Quantitation of total vitamin D2 and D4 in UV-exposed mushrooms using HPLC with UV detection after novel two-step solid phase extraction.

A robust method for quantitation of total vitamin D2 and D4 in mushrooms by high performance liquid chromatography with UV detection (HPLC-UV) was developed to analyze mushrooms exposed to UV light. A two-step solid phase extraction (SPE) (silica, carbon black) removed chromatographic interferences typically resolved only with mass spectrometric detection (LC-MS) and allowed quantitation of all vitamin D and pre-D analytes. The vitamin and pre-vitamin forms of D2, D4 and D3 (internal standard), as well as other photoisomers and sterols were resolved. Results for six types of UV-exposed mushrooms were comparable to LC-MS. Screening of ten additional types of UV-exposed mushrooms without the IS confirmed lack of interference with the IS. The limit of quantification (µg/100 g fresh weight) was 0.4 for vitamin D and 0.9 for pre-vitamin D. Mushrooms do not have to be dried, and separatory funnels and large solvent volumes were also eliminated from sample preparation.

Effect of household cooking on the retention of vitamin D2 and 25-hydroxyvitamin D2 in pulse UV-irradiated, air-dried button mushrooms (Agaricus bisporus).

Vitamin D deficiency has widespread global prevalence. Fresh mushrooms exposed to ultraviolet (UV) radiation generate vitamin D2 which remains after drying. It is not clear if vitamin D2 is retained after rehydration and cooking of dried mushrooms. The aim of this study was to determine the true retention of both vitamin D2 and 25-hydroxyvitamin D2 (25(OH)D2) after cooking UV-irradiated, air-dried, then rehydrated button mushrooms (Agaricus bisporus). Mushrooms were exposed to pulsed UV radiation, then air-dried in a convection oven, followed by rehydration in warm water. Samples were cooked in three different ways: frying (5 min), baking (10 min, 200 °C) and boiling (20 min, 90 °C). Compared to rehydrated, uncooked controls, there was a high retention of D vitamers (≥95%) after cooking. Frying and baking resulted in significantly higher vitamin D2 retention compared to boiling (p < 0.0001). UV-irradiated, dried mushrooms are a valuable source of vitamin D2 after rehydration and cooking.

Development of a two-dimensional liquid chromatography-tandem mass-spectrometry method for the determination of vitamin D2 in mushrooms.

Different foods, especially mushrooms, are a valuable source of vitamin D2. However, published concentrations in mushrooms show large variabilities. One reason for this is certainly the high biological variability caused by growth conditions, and another could also be found in the analytical methodology. Therefore, this study aimed to develop a sensitive and highly selective two-dimensional liquid chromatography mass spectrometry (LC-MS/MS) method for vitamin D2 analysis in mushrooms. After validation, the method was applied to four different mushroom species. The developed method with a one-step extraction procedure showed a limit of detection of 0.01 µg vitamin D2/g dry mass (DM), a limit of quantification of 0.05 µg vitamin D2/g DM, and recovery rates between 87.6 and 94.8%. The total run time including the re-equilibration of the columns for the next injection was 7.5 min. After adding increased concentrations of pure substance to Pleurotus ostreatus, Lentinula edodes, and brown and white button mushrooms the standard addition plot showed excellent correlation coefficients (R2) of > 0.9994. Mean vitamin D2 concentrations were observed at 0.122 ± 0.007, 0.074 ± 0.005, 0.099 ± 0.007, and 0.073 ± 0.005 µg/g DM. The coefficient of variation (CV) was between 5.1 and 7.6%. This well-optimized, sensitive LC-MS/MS method, with a fast and simple sample preparation and a short run time, can be applied to future studies especially in different mushroom species with variable growing conditions. This will improve our knowledge about the vitamin D2 content in mushrooms.

4D printing induced by microwave and ultrasound for mushroom mixtures: Efficient conversion of ergosterol into vitamin D2.

