (r 2 =.95)

4 VITAMIN E DETERMINATION IN BEEF MUSCLE 2409 Table 2. Comparisons for recovery and first extraction efficiency of a-tocopherol between simplified and Arnold et al. (1993) methods Arnold et al. method a Simplified method a Item With muscle Without muscle With muscle Without muscle Recovery, % b 91.1 ± ± ± ± 2.5 First extraction efficiency, % c 87.3 ± 1.7 d 78.9 ± 3.4 e 88.9 ± 1.4 d 81.9 ± 1.6 e a Numbers of observation were 22 and 11 for with muscle and without muscle, respectively. Standard errors are shown. b Calculated as quotient ( 100) of incremental a-tocopherol concentration resulting from addition of 3 mg of a-tocopherol and two isooctane extractions divided by the amount of added tocopherol; see Eq. [2] and [3] in text. c Calculated as quotient ( 100) of the a-tocopherol concentration resulting from a single isooctane extraction divided by the a-tocopherol concentration resulting from the pooling of two isooctane extracts; see Eq. [1] in text. d,e Means with different superscripts differ ( P <.05). and the equation is y = x, where y and x represent the concentration from simplified and Arnold et al. (1993) methods, respectively. The single isooctane extraction of the simplified procedure recovered 95% of the a-tocopherol determined by two extractions with the Arnold et al. (1993) procedure. Sensitivity of the simplified method was down to 1 mg of a-tocopherol g 1 muscle. Discussion Initial efforts to accomplish our goals involved methods to indirectly estimate muscle a-tocopherol concentration based on ability of the muscle sample to resist lipid oxidation accelerated by the addition of ferric ions. The thiobarbituric acid-reactive substances ( TBARS) value was an imprecise indicator of muscle a-tocopherol concentration in this circumstance. The a-tocopherol concentration in RCDM accounted for 76% of the variation in TBARS values for 72 university-fed steers, but only 32% of the variation in 208 industry-fed steers (Liu, Scheller, and Schaefer, unpublished results). Subsequent work focused on simplified procedures for the direct determination of muscle a-tocopherol concentration. a-tocopherol determinations are now commonly accomplished via HPLC with fluorescence detection because this approach is sensitive and specific (Lang et al., 1992). Normal phase chromatography is preferred over reverse phase in this application because the analyte of interest, a-tocopherol, is eluted before g-tocopherol (Lang et al., 1992), which is found in some skeletal muscle samples (Liu and Schaefer, unpublished results) by the method of Arnold et al. (1993). Bourgeois (1992) has addressed the comparison of normal and reverse phase approaches and favors normal phase where a chromatograph is dedicated to long-term conduct of the same analytical method. Emphasis was placed on simplification of sample preparation, saponification, and extraction steps. Muscle subsamples have been obtained previously from frozen, pulverized (Arnold et al., 1993; Liu et al., 1994) or fresh, homogenized (Pfalzgraf et al., 1995) samples. The lack of difference in CV between the Arnold et al. (1993) and simplified procedures and the similarity of these CV with that of Pfalzgraf et al. (1995) for muscle (4.2%) suggest that muscle disruption prior to subsampling is not necessary to achieve low CV. Saponification is necessary to release a- tocopherol from its membranal location. The simplified procedure uses a heated, strong basic solution to accomplish muscle dissolution. Pfalzgraf et al. (1995) succeeded in avoiding sample transfer and in using a Figure 1. Regression relationship between a- tocopherol concentrations determined by the simplified procedure or Arnold et al. (1993) procedure (n = 51).

5 2410 LIU ET AL. single extraction step to achieve 103% recovery of a- tocopherol added to pork muscle. Their analysis was done on an aliquot obtained directly from the organic phase extract without prior evaporation of the extract and redissolution. The same strategy was followed here, but a-tocopherol recovery was lower. When the steps of evaporation and redissolution are circumvented by the simplified method, g-tocopherol is not detectable and thus does not necessitate delay of subsequent HPLC injections. Emphasis was also placed on analytical capacity. The simplified method involves fewer procedural steps than the Arnold et al. (1993) method (Table 1), equipment that is amenable to bulk handling of samples (e.g., tube racks for saponifying incubation and 19-tube vortexer), and a shorter HPLC run time than that reported by Arnold et al. (1993) and Pfalzgraf et al.(1995). In fact, the capacity-limiting step in the simplified procedure is the interval between HPLC injections. Further minimization of the 7-min interval reported here should be possible with a faster mobile phase flow rate, although a faster flow rate necessitates a larger injection volume. The simplified procedure is estimated to allow analysis of 100 muscle samples in duplicate daily by one full-time technician. The Arnold et al. (1993) method allowed analysis of 40 muscle samples in duplicate daily and required 2.5 full-time technicians. Implications A simplified a-tocopherol method has been developed for application to intact, fresh beef muscle. It has more than double the analytical capacity of the Arnold et al. (1993) method without compromising precision of determination. Utilization of this technology in packing plants could accelerate implementation of vitamin E technology in the U.S. beef industry. Literature Cited Arnold, R. N., K. K. Scheller, S. C. Arp, S. N. Williams, and D. M. Schaefer Dietary a-tocopheryl acetate enhances beef quality in Holstein and beef breed steers. J. Food Sci. 58:28. Bourgeois, C Determination of Vitamin E: Tocopherols and Tocotrienols. pp 14 and 65. Elsevier, New York. Getty, R The Anatomy of the Domestic Animals (5th Ed.). p 809. Saunders, Philadelphia, PA. Hill, G. M., and S. E. Williams Vitamin E in beef nutrition and meat quality. Proc. 54th Minnesota Nutr. Conf. p 197. Lang, J. K., M. Schillaci, and B. Irvin Vitamin E. In: A. P. De Leenheer, W. E. Lambert, and H. J. Nelis (Ed.) Modern Chromatographic Analysis of Vitamins (2nd Ed.). p 153. Marcel Dekker, New York. Liu, Q., M. C. Lanari, and D. M. Schaefer A review of dietary vitamin E supplementation for improvement of beef quality. J. Anim. Sci. 73:3131. Liu, Q., K. K. Scheller, D. M. Schaefer, S. C. Arp, and S. N. Williams Dietary a-tocopheryl acetate contributes to lipid stability in cooked beef. J. Food Sci. 59:288. Pfalzgraf, A., H. Steinhart, and M. Frigg Rapid determination of a-tocopherol in muscle and adipose tissue of pork. Z. Lebensm.-Unters.-Forsch. 200:190. SAS SAS User s Guide: Statistics. SAS Inst. Inc., Cary, NC. Schaefer, D. M., Q. Liu, C. Faustman, and M. C. Yin Supranutritional administration of vitamins E and C improves oxidative stability of beef. J. Nutr.125:1792S. Sherbeck, J. A., D. M. Wulf, J. B. Morgan, J. D. Tatum, G. C. Smith, and S. N. Williams Dietary supplementation of vitamin E to feedlot cattle affects beef retail display properties. J. Food Sci. 60:250.