A) THEORY. 1) Vitamin A

Transcription

1 Simultaneous Determination of 13-Cis and All-Trans Vitamin A Palmitate, Vitamin A Acetate, Alpha Vitamin E Acetate, and Alpha Tocopherol by HPLC and Column Switching A) THEORY VitE-18 1) Vitamin A Retinyl palmitate occurs as a yellow to yellow-red, amorphous or crystalline solid and retinyl acetate occurs as a pale yellow prismatic crystalline solid. Vitamin A palmitate and vitamin A acetate may be nearly odorless or have a slight fishy odor but do not smell rancid. The vitamin A palmitate melting point is C while the vitamin A acetate melting point is C. Both vitamin A palmitate and vitamin A acetate are more stable to oxidation than the free alcohol, retinol, and are unstable in the presence of alkali due to hydrolysis. All-trans vitamin A palmitate and all-trans vitamin A acetate, like the free alcohol, are susceptible to cis-trans isomerization. The 13-cis isomer is the most common cis isomer and has 75% of the biological potency of the all-trans isomer. One microgram of all-trans vitamin A palmitate is equivalent to International Units (IU). One microgram of vitamin A acetate is equivalent to IU, and one microgram of the free alcohol is equivalent to 3.33 IU. All-trans vitamin A palmitate is practically insoluble in water or glycerol and is soluble in absolute alcohol, methanol, chloroform, ether, fats, and oils. All-trans vitamin A acetate is also insoluble in water and is soluble in ethanol and chloroform. Vitamin A palmitate is the predominant storage form of vitamin A in the liver. The absorption maximum for vitamin A palmitate and vitamin A acetate is 325 nm. The vitamin A palmitate chemical formula is C 36 H 60 O 2 and its molecular weight is The vitamin A palmitate structural formula is: H 3 C CH 3 CH 3 CH 3 O O C (CH 2 ) 14 CH 3 CH 3 Vitamin A Palmitate The vitamin A Acetate chemical formula is C 22 H 32 O 2 and its molecular weight is The vitamin A acetate structural formula is: Vitamin A Acetate 2) Vitamin E Vitamin E acetate (dl- -tocopheryl acetate or all-rac- -tocopheryl acetate) and alpha tocopherol occur as colorless or pale yellow, nearly odorless, clear viscous liquids. Unlike the free alcohol, vitamin E acetate is practically unaffected by the oxidizing influence of air and visible light or by alkali. Alpha tocopherol is stable to heat and basic conditions when oxygen is not present, but is slowly oxidized, especially in the presence of certain metals (Fe, Cu, Ag, etc.) and darkens upon exposure to light. Vitamin E acetate and alpha tocopherol are practically insoluble in water, freely soluble in oils, fats, acetone, alcohol, chloroform, ether, and other fat solvents. Vitamin E acetate and alpha tocopherol are optically active. The d-form of -tocopheryl acetate has a

