Application of ultra-high-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in infant formula and adult/pediatric nutritional formula: First Action 2011.11

In an effort to measure vitamin D, ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was applied to samples. The use of UHPLC-MS/MS decreased the run time by 50%. The UHPLC-MS/MS achieved equal or better separation efficiency with complex food matrixes compared to HPLC-MS/MS. It was also observed that under the optimized conditions of UHPLC, all previtamins of vitamin D3, D2, and isotope-labeled vitamin D3 were baseline-separated from their corresponding vitamins. The sterol isomers found in complex food matrixes that interfere in the analysis were well separated from the analytes. The accuracy of the method was evaluated by analyzing National Institute of Standards and Technology Standard Reference Material 1849 infant reference material. The average vitamin D3 concentration was 0.251 +/- 0.012 microg/g. This showed excellent agreement with the certified value of 0.251 +/- 0.027 microg/g. The spike recovery study of a commercial infant formula matrix showed a range of recovery from 100 to 108%. The LOQ values determined were 0.0022 and 0.0028 microg/g for vitamins D3 and D2, respectively; LOD values were 0.00065 and 0.00083 microg/g for vitamins D3 and D2, respectively.     

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.     

The vitamin D3 pathway in human skin and its role for regulation of biological processes

The skin is the only tissue yet known in which the complete ultraviolet-B (UV-B)-induced pathway from 7-dehydrocholesterol to hormonally active calcitriol (1alpha,25-dihydroxyvitamin D(3)) occurs under physiological conditions. Epidermal synthesis of calcitriol could be of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and immunocompetent cells. Because of their antiproliferative and prodifferentiating effects, calcitriol and other vitamin D analogs are highly efficient in the treatment of psoriasis vulgaris. The known antipsoriatic effect of UV-B light could, at least in part, be mediated via UV-B-induced synthesis of calcitriol. In addition, mounting evidence indicates that cutaneous vitamin D(3) synthesis is of high importance for the prevention of a broad variety of diseases, including various malignancies. New but controversially discussed sun-protection guidelines were established for the prevention of internal cancers. A better understanding of the metabolism of vitamin D in the skin opens new perspectives for therapeutic applications of vitamin D analogs.     

UVB-induced conversion of 7-dehydrocholesterol to 1 alpha,25-dihydroxyvitamin D3 (calcitriol) in the human keratinocyte line HaCaT

We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D3 (7-dehydrocholesterol, [7-DHC]) which results in the formation of vitamin D3 also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 microM) at 37 degrees C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5-60 mJ/cm2), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125-1500 mJ/cm2). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin D3 in our in vitro model followed by time-dependent isomerization to vitamin D3 and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths > 315 nm generated no or only trace amounts of calcitriol. The conversion of vitamin D3 generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cm2). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.     

Determination of cholecalciferol (vitamin D3) in selected foods by liquid chromatography: NMKL collaborative study

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 microg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSD(R)) values varied between 6.8% (margarine) and 24% (cooking oil).     

新型碳苷糖类维生素D_2衍生物的合成

以D-葡萄糖为原料,经碳苷化反应,酰化反应和脯氨酸-DIPEA催化的aldol反应制得2个碳苷糖[1-(4'-羟基苯基)-4-C-β-四乙酰基葡萄糖基-3-烯-2-酮(5a)和1-(3-羟基苯基)-4-C-β-四乙酰基葡萄糖基-3-烯-2-酮(5b)];5与琥珀酸维生素D2经Steglich酯化反应合成了2个新型碳苷糖类维生素D2衍生物,其结构经1H NMR,13C NMR和HR-ESI-MS表征。     

Provitamin D and vitamin D isomerizations in the mass spectrometer. Translational energy release and collision-induced dissociation studies

Collision-induced dissociation mass-analysed ion kinetic energy spectrometry and translational energy release studies have established that provitamin D₃ (7-dehydrocholesterol), provitamin D₂ (ergosterol), vitamin D₃ and vitamin D₂ isomerizations in the mass spectrometer occur at the fragment ion level leading to [M? H₂O? CH₃]⁺ ions of identical structure. It was found that the reaction, [M]⁺˙→[M? H₂O]⁺˙, plays a central role in these isomerizations.     

