Automation der Bestimmung von Vitamin D in Serum und Plasma

AbstractVitamins play an important role in many processes in the human organism. The detection of insufficient supply of vitamins is therefore of particular importance to avoid significant effects for human health. An increasing number of tests is only possible with suitable automated procedures. For the determination of vitamin D3 and vitamin D2 in serum samples, three methods were automated and compared with regard to their performance. All three methods enable reliable detection of 25(OH)D2 and 25(OH)D3 in serum in the ng/ml range.

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Reversed-Phase Liquid Chromatographic Determination of Vitamin D in Infant Formulas and Enteral Nutritionals

Journal of AOAC International ◽

10.1093/jaoac/75.3.566 ◽

1992 ◽

Vol 75 (3) ◽

pp. 566-571 ◽

Cited By ~ 17

Author(s):

Matthew G Sliva ◽

Astor E Green ◽

James K Sanders ◽

John R Euber ◽

Janice R Saucerman

Keyword(s):

Vitamin D ◽

Vitamin D3 ◽

Reversed Phase ◽

Uv Detection ◽

Acetonitrile Solution ◽

Extraction Column ◽

Vitamin D2 ◽

Infant Formulas ◽

Phase Liquid

Abstract Vitamin D In infant formulas and enteral nutritional products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample Is saponified 30 mln at 60°C and extracted Into 60 mL hexane. The hexane layer is then washed and evaporated to dryness. The sample Is reconstituted and added to a 3 mL silica solid-phase extraction column. Vitamins D2 and D3 are eluted from the column with 7 mL methylene chlorlde-lsopropanol mixture (99.8 + 0.2). The eluant is evaporated to dryness and reconstituted In 1 mLacetonltrlle. The acetonitrile solution is analyzed on a C18 reversed-phase LC column (25 cm x 4.6 mm, 5 μm particle size) with UV detection at 265 nm. Linearity for this method between 8 and 2600 lU/qt has shown a coefficient of determination of 1.000, with method precision ranging from 1 to 6%. Spike recoveries gave a mean of 99.1%. Because this method can quantitate and distinguish between vitamin D2 and vitamin D3 In products, vitamin D2 is used as an Internal standard In quantltatlng vitamin D3, and vice versa. The method Is applicable to milk-, soy-, and protein hydrolysate-based infant formulas and enteral nutritional products, both liquid and powder. The sample throughput is estimated to be 24 per day. An AOAC collaborative study of this method is recommended.

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

Molecules ◽

10.3390/molecules26175182 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5182

Author(s):

Amir Sohail ◽

Asma Al Menhali ◽

Soleiman Hisaindee ◽

Iltaf Shah

Keyword(s):

Vitamin D ◽

Vitamin D3 ◽

Uv Light ◽

Mouse Serum ◽

Vitamin D2 ◽

Vitamin D Metabolites ◽

Serum Samples ◽

Deficient Diet ◽

Ms Analysis ◽

Significant Difference

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.

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Liquid Chromatographic Determination of Vitamin D in Milk and Infant Formula

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/68.2.177 ◽

1985 ◽

Vol 68 (2) ◽

pp. 177-182

Author(s):

David C Sertl ◽

Bruce E Molitor

Keyword(s):

Vitamin D ◽

Infant Formula ◽

Vitamin D3 ◽

Chromatographic Determination ◽

The United States ◽

Vitamin D2 ◽

Relative Standard ◽

Liquid Chromatographic Determination ◽

Liquid Chromatographic

Abstract Vitamin D2 or vitamin D3 is determined by liquid chromatography (LC) in milk and infant formula. Vitamin D is extracted from the saponified sample, passed through an amino-cyano LC cleanup column to remove major interferences, and quantitated using normal phase LC. Within-day precision is 4.5% relative standard deviation (RSD); the overall method RSD (reflecting technician-to-technician, day-today, and within-day variability) is 7.7%. Overspike recoveries averaged 97% for milk, 98% for milk-based infant formula, and 93% for soy-based infant formula. The performance of the method is compared with that of the official AOAC vitamin D method (rat bioassay). The method is applicable to the determination of vitamin D in milk and in the major milk- and soy-based infant formulas available in the United States. The method can quantitate (but not distinguish) either vitamin D2 or vitamin D3. The method is applicable to milk and infant formula samples containing between 100 and 1500 IU vitamin D/L. Sample throughput is between 4 and 8 replicates per da

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Metabolism of vitamin D in patients with primary biliary cirrhosis and alcoholic liver disease

