Metabolic profiling of major vitamin D metabolites using Diels–Alder derivatization and ultra-performance liquid chromatography–tandem mass spectrometry

Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1α,25-dihydroxyvitamin D₃ in human serum (15–60 pg/mL). Here, we demonstrate that liquid–liquid or solid-phase extraction of vitamin D metabolites in combination with Diels–Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)–electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1α,25-dihydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₂, 24R,25-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6–4.8 % and 5–16 % for 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃, respectively, using solid-phase extraction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Study of blood collection and sample preparation for analysis of vitamin D and its metabolites by liquid chromatography–tandem mass spectrometry

The analysis of vitamin D status, with special emphasis on 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, is gaining interest in clinical studies due to the classical and non-classical effects attributed to this prohormone. In this research, the influence of the two steps preceding determination (viz. sample collection and preparation) on the quantitative analysis of vitamin D and its more important metabolites has been studied. Two preparation approaches, deproteination and solid-phase extraction (SPE), have been evaluated in terms of sensitivity to delimit their application, thus establishing that detection of 1,25-dihydroxyvitamin D cannot be addressed by protein precipitation. Concerning sample collection, serum and plasma reported high accuracy (above 83.3%) for vitamin D and metabolites, while precision, expressed as relative standard deviation, was below 12.9% for all analytes in both samples. Statistical analysis revealed that serum and plasma provided similar physiological levels for vitamin D₃, 24,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃, while significantly different levels were obtained for 1,25-dihydroxyvitamin D₃, always higher in plasma than in serum. Sample collection and treatment have proved to be significant in the analysis of vitamin D and its relevant metabolites.     

Identification and Structural Elucidation of Vitamin D3 Metabolites in Human Urine Using LC-MS-MS

An analytical method was developed for the identification of primary vitamin D₃ metabolites in human urine using liquid chromatography tandem mass spectrometry in positive mode. Urine samples were purified using C₁₈ solid-phase extraction cartridges and analytical separations were performed by reversed phase liquid chromatography in gradient mode using ammonium acetate (0.01 mol L^?1) and acetonitrile as the mobile phases. Identification and structural elucidation of the metabolites were carried out by comparison with mass spectral fragmentation behavior of vitamin D₃ and retention characteristics. Three primary urinary vitamin D₃ metabolites were identified as 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and vitamin D₃ sulphate, respectively.     

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis

Blood levels of the vitamin D₃ (D₃) metabolites 25-hydroxyvitamin D₃ (25(OH)D₃), 24R,25-dihydroxyvitamin D₃, and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are recognized indicators for the diagnosis of bone metabolism-related diseases, D₃ deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D₃ metabolites, such as 20-hydroxyvitamin D₃ and lactone D₃, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D₃ metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D₃ (2), 25(OH)D₃-23,26-lactone (6), and 1,25(OH)₂D₃-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)₂D₃-23,26-lactone (7) in human serum.     

Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS

Vitamin D is an important determinant of bone health at all ages. The plasma concentrations of 25-hydroxy vitamin D (25-OH D) and other metabolites are used as biomarkers for vitamin sufficiency and function. To allow for the simultaneous determination of five vitamin D metabolites, 25-OH D₃, 25-OH D₂, 24,25-(OH)₂ D₃, 1,25-(OH)₂ D₃, and 1,25-(OH)₂ D₂, in low volumes of human plasma, an assay using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was established. Plasma samples were spiked with isotope-labeled internal standards and pretreated using protein precipitation, solid-phase extraction (SPE) and a Diels–Alder derivatization step with 4-phenyl-1,2,4-triazoline-3,5-dione. The SPE recovery rates ranged from 55% to 85%, depending on the vitamin D metabolite; the total sample run time was <5 min. Mass spectrometry was conducted using positive ion electrospray ionization in the multiple reaction monitoring mode on a quadrupole–quadrupole-linear ion trap instrument after pre-column addition of methylamine to increase the ionization efficiency. The intra- and inter-day relative standard deviations were 1.6–4.1% and 3.7–6.8%, respectively. The limit of quantitation for these compounds was determined to be between 10 and 20 pg/mL. The 25-OH D results were compared with values obtained for reference materials (DEQAS). In addition, plasma samples were analyzed with two additional Diasorin antibody assays. All comparisons with conventional methods showed excellent correlations (r ² = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.     

