On March 28, 2011, the AOAC Board of Directors

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1 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, INFANT FORMULA AND ADULT NUTRITIONALS Determination of Vitamin A (Retinol) in Infant Formula and Adult Nutritionals by Liquid Chromatography: First Action JONATHAN W. DEVRIES, KARLENE R. SILVERA, and ELLIOT MCSHERRY Medallion Laboratories, 9000 Plymouth Ave North, Minneapolis, MN DAWN DOWELL AOAC INTERNATIONAL, 481 N. Frederick Ave, Gaithersburg, MD During the Standards Development and International Harmonization: AOAC INTERNATIONAL Mid- Year Meeting, held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method for the Determination of Vitamins A (Retinol) and E (alpha- Tocopherol) in Foods by Liquid Chromatography: Collaborative Study, published by Jonathan W. DeVries and Karlene R. Silvera in J. AOAC Int. in After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals. The ERP granted the method First Action status, applicable to determining vitamin A in ready-to-eat infant and adult nutritional formula. In an effort to achieve Final Action status, it was recommended that additional information be generated for different types of infant and adult nutritional formula matrixes at varied concentration levels as indicated in the vitamin A (retinol) SMPR. Existing AOAC LC methods are suited for specific vitamin A analytical applications. The original method differs from existing methods in that it can be used to assay samples in all nine sectors of the food matrix. One sector of the food matrix was powdered infant formula and gave support for the First Action approval for vitamin A in infant and adult nutritional formula. In this method, standards and test samples are saponified in basic ethanol water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm for retinol. Vitamin concentration is calculated by comparison of the peak heights or peak areas of retinol in test samples with those of standards. Submitted for publication October 28, The method was approved by the Expert Review Panel on Infant Formula and Adult Nutritionals as First Action. See Standards News, (2011) Inside Laboratory Management, July/August issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author. Corresponding author s jon.devries@genmills.com DOI: /jaoacint.CS2011_15 On March 28, 2011, the AOAC Board of Directors approved an alternative path to achieve Official First Action status for methods selected and reviewed using the AOAC volunteer consensus standards development processes. Under this path, an Expert Review Panel (ERP) vetted by the Official Methods Board (OMB) reviewed the method for the Determination of Vitamins A (Retinol) and E (alpha-tocopherol) in Foods by Liquid Chromatography: Collaborative Study (1). After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals, and approved this method as Official First Action status for vitamin A in infant formula and adult nutritionals. Methods approved First Action under the alternative path will remain First Action for a period of about 2 years. During this time, methods will be used in laboratories, generating additional information. ERPs will monitor the methods performance, and after about 2 years, will determine whether the methods should be recommended to the OMB for Final Action (2). For First Action adoption, all methods were reviewed by primary, as well as secondary, expert reviewers. Panel members summarized their reviews; the advantages and disadvantages of each method were then discussed thoroughly by the entire panel, stakeholders, and observers present. Methods were evaluated for completeness of validation and likelihood of meeting the SMPRs (applicability to intended use, clarity of the method description, ruggedness, reproducibility, recovery, analytical range, and LOQ). Based on the data presented, the ERP agreed that the method for determining vitamin A (retinol), in addition to being granted Official First Action status, should undergo additional evaluation consisting of data collected from multiple laboratories on a series of infant formula matrixes shared amongst the laboratories and available through AOAC INTERNATIONAL. The protocol for data collection should be guided by the ERP in conjunction with the OMB. With vitamin A being essential for many healthrelated functions, it is important for analytical laboratories to have accurate methods available to determine the concentration of vitamin A in infant formula and adult nutritionals. The method provides a straightforward, modern way to achieve these results using a combination of traditional chemistry and LC technology.