2 melting point of 28 C and the l-form has a melting point of 23 C. Alpha tocopherol has a melting point of C. One milligram of dl- -tocopheryl acetate is equal to 1 International Unit (IU) of vitamin E. One milligram of the d- -tocopheryl acetate, which is more active, is equal to 1.36 IU. One milligram of d- -tocopherol is equivalent to 1.49 IU. The vitamin E acetate chemical formula is C 31 H 52 O 3, its molecular weight is , and its structural formula is: H 3 C C O H 3 C O CH 3 O CH 3 CH 3 CH 3 CH 3 (CH 2 ) 3 CH(CH 2 ) 3 CH(CH 2 ) 3 CH CH CH 3 3 The free alcohol, alpha tocopherol, is found with other tocopherols largely in plant materials. Its chemical formula is C 29 H 50 O2, its molecular weight is and its structural formula is: CH 3 OH CH3 H CH3 H CH 3 CH3 O CH3 CH 3 CH 3 3) Extraction, HPLC Separation, Quantitation Extraction: The entire lipid fraction, including vitamins A and E, is extracted from product with isooctane after products are mixed with methanol, which precipitates proteins and disrupts micelles freeing lipids for extraction. Extracted samples are then centrifuged to separate the isooctane layer from the alcohol water layer. A 20 microliter aliquot of the upper, isooctane layer is injected onto the HPLC system. HPLC Separation: The HPLC system is comprised of two, mid-bore (3.0 mm ID) micro-spherical, totally porous silica columns placed in series and separated by a switching valve. Following sample injection, vitamins A palmitate, vitamin A acetate, and vitamin E acetate are separated from alpha tocopherol and other extraneous compounds present in sample extracts with a 3 cm, 3.0 mm ID column and a 1% methylene chloride, 0.06% isopropanol in iso-octane mobile phase. After vitamin A palmitate, vitamin A acetate, and vitamin E acetate elute from the 3.0 cm column onto a 20 cm, 3.0 mm ID column, a column switch takes place and vitamin A palmitate, vitamin A acetate, and vitamin E acetate are further separated on a 20 cm column and detected by UV at 313 nm (vitamin A palmitate and vitamin A acetate) and 280 nm (vitamin E acetate). Alpha tocopherol is further separated from other extraneous compounds on the 3 cm column and detected by fluorescence at excitation and emission wavelengths of 295 nm 330 nm respectively. Quantitation: The concentration of trans vitamin A palmitate, trans vitamin A acetate, vitamin E acetate, and alpha tocopherol are calculated by the comparison of peak areas or heights of samples to peak areas or heights of standards of known concentration. Quantitation of 13-cis vitamin A, the major cis isomer of vitamin A, is accomplished by using a response factor relative to that for the alltrans isomer. Results of the cis vitamin A determination are added to the all-trans vitamin A results to give total vitamin A results.

3 B) APPARATUS 1) Instrumentation and General Supplies a) HPLC System 1 isocratic pump, 1 programmable pump with flow rates ranging from 0.6 to 1.0 ml/min, 1 six port switching valve, 1 HPLC autosampler capable of 20 L injections, 1 multichannel UV detector (Vitamin A nm and Vitamin E 280 nm), a fluorescence detector (Alpha tocopherol - ex 295 nm, em 330 nm), and data collection system. b) Columns 1 ES Industries Chromegasphere Si-60, 3 m, 3.0 mm x 30 mm, P/N ES Industries Chromegasphere Si-60, 3 m, 3.0 mm x 200 mm, P/N or equivalent. c) Analytical Balance capable of weighing grams. d) Centrifuge capable of holding 50 ml round bottom centrifuge tubes. e) Centrifuge Tubes, Glass - 50 ml and screw caps with Teflon bonded liner. f) Glass transfer pipets g) Graduated Cylinders, assorted sizes h) Pipettor variable volume with tips i) Re-Pipette Heads, assorted sizes j) Stir plate, multi-position with appropriate size test tube rack k) Volumetric flasks, Class A, assorted sizes l) Volumetric Pipets, Class A, assorted sizes m) Vortex Mixer n) Light shields; Clear UV shields with a minimum UV cutoff of 385 nm 2) Chemicals/Standards a) Vitamin A Acetate Standard USP retinyl acetate, official lot with specified potency, or equivalent. Store per label instructions. b) Vitamin A Palmitate Standard Retinyl palmitate, official lot with specified potency, or equivalent. Stored per label instructions. If potency information is not available it can be determined experimentally. See Appendix III. c) Vitamin E Acetate Standard - USP all-rac- -tocopheryl acetate, official lot with specified purity or equivalent. Store per label instructions. d) Alpha-Tocopherol Standard- USP -Tocopherol, official lot with specified purity or equivalent. Store per label instructions. e) Methanol - HPLC (Distilled-in-Glass) grade f) Methylene Chloride (dichloromethane) - HPLC (Distilled-in-Glass) grade g) Isopropanol - HPLC (Distilled-in-Glass) grade h) 2,2,4-trimethylpentane (isooctane) - HPLC (Distilled-in-Glass) grade i) Reagent Alcohol j) Coconut oil, food grade C) STANDARD AND SOLUTION PREPARATION All sources of contamination must be eliminated. This includes reagents, glassware, and all plastics except fluorinated polymers such as Teflon, which do not contain plasticizers. Also, traces of water, except that specifically required during the sample preparation, must be