Clinical Science ◽

10.1042/cs0690561 ◽

1985 ◽

Vol 69 (5) ◽

pp. 561-570 ◽

Cited By ~ 42

Author(s):

E. Barbara Mawer ◽

H. J. Klass ◽

T. W. Warnes ◽

Jacqueline L. Berry

Keyword(s):

Vitamin D ◽

Liver Disease ◽

Primary Biliary Cirrhosis ◽

Alcoholic Liver Disease ◽

Vitamin D3 ◽

Control Group ◽

Biliary Cirrhosis ◽

Vitamin D2 ◽

Dihydroxyvitamin D3 ◽

Poor Renal Function

1. The metabolism of isotopically labelled vitamin D2 and D3 has been investigated in eight patients with primary biliary cirrhosis and in five controls. The concentration of labelled vitamin D2 was lower than that of vitamin D3 in serum of patients with primary biliary cirrhosis on days 1 and 2 after intravenous injection (P < 0.005 and P < 0.05, respectively) but no difference was seen in controls. 2. Similar amounts of labelled 25-hydroxyvitamin D2 and D3 were seen in serum of the control group; the same pattern was observed in the primary biliary cirrhosis group, and no significant differences were observed between the two groups. 3. In both control and primary biliary cirrhosis groups, the serum concentration of labelled 24,25-dihydroxyvitamin D2 exceeded that of 24,25-dihydroxyvitamin D3 (significant for controls on day 2, P < 0.02) but concentrations in the two groups were not different. 4. Concentrations of labelled 25,26-dihydroxyvitamin D3 were significantly higher than those of 25,26-dihydroxyvitamin D2 in the primary biliary cirrhosis group at all times and in the control group on days 2 and 3. Both 25,26-dihydroxyvitamin D2 and D3 were higher in the serum of patients with primary biliary cirrhosis than in controls (significant on day 1, P < 0.05). 5. Urinary excretion over days 0–3 of radioactivity from both vitamins D2 and D3 was significantly higher in the primary biliary cirrhosis group than in controls: 12.03 vs 1.80% for vitamin D2 and 8.98 vs 1.76% for vitamin D3(P < 0.005). Vitamin D2-derived urinary radioactivity in primary biliary cirrhosis correlated strongly with serum bilirubin (P = 0.005). 6. The metabolism of labelled vitamin D3 was studied in seven patients with alcoholic liver disease, three of whom showed low serum concentrations of labelled 25-hydroxyvitamin D3 suggesting impaired hepatic synthesis. The 25-hydroxylation response was quantified as the relative index of 25-hydroxylation and was significantly related to two other indices of liver function. It is concluded that impaired 25-hydroxylation of vitamin D may occur in alcoholic liver disease and results from hepatocellular dysfunction. 7. Less than the predicted amounts of 1,25-dihydroxyvitamin D3 were produced in four of the seven patients with alcoholic liver disease; this defect may be attributable in part to decreased precursor 25-hydroxyvitamin D and to poor renal function.

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Analysis of Fat-Soluble Vitamins. XX. Determination of Vitamin D in Multivitamin Preparations. Second Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/61.3.735 ◽

1978 ◽

Vol 61 (3) ◽

pp. 735-745

Author(s):

Ellen J De Vries ◽

Frits J Mulder ◽

Ben Borsje

Keyword(s):

Vitamin D ◽

Vitamin A ◽

Test Data ◽

Vitamin D3 ◽

Collaborative Study ◽

Three Samples ◽

The Mean ◽

Fat Soluble Vitamins

Abstract The official first action method for determining vitamin D in multivitamin preparations was modified. The method was collaboratively studied by 7 laboratories, using 6 preparations in oil. The preparations consisted of vitamin D at various levels and at various ratios (in w/w) to vitamin A. Three samples contained cholecalciferol and 3 samples contained vitamin D3 from vitamin D3 resin. After outliers were eliminated by the Dixon test, data were analyzed and averages were compared with amounts of vitamin D known to be in each sample. For samples with vitamin D: vitamin A ratios of 1:0.5, 1:5, and 1:10, the mean vitamin D recoveries were 98.8, 94.6, and 90.7%, respectively. The method has been adopted as official final action.

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Determination of Cholecalciferol (Vitamin D3) in Selected Foods by Liquid Chromatography: NMKL Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/86.2.400 ◽

2003 ◽

Vol 86 (2) ◽

pp. 400-406 ◽

Cited By ~ 20

Author(s):

Anders Staffas ◽

Arne Nyman ◽

K Ask ◽

E Hermansson ◽

J S Jacobsen ◽

...