2 2 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 AOAC Official Method Vitamin A (Retinol) in Infant Formula and Adult Nutritionals Liquid Chromatography First Action 2011 (Applicable for the determination of retinol from 7 to 383 g/100 g in ready-to-feed infant formula and adult nutritionals.) Caution: Potassium hydroxide is extremely caustic and can cause severe burns. Protect skin and eyes and wear all personal protective equipment while performing method, which involves the use of flammable liquids and toxic chemicals. Perform saponification behind a barrier when using hot water, steam, or an electric heating mantle. Use an effective fume removal device to remove flammable and toxic vapors/fumes produced during operation of the method. Leave ample headroom in flask and add boiling chips before heating a flammable liquid. See Tables A and B for the results of the interlaboratory study supporting acceptance of the method. A. Principle Standards and test samples are saponified in basic ethanol water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm. Vitamin concentrations are calculated by comparison of peak heights or peak areas of vitamins in test samples with those of standards. B. Apparatus and Materials (a) HPLC system. (1) Pump. High-pressure pump operating continuously at 1.0 to 2.0 ml/min with a flow precision of ±1% or better. (b) Injector. Manual injector or autosampling injector with a 20 μl fixed loop having a typical sampling precision of ±0.25% or better. (c) Chromatography columns. Reversed-phase C18, 10 μm ( mm), capable of separating cis and trans isomers of retinol with a resolution of 1.5 or greater. Cis-retinol typically elutes prior to trans-retinol on columns providing effective separation. (d) Detector. Photometric detector monitoring absorbance at 328 nm. (Alternatively a wavelength of 313 nm can be used.) (e) Recorder, integrator, or data collection system. Compatible with detector used. (f) Erlenmeyer fl asks. Low-actinic, 125 ml, with neck adapted for connecting reflux condenser. (g) Hot plate. With sufficient heating surface area to handle multiple reflux apparatus setups preferred. (h) Refl ux condensers. With adapters (if necessary) to attach 125 ml low-actinic Erlenmeyer flasks and nitrogen lines. (i) Volumetric fl asks. Low-actinic, 100 and 10 ml. (j) Nitrogen blanket apparatus. Supply of nitrogen gas with appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during saponification. C. Reagents (a)(1) Certifi ed vitamin A acetate concentrate. Equivalent to ca 30 mg retinol/g oil. Content certified by U.S. Pharmacopeia (USP; Rockville, MD; or (2) Retinyl palmitate, Table A. Interlaboratory study results for the determination of vitamin A by LC Matrix sector No. Matrix Labs a(b) Mean, μg/100 g Rec., % S R RSD R, % R HorRat 1 Margarine/butter (50/50 mix) 12(0) Margarine/butter (50/50 mix; spiked) 10(2) Chicken gravy (canned; spiked) 9(1) Cheese sauce (spiked) 11(0) Whole egg powder 10(0) Whole egg powder (spiked) 12(0) Multigrain cereal 12(0) Corn cereal 12(1) Corn cereal (spiked) 12(1) Infant formula (powdered) 12(0) NIST SRM (0) Dried nonfat milk 12(0) Dried nonfat milk (spiked) 12(0) Cottage cheese 8(1) Cottage cheese (spiked) 10(2) Canned tuna in oil (spiked) 11(1) Avg ± 13.2 a(b) Number of laboratories: a = number of laboratories retained after outliers were removed, and b = number of outlier laboratories.

3 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, Table B. Interlaboratory study results for the determination of vitamin A by LC (Youden pair statistical treatment) Youden pairs Labs a(b) Mean, μg/100 g s r RSD r, % S R RSD R, % r R HorRat Dried nonfat milk (spiked) and margarine/butter (50/50 mix; spiked) 10(2) Corn cereal and corn cereal (spiked) 10(2) Margarine/butter (50/50 mix) and dried nonfat milk 12(0) Multigrain cereal and infant formula (powdered) 12(0) Cottage cheese (spiked) and NIST SRM (2) Canned tuna in oil (spiked) and cheese sauce (spiked) 11(1) Chicken gravy (canned; spiked) and whole egg powder 8(1) Number of laboratories: a = number of laboratories retained after outliers were removed, and b = number of outlier laboratories. a(b) all-trans. Fluka Chemical Co. (Ronkonkoma, NY, ). Request Certificate of Lot Analysis when ordering. If manufacturer s certification is unavailable, or purity of standards needs to be verified, test vitamin A palmitate purity as follows: Dissolve 50 mg (record to the nearest 0.1 mg) retinol palmitate standard in 2-propanol (UV-spectroscopy grade) in a 500 ml flask and dilute to volume. Dilute 10 ml of this solution to 100 ml with 2-propanol (final concentration is ca 10 mg/l). Measure maximum absorbance obtained at nm using 1 cm path length cell and 2-propanol as a blank. Inject 10 L of solution onto an LC system with a reversed-phase column, a mobile phase of methanol heptane (93 + 7), and detection at the wavelength of the absorbance maximum. Record the peak area of the all-trans retinyl palmitate (PArp) and the total of all peak areas present other than the solvent front (PAt). Calculate the purity of the retinol palmitate as follows: Percent purity = (PArp/PAt) (ABS )/(960 W) where ABS = absorbance maximum; 960 = absorbance of pure retinol palmitate (1% solution in 1 cm cell); W = weight of test portion in mg; and = combined dilution factors, conversion to 1% equivalent solution, and conversion to %. Store retinol palmitate standard at 0 4 C to allow for easier handling while weighing. (b) Acetic acid. Glacial. (c) Methanol. HPLC grade. (d) Ethanol. 