4 avoided. Solutions should be prepared fresh or whenever indications of contamination, chemical degradation, or changes in concentrations are evident. Expiration dates and storage conditions are stated. All solutions listed below may be scaled up for convenience provided good laboratory practices are observed. Preparation should be performed under shielded fluorescent lighting with a minimum UV cutoff of 385 nm. 1) Mobile Phase: 98.94% Isooctane, 1% Dichloromethane, and 0.06% Isopropanol: Add 20 ml dichloromethane and 1.2 ml of isopropanol to a 2 liter flask and dilute to volume with isooctane. Expiration: 2 days. 2) Vitamin A Palmitate Stock Standard Solution (approx IU/mL): Accurately weigh an appropriate amount of vitamin A palmitate standard to give a stock standard concentration of approximately 2600 IU/mL directly into or transfer to a 100 ml volumetric flask with isooctane. Add about 0.10 g (±10%) of coconut oil (as a stabilizer/ preservative for vitamin A). Dissolve and dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 2 months. 3) Vitamin A Acetate Stock Standard Solution (approx IU/mL): Accurately weigh an appropriate amount of vitamin A acetate standard to give a stock standard concentration of approximately 2600 IU/mL directly into or transfer to a 100 ml volumetric flask with ethanol. Add about 0.10 g (±10%) of coconut oil (as a stabilizer/ preservative for vitamin A). Dissolve and dilute to volume with ethanol. Store refrigerated or frozen protected from light. Expiration: 2 months. 4) Vitamin E Acetate Stock Standard Solution (approx. 1 mg/ml): Accurately weigh an appropriate amount of Vitamin E acetate standard to give a stock standard concentration of 1 mg/ml directly into or transfer to a 100 ml volumetric flask. Dissolve and dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 4 months. 5) Alpha Tocopherol Stock Standard Solution (approx. 1 mg/ml ) Accurately weigh an appropriate amount of Alpha-Tocopherol to give a concentration of 1 mg/ml and transfer to a 100 ml volumetric flask. Dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 1 month. 6) Mixed Vitamin Intermediate Standard: Pipet 1.0 ml vitamin A palmitate stock standard solution, 1.0 ml vitamin A acetate stock standard solution, and 25.0 ml vitamin E acetate stock standard solution into a 250 ml volumetric flask containing approximately 0.25 g of coconut oil. Dissolve and dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 2 months. 7) Alpha Tocopherol Intermediate Standard: (approx. 0.2 mg/ml) Pipet 20.0 ml alpha tocopherol stock standard solution into a 100 ml volumetric flask. Dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 1 month. 8) Mixed Vitamin Working Standards: Transfer about 20 ml Mixed Vitamin Intermediate Standard and 15 ml Alpha Tocopherol Intermediate Standard into separate 25 ml volumetric flasks. Stopper and allow to come to room temperature. Store prepared working standards refrigerated or frozen protected from light. Expiration: 2 weeks. a) High Working Standard - Pipet 10.0 ml of mixed vitamin intermediate standard and 5.0 ml of alpha tocopherol intermediate standard into a 50mL volumetric flask containing about 0.50 g of coconut oil. Dissolve and dilute to volume with isooctane.