This study hoped to use microwave and ultrasound combined with 4D printing technology to promote the conversion of ergosterol into vitamin D2 in printing model with mushroom scraps. Under the UV irradiation, the conversion was different in the printed model with different irradiation areas and different physical field pretreatment. Compared with raw materials, vitamin D2 concentrations in the printed models was 4.6 time higher. Vitamin D2 in the product after physical field pretreatment was 2.2-3.8 times higher than that without pretreatment. From partial least square regression (PLS) analysis, irradiation area had the greatest impact while ultrasound treatment had the least. Pretreatment enhanced vitamin D2 content, possibly because pretreatment meant ergosterol was more susceptible to UV radiation, and expansion of the irradiated area increased the beneficial effect. This study established an artificial neural network model to predict ergosterol and vitamin D2 content.

Vitamin D composition of Australian foods.

Australia needs accurate vitamin D food composition data to support public health initiatives. Previously, limitations in analytical methodology have precluded development of a comprehensive database. We used liquid chromatography with triple quadrupole mass spectrometry (LC-QQQ) to analyse 149 composite samples representing 98 foods (primary samples n = 896) in duplicate for vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), vitamin D2, 25(OH)D2. The greatest concentrations of vitamin D3 were found in canned salmon and a malted chocolate drink powder (fortified); chicken eggs and chicken leg meat contained the most 25(OH)D3. Margarine (fortified) and chocolate contained the greatest concentrations of vitamin D2, with smaller amounts found in various meat products. 25(OH)D2 was detected in various foods, including meats, and was quantitated in lamb liver. These data advance knowledge of dietary vitamin D in Australia and highlight the importance of analysis of these four forms of vitamin D to accurately represent the vitamin D content of food.

Discrimination of Milk from Different Animal Species by a Foodomics Approach Based on High-Resolution Mass Spectrometry.

An untargeted foodomics strategy based on ultra-high-performance liquid chromatography coupled with quadrupole orbitrap and chemometrics was used to observe subtle differences in the molecule profiles of raw milk from different animal species (cow milk, goat milk, and water buffalo milk), which could prevent the fraud activities in the dairy industry. In data-dependent acquisition (DIA), spectra for all precursor ions facilitated the comprehensive identification of unknown compounds in untargeted foodomics. Chemometrics techniques were used to analyze large amounts of complex data to observe the separation of different sample groups and find the potential markers of sample groups. Finally, five markers were putatively identified by the potential marker identification workflow. The quantification results showed that ß-carotene was found only in cow milk; ergocalciferol was found only in water buffalo milk; and the contents of nonanoic acid, decanoic acid, and octanoic acid were higher in goat milk than those in cow milk and water buffalo milk. The quantification of ß-carotene enabled the detection of cow milk with a sensitivity threshold of 5% (w/w). This work provided an efficient approach for the discrimination of cow milk, goat milk, and water buffalo milk. Compared with proteomics and genomics, the simpler analytical procedures, lower costs, and higher speed of this work make it of great benefit for routine operations.

[Method Verification for Vitamin D Analysis in Processed Foods Based on the Analytical Manual for the Standard Tables of Food Composition in Japan 2015 (Seventh Revised Edition)].

Considerable amounts of processed foods contain vitamin D (ergocalciferol (D2) and cholecalciferol (D3)) as food additives. For field surveys on food additives, the analytical method for vitamin D should be well-validated. However, the current official method in Japan cannot separately determine the concentrations of D2 and D3, whereas the method for the Standard Tables of Food Composition in Japan 2015 (STFC method) can. Therefore, in this study, we verified the applicability of the STFC method to processed foods. During the course of this research, we added some improvements to the original method. Spike and recovery experiments using vegetable juice, soymilk, and corn flakes as food matrices showed that the recovery rates (relative standard deviation) of D2 and D3 were 103-112% (4.7-12.6%) and 102-109% (2.4-21.8%), respectively, at the estimated method limit of quantification (EMLOQ) level; and 100-110% (4.0-7.4%) and 102-105% (3.8-4.8%), respectively, at 10 times the EMLOQ level. These results indicated that accuracy and precision of the modified STFC method were enough to determine dietary D2 and D3 as endogenous nutrients and/or food additives, and suggested that this method is appropriate for analyzing vitamin D concentrations in processed foods.