95%. (e) Tetrahydrofuran (THF). (f) Hexane. (g) Pyrogallol (pyrogallic acid). Crystals, Sigma-Aldrich, ( CAS or equivalent. (h) Vitamin A mobile phase. Combine 860 ml methanol (HPLC grade) and 140 ml water. Mix well. Stir overnight to degas or mechanically degas prior to use. (i) THF ethanol (50+50). Combine 500 ml THF and 500 ml 95% ethanol. Mix well. (j) Potassium hydroxide solution, 50%. Slowly add 500 g KOH pellets to 500 ml water contained in a 2 L thick-wall Erlenmeyer flask. (Caution: Solution gives off substantial heat while KOH is dissolving; add KOH in 100 g portions while flask is being cooled with cold water. Swirl flask gently to aid in dissolution of KOH. Store in glass container with cork stopper.) (k) Vitamin A working standard (approximately 15 μg/ml). (1) Using USP standard. Weigh 50 mg vitamin A acetate concentrate into a 100 ml low-actinic volumetric flask. Record weight to nearest 0.1 mg. Record concentration in mg/g per USP certification. Add small amount of acetone (<3 ml) to aid dissolution. Dilute to volume with 95% ethanol. Store at 4 C in dark. Solution is stable for 2 weeks. (2) Using retinyl palmitate. Weigh 55 mg retinyl palmitate into a 100 ml low-actinic volumetric flask. Record weight to nearest 0.1 mg. Record purity per supplier certification or purity test. Add pea-sized piece of pyrogallic acid, approximately 50 mg. Dissolve and dilute to volume with hexane. Pipet 5 ml of solution to second 100 ml low-actinic flask and dilute to volume with 95% alcohol. Store at 4 C in dark. Solution is stable for 2 weeks.

4 4 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 D. Extraction and Saponification Turn on hot plate to preheat. Start and adjust cooling water flow to precool reflux condensers. Prepare high standard (approximately 750 ng/ml) by pipeting 5 ml vitamin A working standard into a 125 ml lowactinic Erlenmeyer flask. Add 35 ml of 95% ethanol. Proceed to addition of pyrogallic acid. Prepare intermediate standard (approximately 300 ng/ml) by pipeting 2 ml vitamin A working solution into a second 125 ml low-actinic Erlenmeyer flask. Add 38 ml of 95% ethanol. Proceed to addition of pyrogallic acid. Prepare low standard (approximately 75 ng/ml) by pipeting 0.5 ml vitamin A working standard into a third 125 ml lowactinic Erlenmeyer flask. Add 39.5 ml of 95% ethanol. Proceed to addition of pyrogallic acid. Grind solids to pass a 40 mesh sieve. Blend liquid or wet materials to homogeneity and store 4 C in the dark. To prepare low-fat (<40% fat) test samples, weigh enough test sample ( 5 g) to give approximately 50 μg vitamin A into a 125 ml low-actinic Erlenmeyer flask. For test samples high in sugar, add 3 ml water and disperse the test portion as a slurry. Add 40 ml of 95% ethanol. To prepare high-fat test samples, weigh test sample ( 2 g) to give approximately 50 μg vitamin A into a 125 ml low-actinic Erlenmeyer flask. Add 40 ml of 95% ethanol. Add a pea-sized piece (approximately 50 mg) of pyrogallic acid (antioxidant) to each standard and test flask. Add a glass bead or boiling stone to promote even boiling. Swirl all flasks to ensure that all materials are thoroughly dispersed in the solution. Turn on N flow and ensure N atmosphere for all flasks before and while refluxing. Pipet 10 ml of 50% KOH solution into each flask and immediately place flask on hot plate under reflux condenser. Swirl. Reflux 45 min. Swirl flasks every 10 min. Remove reflux flasks from hot plate, stopper with corks, and quickly cool flasks to room temperature using cold water or ice water. Pipet 10 ml glacial acetic acid into each flask to neutralize the KOH. Mix well and let flasks cool again to room temperature. Quantitatively transfer solution in each flask to a 100 ml low-actinic volumetric flask using THF 95% ethanol ( ). Dilute to volume with the same solvent mixture. Stopper and invert volumetric flask 10 times. Allow flasks to set for at least 1 h at room temperature and preferably overnight in refrigerator to precipitate fatty acid salts formed during saponification. In some cases, centrifugation may reduce settling time. E. Determination Start HPLC system(s) and allow to warm up and equilibrate for a minimum of 30 min with mobile phase flowing at flow rate of 1.0 ml/min. Inject vitamin A standards that have been taken through saponification onto HPLC system. Adjust mobile phase to achieve a resolution of 1.5 or better for cis and trans forms. All-trans-retinol should elute in approximately 9 min (the cisisomer will elute just prior to the all-trans-isomer. Inject high, medium, and low standards. Adjust detector sensitivity