5 b) DO NOT Mid DISTRIBUTE Working Standard - Pipet 4.0 ml of mixed vitamin intermediate standard and 3.0 ml of alpha tocopherol intermediate standard into a 50mL volumetric flask containing about 0.50 g of coconut oil. Dissolve and dilute to volume with isooctane. c) Low Working Standard - Pipet 2.0 ml of mixed vitamin intermediate standard and 1.0 ml of alpha tocopherol intermediate standard into a 50 ml volumetric flask containing about 0.5 g of coconut oil. Dissolve and dilute to volume with isooctane. d) Very Low Working Standard - Pipet 1.0 ml of mixed vitamin intermediate standard and 0.5 ml of alpha tocopherol intermediate standard into a 100 ml volumetric flask containing about 1.0 g of coconut oil. Dissolve and dilute to volume with isooctane. 9) Test Injection Working Solution: Pipet 3.0 ml of the Tocopherol Intermediate Standard into a 100 ml volumetric flask. Add 1.0 ml of Vitamin E acetate stock standard and dilute to volume with isooctane. Store refrigerated or frozen protected from light. Expiration: 6 months. D) PROCEDURE The procedure should be performed under shielded (yellow or clear) fluorescent lighting with a minimum UV cutoff of 385 nm. A complete and rapid vortex must be maintained during all sample mixing steps. If spillage or leakage occurs, a sample must be repeated from the beginning of the procedure. 1) Sample Preparation All samples, liquids and powders, should be as uniform and representative of the product as possible. Mix all products thoroughly before sampling. Powdered products that are not homogeneous at the sub-gram level should be reconstituted first. Sample sizes are chosen so that after dilution with laboratory water, if any, a total sample weight of about 4 g at a total solids value of less than 20% and a maximum oil content of about 250 mg is achieved while maintaining sample preparation concentrations of Vitamin A and Vitamin E within those of the standards. Typically sample sizes are 4.0 grams for ready-to-feed infant formulas and 2.0 grams (with 2.0 ml water) for concentrated infant formulas. Powdered infant formulas may first be reconstituted and treated as a ready-to-feed infant formula or approximately 0.5 grams of the powdered formula may be weighed directly into a 50 ml centrifuge tube and diluted with 3.5 ml laboratory water. a) Nutritional Products (1) Weigh an appropriate amount of sample directly into a 50 ml centrifuge tube and if necessary add enough laboratory water so that the combined weight of the sample and any water is approximately 4 g. Water should be added rapidly to powder samples and the sample should be immediately mixed until all of the powder is dissolved. (2) To each sample, add 25 ml (±10%) methanol using a Re-pipette head or equivalent glassware, cap the centrifuge tube, and vortex immediately and rapidly for at least 30 seconds OR add a stir bar, 25 ml (±10%) methanol and begin stirring at a rate that is fast enough to form a vortex, but does not cause sample to splash out of the centrifuge tube. Methanol should not be added to more than two samples, consecutively, without vortexing or stirring. (3) After the addition of methanol, let samples stand for at least 10 minutes but not more than 40 minutes if vortexing or let samples stir for at least 10 minutes if using a multi-position stir plate. (4) Using a volumetric pipet, add 10.0 ml isooctane to each centrifuge tube. Cap the centrifuge tubes and vortex rapidly for at least 45 seconds OR cap the centrifuge tubes and stir at a rate that

6 is fast enough to form a vortex at least 5 minutes. Isooctane can be added to any number of samples, consecutively, prior to vortexing or stirring. (5) Add 5 ml (±10%) laboratory water to each tube. Cap the centrifuge tubes and vortex rapidly for at least 20 seconds OR stir at a rate that is fast enough to form a vortex for at least 1 minute. Water can be added to any number of samples, consecutively, prior to vortexing. If samples cannot be effectively vortexed or stirred, they can be shaken by hand for at least 20 seconds. (6) A sample must be repeated from the beginning of the procedure if spillage or leakage occurs anytime during steps 1-5. (7) Centrifuge samples until there is a clean phase separation between the isooctane and the watermethanol phase. (8) Pipet about 1 ml of the clear upper layer into appropriate autosampler vials. 2) HPLC Analysis a) Instrumental Operating Parameters (1) System configuration 1 Figure 1 Pump ml/min Autosampler, 20 ul injection Column 1 Chromegasphere Si-60, 3u, 3 x 30 mm Fluorescence Detector Ex: 295mm, Em: 330 nm Column 2 Chromegasphere, Si-60, 3u, 3x 200 mm Pump ml/min UV Detector Vit A: 313 nm Vit E: 280 nm