固相萃取-高效液相色谱法测定钙强化食品中的维生素D

以含有体积分数为20%的0.95 mol/L柠檬酸水溶液的二甲基亚砜作为维生素D的破壁溶液,利用Chromabond XTR固相萃取柱(14500 mg, 70 mL)对样品进行提取和净化,建立了测定钙强化食品中维生素D的固相萃取-高效液相色谱方法。方法的线性范围为0.1～100.0 μg/mL,线性相关系数为0.999。方法的定性检出限为0.01 μg/g,定量检出限为0.03 μg/g。低(0.1 μg/g)、中(0.5 μg/g)、高(1.0 μg/g)三个浓度水平的加标回收率分别为106.2%,99.5%和100.1%,相对标准偏差小于10%。     

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.     

2 alpha-(3-hydroxypropyl)- and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 accessible to vitamin D receptor mutant related to hereditary vitamin D-resistant rickets

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor, which lead to resistance to 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. We found that the A ring-modified analogues, 2alpha-(3-hydroxypropyl)- and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)(2)D(3), (O1C3 and O2C3) can bind better than the natural hormone to the mutant VDR (R274A), which similar to the HVDRR mutant, R274L, had lost the hydrogen bond to the 1alpha-hydroxyl group of 1alpha,25(OH)(2)D(3).     

Synthesis of (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3: compounds to probe vitamin D conformation in receptor complex by 19F-NMR

To study the interaction of vitamin D with its receptor by 19F-NMR, (5Z,10Z)- and (5Z,10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 were synthesized starting from vitamin D2 via electrophilic fluorination of vitamin D-SO2 adducts as the key step. Regio- and stereoselective electrophilic fluorination at C(19) of vitamin D-SO2 adducts was achieved under the conditions using (PhSO2)2NF and bulky bases. The stereochemistry of the addition and elimination of SO2 of various vitamin D derivatives was studied in detail. SO2 causes Z-E isomerization of the 5,6-double bond of vitamin D and adds to the resulting (5E)-isomer from the sterically less hindered side opposite to the substituent at C(1). Elimination of SO2 from 19-substituted vitamin D-SO2 adducts proceeded exclusively in a suprafacial manner with respect to the diene part under either thermal or reductive conditions. Dye-sensitized photochemical isomerization of 19-fluorovitamin D derivatives was studied in detail. The rapid isomerization at the 5,6-double bond was followed by the slow isomerization at the 10,19-double bond to yield the (5E,10Z)-isomer (by nomenclature of the 1-OH derivatives) as the major product. (10Z)- and (10E)-19-Fluorovitamin Ds were also interconverted thermally probably via the corresponding previtamin D by 1,7-sigmatropic isomerization.     

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.     

A concise and efficient route to 2alpha-(omega-hydroxyalkoxy)-1alpha,25-dihydroxyvi tam in D3: remarkably high affinity to vitamin D receptor

[reaction: see text]A convenient and potentially valuable synthetic approach to the novel 2alpha-functionalized 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] derivatives (1a-c), which are the C2-epimer of ED-71 and its analogues, has been developed. The C2alpha-modified ring A precursors (1,7-enynes 16, n = 0, 1, and 2) were constructed stereoselectively starting from D-glucose in high yield. In the synthesized 2alpha-(omega-hydroxyalkoxy)-1alpha,25(OH)2D3 derivatives, 1a and 1b showed a greater binding affinity to vitamin D receptor (VDR), up to 1.8 times that of the native hormone.     

A novel anti-HBeAg homolignan, taiwanschirin D from Kadsura matsudai

A novel C19 homolignan, taiwanschirin D (1), possessing a 3,4-(1-[(Z)-2-methoxy-2-oxoethylidenel)pentano (2,3-dihydrobenzo[b]furan)-3(2-oxoacetate) skeleton, was isolated from the stem of Kadsura matsudai Hayata. Its structure was determined from physical and spectral data including 2D NMR spectra. The Anti-HBeAg test revealed that taiwanschirin D (1) had moderate activity at a concentration of 94.3 microM (50 microg/ml).     