to give peak heights of 50 90% of full scale at the high standard. Repeat injection of standard until peak height(s) are reproducible. Inject test solutions. Intersperse with standard solution injections after every nine tests. [If retinol in test exceeds the peak height of the high standard by more than 25%, dilute test solutions using a solution of 10 ml 50% KOH, 40 ml of 95% ethanol, 10 ml glacial acetic acid, and 40 ml THF 95% ethanol ( ).] F. Calculations Calculate μg/g vitamin A (as retinol) as follows: Measure the peak heights or areas of the standards. (1) Using USP standard. Determine the response factor for vitamin A (RF A ) using the following calculation: RF A = mg std ml std concn std /PkHT std where PkHT std = peak height or area of standard from chromatogram; ml std = ml of working standard used in procedure; concn std = concentration of USP vitamin A (as retinol) per USP certification (mg/g); mg std = mg of USP standard weighed in reagents section; = combined dilution factors for vitamin A standard. (2) Using retinyl palmitate. Determine RF A using the following calculation: RF A = mg std ml std purity std /PkHT std 200 where purity std = percent purity certified by supplier or determined, divided by 100; mg std = mg of retinyl palmitate weighed; PkHT std = peak height or area of standard from chromatogram; ml std = ml of working standard used in procedure; = ratio of retinol to retinyl palmitate molecular weights; and 200 = combined dilution factors/ conversion from mg to mg. The RF A values of the low, medium, and high standards should agree with each other within 3% relative since the detector response should be linear across this concentration range. Use an average of RF A values calculated from high, medium, and low standards for test sample quantitation. Measure the peak heights or areas corresponding to retinol (vitamin A) in the test sample extracts. The 13-cis isomer of retinol (eluting immediately preceding the all-trans-isomer) might be present in some test samples. Measure the 13-cis peak also. Multiply the height or area of the 13-cis retinol peak by 1.08 (to compensate for difference in absorbance compared to the trans-isomer). Add the corrected peak height or area for the 13-cis isomer to that of the all-trans isomer to give total test sample peak height or area. Calculate the concentration of vitamin A (in μg/100 g as retinol) using the following equation: Vitamin A, μg/100 g (as retinol) = RF A PkHT SPLE /W where RF A = response factor for vitamin A; PkHT SPLE = total test sample peak height or area of all-trans and 13-cis retinol; = dilution volume of test portion, ml conversion to 100 g portion; and W = weight of test portion, g. References: J. AOAC Int. 85, 424(2002); in press(2012)

5 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, Table 1. Samples selected for collaborative study Matrix sector Test samples Protein, % Fat, % Carbohydrate, % 1 Margarine/butter (50/50 mix) Chicken gravy (canned) Cheese sauce Whole egg powder Multigrain cereal Corn cereal Infant formula (powdered) NIST SRM Baked beans with franks (60/40, w/w) Dried milk Full fat soy fl our Cottage cheese Canned tuna in oil Results and Discussion The original study data presented in Tables A and B collected for vitamins A and E in nine food matrixes. The data were presented for review by the ERP to determine the method s appropriateness for use to determine the vitamin A content in infant and adult nutritional formula. The study data presented was reported from 13 laboratories, with one laboratory report showing a systematic high bias in relation to that of the other laboratories. The cause of this bias was determined to be a result of not properly following the protocol. Therefore, the results were omitted from the calculations. Tabulated results are found in Table A for vitamin A from the remaining 12 laboratories. The test samples for chicken gravy, baked beans with franks, cheese sauce, and tuna (canned) did not provide data sufficient for statistical review. In addition, soy flour (full fat) does not have a measurable quantity of retinol because it is plant-based. The decision to include it in the original study was made based on theoretical data for content of vitamin A. Because the typical operating LOD for retinol on LC is 15 μg/100 g for most laboratories, it is possible that the level of retinol is <15 μg/100 g, or that the detectors used by the laboratories were not tuned adequately to a sufficiently low S/N to detect the low levels of retinol present. Because of the expected low levels of retinol, one additional test sample was included for each of the five food sectors to obtain valid data. It was decided to apply the Dixon test to the individual test samples that had sufficient data points to obtain betweenlaboratory statistical data based on similar analyte level. As seen in Table A, the results are good except over sector 4 (see Table 1), which is represented by whole egg samples (fat, 33 67%; protein, 33 67%; carbohydrates, 0 33%). Based on the HorRat results, the RSD at