7 (2) System configuration 2 Figure 2 Pump ml/min Autosampler, 20 ul injection Column 1 Chromegasphere Si-60, 3u, 3 x 30 mm Fluorescence Detector Ex: 295mm, Em: 330 nm Column 2 Chromegasphere, Si-60, 3u, 3x 200 mm Pump ml/min UV Detector Vit A: 313 nm Vit E: 280 nm b) System settings Run time: 30 minutes Pump 1 flow rate: 0.6 ml/min 0-11 min., 1.0 ml/min min, 0.6 ml/min min Pump 2 flow rate: 0.6 ml/min Injection volume: 20 ul Multichannel UV Detector settings: Channel nm, Channel nm. Fluorescence Detector settings: Excitation: 295 nm, Emission: 330 nm. c) System startup (1) At system startup, turn on the pumps and detector. Allow mobile phase to pass through each column for at least one-half hour. After the system has equilibrated, auto-zero all detectors before beginning an analysis. d) Column Switch Time Determination (1) After the system has equilibrated, the column switch-time is determined by injecting the Test Injection working solution onto the 3x30 mm column with the system in configuration 2. In this configuration, the standard is only going through the 3x30 mm column. See Figure 2, HPLC Analysis, Instrumental Operating Parameters (2) After determining the retention time of vitamin E acetate and -tocopherol peaks from the 3 x 30 mm column, set the column switch time to approximately halfway between when the vitamin E peak returns to baseline and the beginning of the -tocopherol peak; Figure 3. The resolution (R) between -tocopherol and vitamin E acetate should be 2.5. See Appendix II for an example calculation.

8 Figure 3. Example Test Injection e) Standard, Control, and Sample Injections (1) Standard and sample run times are approximately 30 minutes. Typical retention times are 5.7 minutes and 7.2 minutes for 13-cis and all-trans vitamin A palmitate, 15.0 minutes and 19.0 minutes for 13-cis and all trans vitamin A acetate, 23.5 minutes for vitamin E acetate, and 4 minutes for - tocopherol. See Appendix I for example chromatograms. These times may vary somewhat from column to column or system to system. (2) Confirm that the column switch time is set correctly according to the test injection. Perform 3 consecutive injections of the High Working Standard and confirm that peak responses have an RSD of 2.0%. (3) Inject the remaining working standards, a control sample, samples preparations, and a second set of standards. The column switch time should be checked before and after each set of standard and sample injections or every 24 hours or when the mobile phase is changed. E) CALCULATIONS 1) Chromatography Visually inspect each standard and sample chromatogram and verify that the vitamin A and vitamin E peaks are adequately resolved. Compare the vitamin A and vitamin E standard responses to the vitamin A and vitamin E sample responses and verify that the sample responses are within the range of the standard responses. Calculate the resolution (R) of the 13-cis vitamin A and all-trans vitamin A peaks. Resolution of these isomers must be > See Appendix II for example calculations. 2) Preparation of HPLC Vitamin Standard Curves a) Working Standard Concentration Calculations The concentrations of all-trans vitamin A palmitate, vitamin A Acetate, vitamin E acetate, and alpha tocopherol in each of the working standards are calculated from the amounts of standard weighed and the declared or determined potencies or purities of the standards using the following formula C= (Wt x P)/D Where C= vitamin A or E concentration in IU/mL or mg/ml Wt=the weight of reference standard weighed in grams. P=standard potency or purity in IU/g or mg/g