[Biotin analytical interference on Siemens immunoanalysis on Dimension EXL®: impact on cardiac troponin I and total vitamin D (D2+D3) assays]. / Étude de l'interférence analytique de la biotine avec les immunoanalyses Siemens sur l'automate Dimension EXL® : impact sur les dosages de troponine I cardiaque et de vitamine D totale (D2+D3).

Several hyperthyroidism misdiagnoses cases have been recently described due to biotin intake. Biotin used in immuno-analysis assays which rely on biotin/streptavidin binding properties. In these assays, high plasmatic biotin levels can lead to major analytical interferences resulting in falsely higher (competition tests) or falsely reduced determinations (for sandwiches assays). We performed a simulation test of biotin intake with patient's samples. We studied the effect of biotin on cardiac troponin I and total vitamin D (D2+D3) assays that are using biotin-streptavidin binding on Dimension EXL®. Increasing doses of biotin were added (28 samples for each parameter) before the assays. The results evidenced a significant negative interference of biotin on cardiac troponin I determinations for concentrations of 100 ng/mL and above, with a total loss of signal for higher biotin additions. Such interference may lead to inappropriate therapeutic decisions. Positive interferences were observed on total vitamin D (D2+D3) with less impact for therapeutic decisions.

Effects of vitamin D2-fortified shiitake mushroom on bioavailability and bone structure.

Bioavailability and bone loss inhibitory effects of vitamin D2 derived from UV-irradiated shiitake mushroom were determined in vivo. The effect of the absence of ovaries on the bioavailability of vitamin D2 and bone structure was also investigated. Sham operated (sham) and ovariectomized (OVX) rats were divided in 3 groups according to their diets, i.e. control: only vitamin D-deficient diets; UV(X): vitamin D-deficient diets with non-irradiated mushroom powder; UV(O): vitamin D-deficient diets with irradiated mushroom powder. The obtained results showed that vitamin D2 from shiitake mushroom was able to increase bone mineral density and trabecular bone structure of femur bone as well as its bioavailability. The absence of estrogen induced adverse effects not only on bioavailability of vitamin D2 but also on trabecular bone. In conclusion, vitamin D2-fortified shiitake mushroom might help postmenopausal women increase vitamin D2 bioavailability and retard trabecular bone loss. Abbreviations: OVX: ovariectomized; 25(OH)D: 25-hydroxyvitamin D; 1,25(OH)2D: 1,25-dihydroxyvitamin D; BMD: bone mineral density; micro-CT: micro computed tomography; RSM: response surface methodology; RP-HPLC: Reverse phase-high performance liquid chromatography; MS/MS: tandem mass spectrometry; E2: estradiol; NTx: N-terminal telopeptide of type I collagen; BV/TV: bone volume/total volume; BS/BV: bone surface/bone volume; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation.

Single-Laboratory Validation of a Method for Determination of Ergocalciferol in Protein Drink Powders and Tablets by LC-MS/MS.

Background: Currently, there is a lack of validation studies available in the literature for the determination of ergocalciferol, especially for those using a direct extraction technique. The current official methodologies for the quantification of ergocalciferol require saponification, liquid-liquid extraction, or both, thus requiring experienced technicians and specialized reflux equipment. This work provides a method that is more easily accessible to laboratories without these resources while still achieving the robustness needed for a successful validation of low levels of ergocalciferol in complex matrixes. Objective: A single-laboratory validation study was conducted for a rapid quantification method of ergocalciferol in protein drink powders and tablets. Methods: The method uses an LC-MS/MS with multimode source utilizing atmospheric pressure chemical ionization positive ionization mode. For both protein drink powders and tablets, the procedure consisted of a liquid extraction step using dimethyl sulfoxide and methanol. Isotopically labeled ergocalciferol was used as an internal standard to correct for signal depression caused by matrix interference. Results: This LC-MS/MS method was found to be accurate, precise, linear (from 0.01 to 0.3 µg/mL), rugged, and suitable for protein drink powders and tablets. Conclusions: The method was validated and is suitable for accurate quantification of ergocalciferol in tablet and protein powder products. Highlights: This work provides a validated method for accurate quantification of ergocalciferol in complex matrixes using a direct extraction technique. This may benefit quality control laboratories in the food and nutraceutical industries, where simple and efficient methodology is key to optimal functioning.