various concentrations is in agreement relative to the analyte levels. The analytical variability of the whole egg sample cannot be specifically attributed to the particular fat, carbohydrate, or protein ratio. Possible explanations include difficulty in digesting the matrix and extract, or homogeneity was not achieved for the whole egg powder sample. It is plausible to exclude issues involving protein content based on satisfactory results from test samples in adjacent sectors 7 and 8 that have similar protein content. In the original study, Youden pair statistics was used since the method was applicable to all foods and a similar variance was expected. This approach was applied to the data to calculate the variability between laboratories for this method. Youden pairs were established based on samples in which the analyte levels were closest. As shown in Table B, the 14 test samples were combined to form seven pairs. The results show that the within-laboratory variability for each sample pair is less than the variability between laboratories. The recovery of the method was assessed by spiking eight samples with a vitamin concentration range. National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1846 was also included in the sample set. This approach allowed a recovery sample for each sector of the food analysis triangle. As shown in Tables A and B, the average recovery for the method was 100 ± 13%. This is in line with the expected recovery variability based on the expected reproducibility RSD (RSD R ) of approximately 13% (based on HorRat values in Tables A and B). Accuracy of the method was determined by including a powdered, milk-based infant formula (NIST SRM 1846). This sample was included as an unknown sample. The NIST Certificate of Analysis gives the noncertified vitamin A content of NIST SRM 1846 as 584 ± 68 μg/100 g, 95% uncertainty range. The reported study results showed a recovery of 79.5%, with the results being 464 ± 31 mg/100 g, 95% uncertainty range. SRM 1846 packets were obtained approximately 6 months prior to the study beginning. The packets were stored unopened in a dark cabinet in an office. The temperature ranged from 20 to 25 C (68 77 F), as suggested by the instructions received with SRM Because the cis-retinol proved to be somewhat elevated from what might be expected from the SRM, it was decided to investigate possible loss of retinol during storage of the materials. As part of the investigation, new packets of SRM 1846 were purchased and analyzed in a comparison study of the previously procured SRM packets used in the collaborative study (stored an additional 8 months, for a total of 14 months).

6 6 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 Table 2. Collaborative study data for vitamin A Matrix sector Vitamin A samples Spike level, μg/100 g Laboratory Margarine/butter (50/50 mix) Margarine/butter (50/50 mix; spiked) a a Chicken gravy (canned) 10 2 Chicken gravy (canned; spiked) a Cheese sauce Cheese sauce (spiked) Whole egg powder Whole egg powder (spiked) Corn cereal a Corn cereal (spiked) a Multigrain cereal Baked beans with franks (60/40, w/w) Infant formula (powdered) NIST SRM Nonfat dry milk Nonfat dry milk (spiked) Full fat soy fl our Cottage cheese a Cottage cheese (spiked) a a Canned tuna in oil 9 Canned tuna in oil (spiked) a a Dixon outlier.

7 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, A testing schedule was set for the newly obtained SRM 1846 at about 2 weeks, 5 weeks, and 6 months after receipt. Results obtained for the stored NIST sample were 80.7, 78.6, and 81.1%, respectively (average 80.1%), compared to those of the newly obtained NIST samples. The average concentration of the stored NIST samples was 465 mg/100 g; the newly purchased sample average was 580 mg/100 g. The cause for the level reduction of vitamin A in the collaborative study sample of SRM cannot be determined. However, the levels found during this investigation are consistent with the comparative results obtained during the collaborative study. One possible reason for the change in the SRM could be attributed to office environment. The office that was used to store the SRM prior to the collaborative study is climatecontrolled. However, the area of the building adjacent to the office contains a food production pilot plant faculty. This area was periodically heated above 100 F for pest control purposes during weekends. It is possible that the cabinet that housed the SRM sample was exposed to periods of high temperature during SRM sample storage, as the cabinet was against the wall adjacent to the food production facility. Thus, it is possible the SRM sample was exposed to degradation temperature prior to being included in the collaborative study. Collaborative study data for vitamins A and E were received and reviewed during the AOAC Offi cial Methods SM approval process. From the data reported, vitamin A was shown to perform as expected (Table 2). References (1) DeVries, J., & Silvera, K. (2002) J. AOAC Int. 85, (2) Sullivan, D. (2012) J. AOAC Int. 95, in press