9 D= the dilution volume in ml. Dilution volumes for all of the standards are listed in the Table I below. Table I: Standard Dilution Volumes Vitamin High Working Standard Dilution Volume Middle Working Standard Dilution Volume Low Working Standard Dilution Volume Very Low Working Standard Dilution Volume Vitamin A Palmitate 125, , ,000 2,500,000 Vitamin A Acetate 125, , ,000 2,500,000 Vitamin E Acetate 5,000 12,500 25, ,000 Alpha-Tocopherol 5,000 8,333 25, ,000 b) Standard curves are prepared for all-trans vitamin A palmitate, all-trans vitamin A acetate, vitamin E acetate, and alpha tocopherol by performing a linear least squares routine on standard peak responses (peak areas for vitamin E and tocopherol and peak areas or heights for vitamin A palmitate and vitamin A acetate) versus corresponding standard concentrations. The all-trans vitamin A standard curves are used for the quantitation of both 13-cis vitamin A and all-trans vitamin A in samples. The linear correlation coefficients, r, for all standard curves must be and the difference in peak responses of the same standards injected before and after a set of samples must be 4.0%. c) Vitamin A Calculations Since 13-cis vitamin A palmitate and 13-cis vitamin A acetate standards are not available, the all-trans vitamin A palmitate and all-trans vitamin A acetate standard curves can also be used for the determination of 13-cis vitamin A palmitate and 13-cis vitamin A acetate using the following correction factors. Using peak heights: Corrected 13-cis vitamin A peak height response = 13-cis vitamin A peak height response RT RT OR Using peak areas: E all Corrected 13-cis vitamin A peak area response = 13-cis vitamin A peak area response B Where: E all and E 13 are the molar absorptivity of all-trans and 13-cis vitamin A, respectively, at the wavelength employed for the determination. This ratio has been estimated to be B is the biological potency of 13-cis vitamin A relative to that of all-trans vitamin A (0.75). RT all and RT 13 are the retention times of all-trans and 13-cis vitamin A respectively E all E E all 13 B d) Sample Concentration Calculations The vitamin A and vitamin E concentrations for products can be calculated from the following equation: C 10 df 1000 Vitamin Concentration (IU/kg or mg/kg) = W Where:

10 C = the vitamin concentration of the sample preparation obtained from the vitamin A and E standard curves in IU/mL or mg/ml. For total vitamin A, the sum of the trans vitamin A concentration and the 13-cis vitamin A concentration are added together. W = the sample weight in grams. 10 = the sample dilution volume in ml. 1000= the conversion of grams to kilograms, and df* is the dilution factor of the reconstituted powder (if applicable): weight product weight water * df weight of product e) Units conversions 1) Vitamin A results can be converted from weights to International Units (IU) or Retinol Equivalents (RE) using the following conversion factors: (a) All-trans retinyl acetate - 1 mcg of all-trans retinyl acetate is equivalent to IU or RE (b) All-trans retinyl palmitate 1 mcg all-trans retinyl palmitate is equivalent to IU or RE. 2) Vitamin E results can be converted from weights to IU or Tocopherol Equivalents (TE) using the following conversion factors: (a) All-rac alpha tocopheryl acetate 1 mg is equivalent to 1 IU or TE (b) RRR alpha tocopheryl acetate 1 mg is equivalent to 1.36 IU or TE (c) All-rac alpha tocopherol 1 mg is equivalent to 1.1 IU or TE (d) RRR alpha tocopherol 1 mg is equivalent to 1.49 IU or 1.0 TE

11 APPENDIX I Example Chromatograms Mixed Working Standard Injections Channel 1, UV Vitamin A Palmitate and Vitamin A 313 nm AU TRANS A TRANS A Acetate Channel 2, UV Vitamin E 280 nm VIT E mv Channel 3, Fluorescence alpha Ex: 295 nm Em: 330 nm A-Tocopherol FU SRM 1849 Channel 1, UV Vitamin A 313 nm TRANS A AU CISA Channel 2, UV Vitamin E 280 nm VIT E mv

12 Channel 3, Fluorescence alpha Ex: 295 nm Em: 330 nm A-Tocopherol FU Infant Formula Powder Channel 1, UV Vitamin A nm TRANS A Acetate AU CIS A Acetate Channel 2, UV Vitamin E 280 nm VIT E mv Channel 3, Fluorescence alpha Ex: 295 nm Em: 330 nm A-Tocopherol FU

13 APPENDIX II Resolution Calculation R = 2 (t 2 - t 1 ) (W 2 -W 1 ) Where: R = Resolution t 1 = retention time of peak 1 in minutes, t 2 = retention time of peak 2 in minutes, W 1 = peak width of peak 1 in minutes, W 2 = the peak width of peak 2 in minutes. In the example above, t 1 = 5.65 minutes, t 2 = 7.10 minutes, W 1 = minutes, W 2 = minutes, and R = 4.17.