Cocoa and chocolate are sources of vitamin D2.

Cocoa beans are susceptible to fungal contamination and often contain substantial amounts of ergosterol, the precursor to vitamin D2. We hypothesized that sun-drying the fermented cocoa beans might lead to the conversion of ergosterol to vitamin D2. We quantified vitamin D in cocoa and cocoa-based foods by liquid chromatography-tandem mass spectrometry. Here, we show that cocoa beans from different growing regions contain vitamin D2. Particularly high vitamin D2 content was found in cocoa powder and butter. Among the chocolates, dark chocolate had the highest vitamin D2 content (ranging from 1.90 to 5.48 µg/100 g), white chocolate had the lowest vitamin D2 content (ranging from 0.19 to 1.91 µg/100 g), and chocolate nut spreads had a comparatively low vitamin D2 content, with an average of 0.15 µg/100 g. Thus, cocoa and chocolate are clearly a dietary source of vitamin D, therefore, it is necessary to update food composition databases.

Evidence of vitamin D synthesis in insects exposed to UVb light.

Vertebrates obtain the prohormone vitamin D primarily by endogenous cutaneous synthesis under ultraviolet b (UVb) exposure. To date, endogenous synthesis of vitamin D in insects has never been investigated. In an initial experiment, we exposed four insect species which differ in ecology and morphology (migratory locusts, house crickets, yellow mealworms and black soldier fly larvae (BSFL)) to a low irradiance UVb source. In a second experiment we exposed these species to a higher UV irradiance, and in a third we tested the effect of exposure duration on vitamin D concentrations in yellow mealworms. Low irradiance UVb tended to increase vitamin D3 levels in house crickets, vitamin D2 levels in BSFL and vitamin D2 and D3 in yellow mealworms. Higher UVb irradiance increased vitamin D3 levels in all species but BSFL. Both BSFL and migratory locusts had increased vitamin D2 levels. Longer UVb exposure of yellow mealworms increased vitamin D2 and increased vitamin D3 until a plateau was reached at 6400 IU/kg. This study shows that insects can synthesize vitamin D de novo and that the amounts depend on UVb irradiance and exposure duration.

The use of synthetic and natural vitamin D sources in pig diets to improve meat quality and vitamin D content.

This study investigated the effects of synthetic and natural sources of vitamin D biofortification in pig diets on pork vitamin D activity and pork quality. One hundred and twenty pigs (60 male, 60 female) were assigned to one of four dietary treatments for a 55 d feeding period. The dietary treatments were (1)50 µg vitamin D3/kg of feed; (2)50 µg of 25-hydroxvitamin D3/kg of feed (25-OH-D3); (3)50 µg vitamin D2/kg of feed; (4)50 µg vitamin D2-enriched mushrooms/kg of feed (Mushroom D2). The pigs offered the 25-OH-D3 diet exhibited the highest (P < 0.001) serum total 25-hydroxyvitamin D concentration and subsequently exhibited the highest (P < 0.05) Longissimus thoracis (LT) total vitamin D activity. Mushroom D2 and 25-OH-D3 supplementation increased pork antioxidant status. The vitamin D2-enriched mushrooms improved (P < 0.05) pig performance, carcass weight and LT colour. In conclusion, 25-OH-D3 is the most successful source for increasing pork vitamin D activity, while Mushroom D2 may be a new avenue to improve animal performance and pork quality.