Sunbeds as Vitamin D Sources

The objectives of this work were: (1) To determine whether repeated exposures to small doses from a commercial sun bed (Wolff Solarium Super Plus 100 W) over 5 weeks gave less vitamin D than repeated exposures to twice as large, but still nonerythemogenic, doses. (2) To investigate whether the contribution to the vitamin D status from such sessions of exposures was dependent on the baseline status before the start of the sessions. (3) To determine the decay rate of the induced increment of vitamin D. The sun bed sessions raised the 25-hydroxyvitamin D levels from typical winter values to typical summer values. The mean value after exposure being 80 n m (±14) and the increase being 15 n m on average. Persons with the lowest initial levels got the largest increase. The level in this group was back to the pre-exposure level after 2–4 weeks. To maintain a summer level through the winter, when no vitamin D is produced by the sun in northern countries, one should consider increasing the recommended intake of vitamin D intake significantly, or encouraging the population to get moderate, nonerythemal sun bed exposures.     

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.     

Synthesis of vitamin D(3) and calcitriol dimers as potential chemical inducers of vitamin D receptor dimerization

The design and synthesis of vitamin D(3) dimers 2 and 3 and 1 alpha, 25-dihydroxyvitamin D(3) (calcitriol) dimers 4 and 5 are described. The dimers were designed with a view to doubly binding the vitamin D receptor (VDR) and inducing the receptor homodimerization. In the dimers the units are linked through the C-11 position in ring C by an alkyl side chain of six or 10 carbon atoms, far from the hydroxy groups responsible for the VDR binding. The linker is formed by olefin metathesis of an olefinic side chain at the C-11 position introduced by stereoselective cuprate addition. The synthesis, which is both short and convergent, uses the Wittig-Horner approach to construct the vitamin D triene system and allows the preparation of dimers with a linker of modulated length with the purpose of optimizing the vitamin D(3)-VDR interaction.     

Alpha-ribazole, a fluorescent marker for the liquid chromatographic determination of vitamin B12 in foodstuffs

A method to determine the contents of free vitamin B12 in various foods by reversed phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by passage through an immunoaffinity column and a pre-column conversion of vitamin B12 into the fluorescent alpha-ribazole (successive treatments of the extract with 2.5 M sodium hydroxide (at 100 degrees C for 15 min) and alkaline phosphatase (7.5 U) at 37 degrees C and pH 8 for 16 h). An enzymatic hydrolysis prior to the purification step (pepsin at 37 degrees C and pH 4 for 3 h) made it possible to release the vitamin B12 bound to proteins and thus to obtain the total vitamin B12 contents of these foodstuffs. The method proposed for the determination of free and bound vitamin B12 gives a good recovery rate (95-100%) and a satisfactory repeatability (R.S.D.r between 1.0 and 5.4%). Owing to its low quantification limit (3 ng g(-1)) and the good resolution of the alpha-ribazole peak, it could most probably be applied to the determination of this vitamin in any foodstuff.     

Solar radiation, vitamin D and survival rate of colon cancer in Norway

Solar radiation contributes significantly to the status of serum calcidiol (25-hydroxyvitamin D3, 25-(OH)D3) in humans, even at the high latitudes of northern Norway. Thus, in late summer the serum concentration of calcidiol is roughly 50% larger than that in late winter, when the solar radiation in Norway contains too little ultraviolet radiation to induce any synthesis of vitamin D3 in human skin. This seems to influence the prognosis of colon cancer. We here report that the survival rate of colon cancer in men and women, assessed 18 months after diagnosis, is dependent on the season of diagnosis. A high serum concentration of calcidiol at the time of diagnosis, i.e. at the start of conventional therapy, seems to give an increased survival rate. This agrees with cell and animal experiments reported in the literature, as well as with epidemiological data from some countries relating colon cancer survival with latitude and vitamin D3 synthesis in skin. One possible interpretation of the present data is that, the level of calcidiol, or its derivative calcitriol (1alpha,25-dihydroxyvitamin D3, 1alpha,25-(OH)2D3), may act positively in concert with conventional therapies of colon cancer.