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.

[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.

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.

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.

Effects of vitamin D2-fortified bread v. supplementation with vitamin D2 or D3 on serum 25-hydroxyvitamin D metabolites: an 8-week randomised-controlled trial in young adult Finnish women.

There is a need for food-based solutions for preventing vitamin D deficiency. Vitamin D3 (D3) is mainly used in fortified food products, although the production of vitamin D2 (D2) is more cost-effective, and thus may hold opportunities. We investigated the bioavailability of D2 from UV-irradiated yeast present in bread in an 8-week randomised-controlled trial in healthy 20-37-year-old women (n 33) in Helsinki (60°N) during winter (February-April) 2014. Four study groups were given different study products (placebo pill and regular bread=0 µg D2 or D3/d; D2 supplement and regular bread=25 µg D2/d; D3 supplement and regular bread=25 µg D3/d; and placebo pill and D2-biofortified bread=25 µg D2/d). Serum 25-hydroxyvitamin D2 (S-25(OH)D2) and serum 25-hydroxyvitamin D3 (S-25(OH)D3) concentrations were measured at baseline, midpoint and end point. The mean baseline total serum 25-hydroxyvitamin D (S-25(OH)D=S-25(OH)D2+S-25(OH)D3) concentration was 65·1 nmol/l. In repeated-measures ANCOVA (adjusted for baseline S-25(OH)D as total/D2/D3), D2-bread did not affect total S-25(OH)D (P=0·707) or S-25(OH)D3 (P=0·490), but increased S-25(OH)D2 compared with placebo (P<0·001). However, the D2 supplement was more effective than bread in increasing S-25(OH)D2 (P<0·001). Both D2 and D3 supplementation increased total S-25(OH)D compared with placebo (P=0·030 and P=0·001, respectively), but D2 supplementation resulted in lower S-25(OH)D3 (P<0·001). Thus, D2 from UV-irradiated yeast in bread was not bioavailable in humans. Our results support the evidence that D2 is less potent in increasing total S-25(OH)D concentrations than D3, also indicating a decrease in the percentage contribution of S-25(OH)D3 to the total vitamin D pool.

Impact on Vitamin D2, Vitamin D4 and Agaritine in Agaricus bisporus Mushrooms after Artificial and Natural Solar UV Light Exposure.

Commercial mushroom production can expose mushrooms post-harvest to UV light for purposes of vitamin D2 enrichment by converting the naturally occurring provitamin D2 (ergosterol). The objectives of the present study were to artificially simulate solar UV-B doses occurring naturally in Central Europe and to investigate vitamin D2 and vitamin D4 production in sliced Agaricus bisporus (button mushrooms) and to analyse and compare the agaritine content of naturally and artificially UV-irradiated mushrooms. Agaritine was measured for safety aspects even though there is no rationale for a link between UV light exposure and agaritine content. The artificial UV-B dose of 0.53 J/cm(2) raised the vitamin D2 content to significantly (P < 0.001) higher levels of 67.1 ± 9.9 µg/g dry weight (DW) than sun exposure (3.9 ± 0.8 µg/g dry DW). We observed a positive correlation between vitamin D4 and vitamin D2 production (r(2) = 0.96, P < 0.001) after artificial UV irradiation, with vitamin D4 levels ranging from 0 to 20.9 µg/g DW. The agaritine content varied widely but remained within normal ranges in all samples. Irrespective of the irradiation source, agaritine dropped dramatically in conjunction with all UV-B doses both artificial and natural solar, probably due to its known instability. The biological action of vitamin D from UV-exposed mushrooms reflects the activity of these two major vitamin D analogues (D2, D4). Vitamin D4 should be analysed and agaritine disregarded in future studies of UV-exposed mushrooms.

Determination of vitamins D2 and D3 in selected food matrices by online high-performance liquid chromatography-gas chromatography-mass spectrometry (HPLC-GC-MS).