Stability of vitamin D3 and vitamin D2 in oil, fish and mushrooms after household cooking.

Information on the retention of vitamin D in food following household cooking is scarce. So far the retention of its metabolites vitamin D3, vitamin D2, and 25-hydroxyvitamin D3 has shown that the type of food and the cooking method are the essential determinants, and there is no significant difference between the metabolites. We investigated the retention of vitamin D3 and vitamin D2 in sunflower oil, vitamin D3 in rainbow trout, and vitamin D2 in button mushrooms. The investigated cooking methods were boiling at different pH, steam cooking, microwave cooking, pan-frying, and oven baking. There was no difference between the retention of vitamin D3 and vitamin D2 added to sunflower oil, which ranged from 70 to 99%. In rainbow trout, the retention of vitamin D3 at 85-114% was not significantly different from 100%, except for panfrying at 85%. However, the retention of vitamin D2 in mushrooms at 62-88% was significantly different from 100% (p ≤ 0.05).

Analysis of Vitamin D2 and Vitamin D3 in Infant and Adult Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: A Multilaboratory Testing Study.

A multilaboratory testing study was conducted on AOAC First Action Method 2016.05 "Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry." Nine laboratories participated in the analysis of duplicate samples of 20 nutritional products. The samples were saponified at high temperature with lipid-soluble components extracted into isooctane; an aliquot was washed and vitamin D derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to form a high-molecular mass, easily ionizable adduct, extracted into acetonitrile and analyzed by reversed-phase LC-tandem MS. Stable isotope-labeled internal standards were used for quantitation to correct for losses in extraction and variation in derivatization and ionization efficiencies. Acceptable precision as RSD was demonstrated; repeatability ranged from 1.9 to 5.8% RSDr and reproducibility values ranged from 6.4 to 12.7% RSDR, with samples meeting the precision limits specified in the vitamin D Standard Method Performance Requirements and the guidelines recommended for the Horwitz ratio. Method accuracy was assessed using NIST 1849a Standard Reference Material, with a P-value of 0.32, indicating an absence of bias against the certified value. As expected, placebo samples not fortified with vitamin D returned negligible results.

Culinary-Medicinal Mushroom Products as a Potential Source of Vitamin D.

The incidence of vitamin D deficiency has increased in recent years, mainly in Europe. The consumption of processed mushrooms may play an important role in preventing diseases associated with vitamin D deficiency. We determined the effects of 2 kinds of freezing (blast, cryogenic), canning (mild and strong brine), and drying (air-drying, freeze-drying) on the retention of vitamin D2 and ergosterol in Agaricus bisporus. Fresh and processed A. bisporus mushrooms can be a good dietary source of vitamin D2. After 12 months of storage, canned mushrooms retained the largest amount of vitamin D2 and ergosterol, whereas the smallest amount was retained in dried mushrooms. Cryogenic freezing resulted in higher levels of vitamin D2, whereas ergosterol levels were higher using air-blast freezing. The drying method had a significant effect only on ergosterol levels, which were higher in the case of freeze-drying. Room temperature gave the best results for storing dried mushrooms. In canned mushrooms, the type of brine had an effect only on levels of vitamin D2; retention was higher using the strong brine. Retention of vitamin D2 was higher at cool temperatures, whereas room temperature resulted in higher retention of ergosterol in the canned products.

Vitamin D2 Stability During the Refrigerated Storage of Ultraviolet B-Treated Cultivated Culinary-Medicinal Mushrooms.