14 APPENDIX III To determine to potency of the vitamin A palmitate standard: 1. Quantitatively transfer 0.1 g retinyl palmitate to a 100 ml volumetric flask and dissolve in and dilute to volume with hexane. 2. Pipet 2 ml of solution prepared in step 1 into a 250 ml volumetric flask and dilute to volume with hexane. 3. Pipet 2 ml of the solution prepared in step 2 into a 50 ml volumetric flask and dilute to volume with hexane. 4. Transfer a portion of this solution into a 1 cm cell path length cuvette and measure absorbance at 325 nm. 5. Calculate the concentration of vitamin A in mg/g using the following equation: Where: C std = A 325 x ε x b x wt C std = the concentration of retinyl palmitate in the vitamin A palmitate standard in mg/g; A 325 = absorbance of the solution prepared in step 3 at 325 nm; ε = the extinction coefficient of retinyl palmitate in hexane at 325 nm, 975 dl/g.cm; b = the cell path length, 1 cm wt= weight of standard weighed = conversion factor for g/dl to mg/g. To convert standard potency of vitamin A palmitate from mg/g to IU/g, multiply the standard potency by 1817 IU/mg.

15 APPENDIX IV: ACCURACY AND PRECISION DATA Estimated quantitation and detection limits are summarized in Table II below. Table II: Quantitation and Detection Limits Analyte Detection Limit with Standards Quantitation Limit with Standards Quantitation Limit in Product Vitamin A Palmitate 9.7 IU/L or 5.3 mcg/l 32 IU/L or 18 mcg/l 80 IU/L or 44 mcg/l Vitamin A Acetate 25 IU/L or 8.6 mcg/l 83 IU/L or 29 mcg/l 207 IU/L or 71 mcg/l Vitamin E Acetate 0.29 mg/l 0.95 mg/l 2.4 mg/l Alpha Tocopherol <0.018 mg/l <0.059 mg/l <0.15 mg/l Table III: Accuracy Data Generated with Individual Vitamin A and E and Tocopherol Methods Product Type Vitamin A Palmitate % Recovery Vitamin E Acetate % Recovery -Tocopherol % Recovery Milk Based Infant 99.1% 99.5% Formula RTF Liquid n=18 n=18 Milk Based High Calorie 99.5% 99.8% Infant Formula RTF n=12 n=12 Soy Based Infant Formula 98.2% 99.5% Powder n=12 n=12 Milk and Soy Based Infant 99.6% 100.0% and Pediatric RTF Liquid n=18 n=18 Whey Infant Formula Powder 98.6% n= % n= % n=3 Adult Nutritional Beverage 98.7% n= % n= % n=3 Liquid Medical Food 98.8% n= % n= % n=3 Powdered Hydrolyzed Protein/Free Amino Acid 97.3% n= % n= % n=3 Medical Food = Not Applicable Table IV: Intermediate Precision Data Generated with combined Vitamin A, Vitamin E and Tocopherol Method Product Type Vitamin A Vitamin A Vitamin E Acetate* -Tocopherol* Palmitate* Acetate* SRM IU/kg 1.73% RSD 363 mg/kg 0.98% RSD 37.1 mg/kg 3.65% RSD Whey based Infant Formula Powder IU/kg 1.83% RSD 87.2 mg/kg 1.82% RSD 16.2 mg/kg 2.00% RSD Milk based Pediatric Ready to Feed Liquid 2890 IU/kg 0.90 % RSD 23.7 mg/kg n=5 2.00% RSD 8.63 mg/kg 1.00% RSD Free Amino Acid Infant and Pediatric Formula Powder IU/kg 0.44% RSD 179 mg/kg 1.52% RSD 47.6 mg/kg 1.71% RSD Protein Free Infant and Pediatric IU/kg 264 mg/kg 62.8 mg/kg