An online normal-phase liquid chromatography-gas chromatography-mass spectrometry (HPLC-GC-MS) method was developed for the determination of vitamins D2 and D3 in selected food matrices. Transfer of the sample from HPLC to GC was realized by large volume on-column injection; detection was performed with a time-of-flight mass spectrometer (TOF-MS). Typical GC problems in the determination of vitamin D such as sample degradation or sensitivity issues, previously reported in the literature, were not observed. Determination of total vitamin D content was done by quantitation of its pyro isomer based on an isotopically labelled internal standard (ISTD). Extracted ion traces of analyte and ISTD showed cross-contribution, but non-linearity of the calibration curve was not determined inside the chosen calibration range by selection of appropriate quantifier ions. Absolute limits of detection (LOD) and quantitation (LOQ) for vitamins D2 and D3 were calculated as approximately 50 and 150 pg, respectively. Repeatability with internal standard correction was below 2 %. Good agreement between quantitative results of an established high-performance liquid chromatography with UV detection (HPLC-UV) method and HPLC-GC-MS was found. Sterol-enriched margarine was subjected to HPLC-GC-MS and HPLC-MS/MS for comparison, because HPLC-UV showed strong matrix interferences. HPLC-GC-MS produced comparable results with less manual sample cleanup. In summary, online hyphenation of HPLC and GC allowed a minimization in manual sample preparation with an increase of sample throughput.

Sensitive method for the determination of paricalcitol by liquid chromatography and mass spectrometry and its application to a clinical pharmacokinetic study.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of paricalcitol (PAR) in human plasma (500 µL) using paricalcitol-d6 (PAR-d6 ) as an internal standard (IS) as per regulatory guidelines. A liquid-liquid extraction method was used to extract the analyte and IS from human plasma. Chromatography was achieved on Zorbax SB C18 column using an isocratic mobile phase in a gradient flow. The total chromatographic run time was 6.0 min and the elution of PAR and PAR-d6 occurred at ~2.6 min. A linear response function was established for the range of concentrations 10-500 pg/mL in human plasma. The intra- and inter-day accuracy and precision values for PAR met the acceptance criteria. The validated assay was applied to quantitate PAR concentrations in human plasma following oral administration of 4 µg capsules to humans.

Mechanism of error caused by isotope-labeled internal standard: accurate method for simultaneous measurement of vitamin D and pre-vitamin D by liquid chromatography/tandem mass spectrometry.

RATIONALE: Bias of up to 25% has been observed for vitamin D3 and D2 samples exposed to heating during sample preparation, even when isotope-labeled internal standards are used. The goals of this study were to identify the mechanism of the positive bias observed in measuring vitamin D3 and D2 by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and determine a way to eliminate the error source. METHODS: Several internal standards with varying locations of labeling were used for comparison in this study. Additionally, different temperatures (25, 37, 55, and 75 °C) and different treatment times were investigated for sample preparation and a LC/MS/MS method capable of simultaneously measuring vitamin D and pre-vitamin D was developed. RESULTS: It was demonstrated that the different conversion behaviors of the analyte and the internal standard were the cause of the positive bias. This bias was eliminated when internal standards with labeling remote from the double-bond area of the molecules were used. Additionally, sample preparation was shortened from overnight saponification at room temperature to 0.5 h at 75 °C. CONCLUSIONS: The use of an internal standard with labeling remote from the conjugated area eliminated the error source and gave accurate correction at all of the temperatures investigated. Heating may be used for rapid sample preparation as an alternative to overnight saponification at room temperature. This work not only describes the mechanism of an inaccurate internal standard correction, but also establishes a rapid LC/MS/MS method for simultaneous measurement of vitamin D and pre-vitamin D.

Dietary vitamin D2--a potentially underestimated contributor to vitamin D nutritional status of adults?

It has been suggested that vitamin D2 is not very prevalent in the human food chain. However, data from a number of recent intervention studies suggest that the majority of subjects had measurable serum 25-hydroxyvitamin D2 (25(OH)D2) concentrations. Serum 25(OH)D2, unlike 25(OH)D3, is not directly influenced by exposure of skin to sun and thus has dietary origins; however, quantifying dietary vitamin D2 is difficult due to the limitations of food composition data. Therefore, the present study aimed to characterise serum 25(OH)D2 concentrations in the participants of the National Adult Nutrition Survey (NANS) in Ireland, and to use these serum concentrations to estimate the intake of vitamin D2 using a mathematical modelling approach. Serum 25(OH)D2 concentration was measured by a liquid chromatography-tandem MS method, and information on diet as well as subject characteristics was obtained from the NANS. Of these participants, 78.7 % (n 884) had serum 25(OH)D2 concentrations above the limit of quantification, and the mean, maximum, 10th, 50th (median) and 90th percentile values of serum 25(OH)D2 concentrations were 3.69, 27.6, 1.71, 2.96 and 6.36 nmol/l, respectively. To approximate the intake of vitamin D2 from these serum 25(OH)D2 concentrations, we used recently published data on the relationship between vitamin D intake and the responses of serum 25(OH)D concentrations. The projected 5th to 95th percentile intakes of vitamin D2 for adults were in the range of 0.9-1.2 and 5-6 µg/d, respectively, and the median intake ranged from 1.7 to 2.3 µg/d. In conclusion, the present data demonstrate that 25(OH)D2 concentrations are present in the sera of adults from this nationally representative sample. Vitamin D2 may have an impact on nutritional adequacy at a population level and thus warrants further investigation.