The effects of ultraviolet B (UVB) irradiation on the synthesis of vitamin D2 and its stability during refrigerated storage was determined in fresh cultivated culinary-medicinal mushrooms (Agaricus bisporus, Pleurotus ostreatus, and Lentinus edodes) after harvest. The irradiated mushrooms were stored at 4°C for up to 10 days. The concentrations of vitamin D2 and ergosterol were determined using ultrahigh-performance liquid chromatography/tandem mass spectrometry. The cultivated mushrooms not treated with UVB were devoid of vitamin D2. After UVB irradiation, we obtained mushrooms with a large amount of ergocalciferol. A. bisporus showed the lowest vitamin D2 content (3.55 ± 0.11 µg D2/g dry weight); P. ostreatus contained 58.96 ± 1.15 µg D2/g dry weight, and L. edodes contained 29.46 ± 2.21 µg/g dry weight. During storage at 4°C, the amount of vitamin D2 was gradually decreased in P. ostreatus and L. edodes, whereas in A. bisporus vitamin D2 gradually increased until the sixth day, then decreased. Mushrooms exposed to UVB radiation contain a significant amount of vitamin D2 and are therefore an excellent food source of vitamin D.

Compositional Differences of the Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), under Three Types of Light Conditions.

The focus of this study was to investigate the effect of light on the cultivation and the amounts of bio-active components in Flammulina velutipes. The mushrooms were cultivated under fluorescent tube (T8) grow lights, lightemitting diodes (LEDs), and cold-cathode fluorescent lamps. The biological efficiency of the T8 lights was the highest, at 92%. The crude fat content, crude fiber content, polysaccharide content, and ergosterol content were highest under the LEDs, at 2.9 g/100 g, 7.9 g/100 g, 3.9 g/100 g, and 1.4 mg/g, respectively. Moreover, vitamin D2 (1.9 µg/g) was generated only under light from LEDs. Principal component analysis showed that F. velutipes cultivated under the 3 different lighting conditions showed different profiles for proximate composition, nutritional compounds, and principal fatty acids.

DOES VITAMIN D METABOLITE MEASUREMENT HELP PREDICT 25(OH)D CHANGE FOLLOWING VITAMIN D SUPPLEMENTATION?

OBJECTIVE: Variability in 25-hydroxyvitamin D (25(OH)D) change following vitamin D supplementation exists. Vitamin D metabolite measurement might assist in predicting 25(OH)D response and also contribute to defining vitamin D adequacy. This study assessed utility of vitamin D metabolite measurements to predict 25(OH)D response and explored the relationship between parathyroid hormone (PTH) and a "vitamin D composite index" comprised of the sum of serum 25(OH) D, cholecalciferol (vitamin D3) and 24,25 dihydroxyvitamin D (24,25(OH)2D). METHODS: Sixty-two postmenopausal women were randomized to daily vitamin D3 1,800 IU or placebo for 4 months. Blood was drawn at baseline and after 1 and 4 months. Serum 25(OH)D, vitamin D3, and 24,25(OH)2D were measured by liquid chromatography tandem mass spectroscopy. Free 25(OH)D and PTH were measured by enzyme-linked immunosorbent assay. Repeated measures analysis of variance and regression analyses were performed. RESULTS: Baseline 25(OH)D was positively correlated (P<.05) with vitamin D3, 24,25(OH)2D and free 25(OH) D. Daily vitamin D supplementation increased all metabolites (P<.001). Substantial individual variability in 25(OH) D change at 4 months was observed but was unrelated to baseline vitamin D3, 25(OH)D or 24,25(OH)2D. Only body mass index, body weight, and body fat mass was associated with 25(OH)D change at 4 months. The vitamin D composite score was associated with serum PTH, but this association was similar to that observed with 25(OH) D alone. CONCLUSION: This study does not support measurement of vitamin D metabolites in a composite index to assist in prediction of 25(OH)D response to supplementation. Overweight individuals have less robust 25(OH) D response to supplementation, but variability precludes prediction of the result following daily supplementation. ABBREVIATIONS: BMI = body mass index DXA = dual-energy X-ray absorptiometry LC-MS/MS = liquid chromatography tandem mass spectroscopy 25(OH)D = 25-hydroxyvitamin D 24,25(OH)2D = 24,25 dihydroxyvitamin D PTH = parathyroid hormone vitamin D3 = cholecalciferol.

Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.

A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the vitamin D to form a high-molecular-mass, easily ionizable adduct. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.