16 Formula DO Powder NOT DISTRIBUTE 1.45% RSD Milk based Starch containing Infant 3060 IU/kg Formula Ready to Feed Liquid Hydrolyzed casein Infant Formula Powder Hydrolyzed Whey Pediatric Ready to Feed Liquid Hydrolyzed casein Infant Formula Ready to Feed Liquid Hydrolyzed Whey Infant Nutritional Powder Free Amino Acid Infant and Pediatric Nutritional Powder Milk based Infant Formula Powder High protein, very low fat casein based Infant Formula Powder Soy based Infant Formula Concentrated Liquid High protein, high fat Milk based Adult Nutritional Ready to Feed Liquid High protein, high fat casein based Adult Nutritional Ready to Feed Liquid High protein, moderate fat hydrolyzed casein Adult Nutritional Ready to Feed Liquid High protein, high fat casein based Adult Nutritional Ready to Feed Liquid Moderate protein, low fat hydrolyzed whey and casein Adult Nutritional Ready to Feed Liquid High protein, high fat casein based Adult Nutritional Ready to Feed Liquid Moderate protein, moderate fat casein and soy based Adult Nutritional Ready to Feed Liquid Milk based Adult Nutritional Pudding High protein, low fat Hydrolyzed Protein & Free Amino Acid Adult Nutritional Powder High protein, very low fat Hydrolyzed Protein & Free Amino Acid Adult Nutritional Powder Moderate fat and protein milk and soy based Adult Nutritional Ready to Feed Liquid 2.98% RSD IU/kg 0.67% RSD 2430 IU/kg 1.56% RSD 2830 IU/kg 0.84% RSD IU/kg 2.19% RSD 9960 IU/kg 0.56% RSD IU/kg 1.70% RSD 4270 IU/kg 1.75% RSD 4460 IU/kg 0.98% RSD 6240 IU/kg 0.73% RSD 4070 IU/kg 1.71% RSD 6560 IU/kg 1.07% RSD 3460 IU/kg 1.46% RSD 6510 IU/kg 2.09% RSD 6380 IU/kg 1.26% RSD 7180 IU/kg 2.01% RSD IU/kg 3.29% RSD 8880 IU/kg 6.30% RSD 6740 IU/kg 1.72% RSD 1.50% RSD 23.9 mg/kg 2.94% RSD 195 mg/kg 0.59% RSD 25.3 mg/kg 1.71% RSD 22.2 mg/kg 1.24% RSD 54.5 mg/kg 5.61% RSD IU/kg 2.75% 224 mg/kg 2.74% RSD 79.6 mg/kg 1.07% RSD 787 mg/kg 1.34% RSD 16.0 mg/kg 0.93% RSD 78.1 mg/kg 1.37% RSD 62.5 mg/kg 1.11% RSD 176 mg/kg 0.83% RSD 253 mg/kg 0.55% RSD 151 mg/kg 1.62% RSD 52.3 mg/kg 1.60% RSD 50.4 mg/kg 1.30% RSD 80.2 mg/kg 1.44% RSD 92.1 mg/kg 1.26% RSD 174 mg/kg 1.24% RSD 57.2 mg/kg 1.24% RSD 1.14% RSD 7.60 mg/kg 3.23% RSD 66.9 mg/kg 1.30% RSD 5.45 mg/kg 1.74% RSD 6.83 mg/kg 1.97% RSD 65.8 mg/kg 2.72% RSD 55.5 mg/kg 2.51% RSD 36.5 mg/kg 1.09% RSD 1.87 mg/kg 4.25% RSD 14.3 mg/kg 6.42% RSD 38.1 mg/kg 1.99% RSD 15.6 mg/kg 1.16% RSD 8.56 mg/kg 1.72% RSD 7.63 mg/kg 1.44% RSD 23.3 mg/kg 0.99% RSD 22.5 mg/kg 1.54% RSD 7.34 mg/kg 1.79% RSD 2.61 mg/kg 2.28% RSD 9.66 mg/kg 4.22% RSD 1.41 mg/kg 8.26%RSD 6.99 mg/kg 1.45% RSD

17 = not DO applicable NOT DISTRIBUTE * Results are for products as is.