Extract-filter-shoot liquid chromatography with mass spectrometry for the analysis of vitamin D2 in a powdered supplement capsule and standard reference material 3280.

An "extract-filter-shoot" method for the analysis of vitamin D2, ergocalciferol, in a dry powdered dietary supplement capsule containing rice flour excipient and in a National Institute of Standards and Technology standard reference material 3280 is reported. Quantification of vitamin D2 was done by atmospheric pressure chemical ionization mass spectrometry using selected ion monitoring, two transitions of selected reaction monitoring, and extracted ion chromatograms from full scans. UV detection was used for the quantification of Vitamin D2 in the dry powder capsule, whereas interfering species rendered UV detection unreliable for standard reference material 3280. Average values for standard reference material 3280 ranged from 8.27 ± 0.58 to 8.33 ± 0.57 µg/g using internal standard calibration and response factor approaches, compared to the previous National Institute of Standards and Technology internal value for vitamin D2 of 8.78 ± 0.11 µg/g, and the recently updated reference value of 8.6 ± 2.6 µg/g. The powdered supplement capsule was found to contain 28.19 ± 0.35 to 28.67 ± 0.90 µg/capsule for a capsule labeled to contain 25.00 µg. The triacylglycerol composition of the rice flour excipient in the powdered supplement capsule determined by atmospheric pressure chemical ionization mass spectrometry is also reported.

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.

Triple quadrupole liquid chromatography-mass spectrometry-mediated evaluation of vitamin D2 accumulation potential, antioxidant capacities, and total polyphenol content of white jelly mushroom (Tremella fuciformis Berk.).

Tremella fuciformis Berk. (TF), or the white jelly mushroom, is well known for its myriad of pharmacological properties, such as immunomodulatory, anti-inflammatory, antidiabetic, antitumor, and antioxidant activities, and hypocholesterolemic and hepatoprotective effects that boost human health. Most of the studies of TF are concentrated on its polysaccharide (glucuronoxylomannan) composition, which is responsible for its pharmacological as well as rheological properties. It is well established that mushrooms are a great source of dietary vitamin D due to the presence of ergosterol in their cell membrane. There is a lack of published data on TF as a source of vitamin D2. Therefore, this study aimed to evaluate the vitamin D2 composition of the fruiting bodies of TF using triple quadrupole liquid chromatography-mass spectrometry (LC-MS/QQQ). The results showed highest vitamin D2 content (292.02 µg/g dry weight) in the sample irradiated with ultraviolet B (UVB; 310 nm) for 180 min as compared with the control group (52.47 µg/g dry weight) (P ≤ 0.001). The results showed higher accumulation potential of vitamin D2 in TF as compared with published data available for other extensively studied culinary mushrooms, such as Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Cordiceps militaris, and Calocybe indica. Moreover, the impact of UV treatment on antioxidant capacities and total polyphenol content of TF was also studied. The accumulation potential of vitamin D in TF reveals a novel commercial source for this nutrient.

Simultaneous determination of vitamins D2 and D3 by electrospray ionization LC/MS/MS in infant formula and adult nutritionals: First Action 2012.11.

A method was developed for the analysis of vitamins D2 and D3 in a variety of nutritional products. To extract vitamins D2 and D3 from products containing substantial amounts of fat, a saponification with alcoholic potassium hydroxide is required to release the vitamin D. Trideuterium-labeled vitamin D is added to the sample prior to saponification, and quantitation is achieved using linear regression of the ratio of peak response for 2H3-D and vitamin D. Acceptable linearity was achieved between 0.6 and 27 microg/100 g with a correlation requirement of >0.999. The method detection limit of 0.02 microg/100 g was verified by spiking placebo products carried through the saponification and extraction steps of the method. At the quantitation limit (0.12 microg/100 g), the signal was easily distinguished from the background. Vitamin D3 spike recoveries ranged from 107 to 119% at the low level and 104 to 116% at the high-level spike. Vitamin D2 recoveries were 105 to 116% and 91 to 110% for the low- and high-level spikes, respectively. SRM 1849a has a certified concentration of 11.1 +/- 1.7 microg/100 g; using this standard reference material, the range of 9.4 to 12.8 microg/100 g was met on each of the 6 days. Method repeatability, determined in 12 vitamin D3 product matrixes over 6 days, ranged from 3.9 to 48%. The adult nutrition-milk protein sample was the most notable; it failed within-day, as well as day-to-day, precision requirements. There was no attempt to optimize the sample preparation to accommodate any problem matrix.