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Fish Oil ◽

Vitamin D3 ◽

Collaborative Study ◽

The Other ◽

Vitamin D2 ◽

Cooking Oil ◽

Relative Standard

Abstract 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 μg/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 (RSDR) values varied between 6.8% (margarine) and 24% (cooking oil).

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Vitamin D – Funktionen und Versorgung

Ernährung & Medizin ◽

10.1055/s-0043-108869 ◽

2017 ◽

Vol 32 (02) ◽

pp. 57-58

Author(s):

Tilman Grune

Keyword(s):

Vitamin D ◽

Vitamin D3 ◽

Vitamin D2

Die Bezeichnung Vitamin D ist ein Sammelbegriff für verschiedene Substanzen, die aus Cholesterol (Vitamin-D3-Derivate) und Ergosterol (Vitamin-D2-Derivate) synthetisiert werden. Der Stoffwechsel des Vitamin D und die Bildung seiner Wirk- und Transportformen ist ausführlich beschrieben [1, 2] und soll deshalb hier nicht erwähnt werden – nur an die Notwendigkeit von UV-Strahlung für einen zentralen Syntheseschritt in der Haut sei erinnert. Da diese UV-Strahlung offensichtlich nicht unter allen Umständen ausreicht, ist Vitamin D ein essenzieller Nahrungsbestandteil.

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Determination of Vitamin D and Its Metabolites in Human Brain Using an Ultra-Pressure LC–Tandem Mass Spectra Method

Current Developments in Nutrition ◽

10.1093/cdn/nzz074 ◽

2019 ◽

Vol 3 (7) ◽

Cited By ~ 3

Author(s):

Xueyan Fu ◽

Gregory G Dolnikowski ◽

William B Patterson ◽

Bess Dawson-Hughes ◽

Tong Zheng ◽

...

Keyword(s):

Vitamin D ◽

Corpus Callosum ◽

Human Brain ◽

Vitamin D3 ◽

Temporal Cortex ◽

National Development ◽

Brain Regions ◽

Accurate Information ◽

Vitamin D Metabolites

ABSTRACTBackgroundLow serum total 25-hydroxyvitamin D3 [25(OH)D3] concentrations have been associated with cognitive impairment. However, it is unclear if serum 25(OH)D3 concentrations are a valid indicator of the concentrations of vitamin D and its metabolites in human brain.ObjectivesThe aim of this study was to develop and validate a method to quantify vitamin D3, 25(OH)D3, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in human brain.MethodsThe assay developments were performed using porcine brains. Liquid extraction was used in homogenized samples (∼0.1 g each) prior to analysis by LC-MS/MS with electrospray ionization following derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. This method was then applied to the determination of vitamin D and its metabolites in a whole human brain obtained from the National Development and Research Institutes.ResultsThe method showed good linearity of vitamin D3, 25(OH)D3, and 1,25(OH)2D3 over the physiological range (R2 = 0.9995, 0.9968, and 0.9970, respectively). The lowest detection limit for vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in porcine brain was 25, 50 and 25 pg/g, respectively. The method was successfully applied to the determination of vitamin D3 and its metabolites in the prefrontal cortex, middle frontal cortex, middle temporal cortex, cerebellum, corpus callosum, medulla, and pons of a human brain. All analyzed human brain regions contained 25(OH)D3, with corpus callosum containing 334 pg/g compared with 158 pg/g in cerebellum. 1,25(OH)2D3 was only detected in prefrontal and middle frontal cortices at a very low level. No vitamin D3 was detected in any examined areas of this single human brain.ConclusionsTo the best of our knowledge, this study is the first report of the measurement of concentrations of vitamin D metabolites in human brain. This validated method can be applied to postmortem studies to obtain accurate information about the presence and role of vitamin D and its metabolites in human brain and neurodegenerative diseases.

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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

Journal of AOAC International ◽

10.5740/jaoacint.16-0160 ◽

2016 ◽

Vol 99 (5) ◽

pp. 1321-1330 ◽

Cited By ~ 5

Author(s):

Brendon D Gill ◽

Grant A Abernethy ◽

Rebecca J Green ◽

Harvey E Indyk

Keyword(s):

Vitamin D ◽

Vitamin D3 ◽

Multiple Reaction Monitoring ◽

Reference Method ◽

Vitamin D2 ◽

Method Performance ◽

Expert Review ◽

Fortified Milk ◽

Expert Review Panel ◽

Single Laboratory

Abstract 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.

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