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.

Determination of vitamins D2 and D3 in infant formula and adult nutritionals by ultra-pressure liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS): First Action 2011.12.

The method for the "Determination of Vitamins D2 and D3 in Infant Formula and Adult Nutritionals by Ultra-Pressure Liquid Chromatography with Tandem Mass Spectrometry Detection (UPLC-MS/MS)" was adopted as AOAC Official First Action during the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held June 29, 2011. During the meeting, an Expert Review Panel (ERP) evaluated the available validation information against standard method performance requirements (SMPRs) articulated by stakeholders. The method, approved by the ERP, is applicable for the determination of vitamin D (total vitamins D2 and D3). A range of products had been tested during a single-laboratory validation study. The products included butter, National Institute of Standards and Technology SRM 1849, eggs, cheese, yogurt, ready-to-eat cereal, bread, mushrooms, and tuna. The testing of the method established linearity in the range of 0.005-50 microg/mL. The recovery range was 93.4-100.9% for vitamin D2 and 102.4-106.2% for vitamin D3. The LOD and LOQ for vitamin D2 were reported as 0.20 and 0.61 microgl100 g, respectively; for vitamin D3, the reported values were 0.47 and 1.44 microg/100 g, respectively. The method met the SMPRs set by the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). It was, therefore, decided that the method was appropriate for Official First Action Method status.

Simultaneous determination of vitamins D2 and D3 by LC-MS/MS in infant formula and adult nutritionals: First Action 2011.13.

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held on June 29, 2011, an Expert Review Panel (ERP) on behalf of AOAC INTERNATIONAL adopted the method "Simultaneous Determination of Vitamins D2 and D3 by LC-MS/MS in Infant Formula and Adult Nutritionals" as an AOAC Official First Action method. Vitamins D2 and D3 are extracted from the sample using pentane-ether; the extract is collected and dried under nitrogen. Vitamin D is separated from interfering compounds using UPLC, and quantitated using tandem mass spectrometry (MS/MS). Preliminary data showed the intermediate precision ranged from 3.34-8.05% and an accuracy range of 98.5-111% over the samples tested for vitamin D3. For vitamin D2, the intermediate precision ranged from 2.37-5.45% and accuracy ranged from 96.4-104% over the four matrixes evaluated. The analytical range for the method is bounded by the concentrations of the working standards, 21-270 ng/mL, and is equivalent to 0.168-2.16 mcg/100 g in ready-to-feed product. The practical method quantitation limit is 0.168 mcg/100 g product with method detection limit of 60 ng/100 g product. The ERP reviewed the data and determined that the performance characteristics of the method met the standard method performance requirements, and therefore the method was granted First Action status.

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.

Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2 , vitamin D3 , 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.

Application of ultra-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in foods and nutritional supplements.

Ultra-performance liquid chromatography (UPLC)/MS/MS was applied to measure vitamin D in various foods and nutritional supplements. The run-time of the chromatographic separation was cut from 20 min in HPLC/MS/MS to 10 min in UPLC/MS/MS, while equal or better separation efficiency was achieved to deal with complex food matrixes. Under the optimized conditions, all the previtamins of vitamin D3, D2, and isotope-labeled vitamin D3 were baseline-separated from their corresponding vitamins. It was also demonstrated that many sterol isomers in complex food matrixes that interfere in the analysis could be well-separated from the analytes. Accuracy of this method was evaluated by analysis of NIST SRM 1849 infant formula reference material. With eight replicates, the average vitamin D3 concentration was 0.251 +/- 0.012 mg/kg, an excellent agreement with the certified value of 0.251 +/- 0.027 mg/kg. In addition, spike recovery from a commercial infant formula matrix was in the range of 100 to 108% for both vitamins D3 and D2 at three spike concentration levels. The spike recovery for an even more complex matrix, pet food, was 101-105%. LOQ values were 0.026 and 0.033 IU/g, or 0.086 and 0.11 IU/mL in solution, for vitamins D3 and D2, respectively. The dynamic range had three orders of magnitude, which made the method flexible and useful to deal with the wide concentration range of vitamin D in various samples. The method was robust based on the results of changing the parameters of LC separation and MS measurement. This accurate and reliable vitamin D method increased instrument efficiency and analysis productivity significantly.