Liquid-Solid Extraction of Vitamin D3 Metabolites from Plasma for Analysis by HPLC,GC/MS and Protein Binding Techniques

Abstract

Liquid-solid extraction of vitamin D₃ metabolites from human plasma using octadecylsilane bonded silica has been studied. Steroid-protein interactions were minimized by diluting the plasma (or serum) with two volumes of saline and passing the solution through the sorbent at 64°C. Highly purified secosteroid fractions were obtained by washing with aqueous methanol, drying the sorbent in situ with a stream of nitrogen for one minute and eluting with mixtures of hexane/chloroform. Recoveries of vitamin D₃ metabolites were essentially quantitative. Applications to the rapid analysis of 25-hydroxy- and 1α, 25-dihydroxy-vitamin D in plasma by high-performance liquid chromatography, gas chromatography-mass spectrometry or by a receptor protein assay are reported.     

Determination of Vitamin D3 and 25-Hydroxyvitamin D3 Using Liquid Chromatography with Amperometric Detection

Abstract

The electrochemical behaviour of vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH D₃) in a high performance liquid chromatography system using amperometric detection is described. Separation is carried out using a C18 reversed-phase column and the optimum mobile phase was a 0.1 M LiClO₄ solution in methanol-water (97:3, v/v) at a flow rate of 1.25 ml/min. 25-OH D₃ and vitamin D₃ were eluted with good resolution at retention times of 3 and 6 minutes respectively, and determined by amperometric detection with a glassy carbon electrode at + 1.050 V (vs Ag/AgCl). Calibration graphs for both substances showed good linearity when amounts of vitamin D₃ between 18 and 312 ng and 27 and 412 ng of 25-OH D₃ were injected. Detection limits of 8 ng (vitamin D₃) and 25 ng (25-OH D₃); relative standard deviations of 3.2% (vitamin D₃) and 5.8% (25-OH D₃) were obtained.     

Liquid chromatography–tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D<Subscript>3</Subscript>: a noninvasive tool for the assessment of vitamin D status

A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the determination of 25-hydroxyvitamin D₃ [25(OH)D₃] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC–MS/MS. The PTAD derivative was much more easily ionized in positive-ESI–MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D₃. Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D₄ was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D₃ using a 1.0-ml sample, and the limit of quantitation for 25(OH)D₃ was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r ² = 0.830) between the serum 25(OH)D₃ level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D₃ level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D₃ level after the supplementation of vitamin D₃.     

Determination of Vitamin D3 Metabolites Using High‐Performance Liquid Chromatography or Immunoaffinity Chromatography

Our investigation of the analysis of vitamin D₃ metabolites has been reviewed. The development of high‐performance liquid chromatographic methods for the quantitative determination of 25‐hydroxyvitamin D₃ 3‐sulfate and 25‐hydroxyvitamin D₃, which are the major circulating metabolites of vitamin D₃ in human serum/plasma, has been described. The developed methods were applied to the determination of the correlation between the concentration of the sulfate and its genin in healthy subjects and patients with chronic renal failure. The development of immunoaffinity chromatography immobilizing the highly specific anti‐1,25‐dihydroxyvitamin D₃ antibody for the pretreatment of radioreceptor assay of 1,25‐dihydroxyvitamin D₃, which is the active metabolite of vitamin D₃, is also described.     

Molecular characterization,modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein’s structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme’s possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D₃, 25-hydroxyvitamin D₃ and 1α-hydroxyvitamin D₃, in which all substrates docked proximally to the enzyme’s heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D₃ from 1α-hydroxyvitamin D₃. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D₃ to calcitriol, or related metabolic products.     

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D₃, and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q₁₀. The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D₃ in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.     

Synthesis of a 1α-C-methyl analogue of 25-hydroxyvitamin D3: interaction with a mutant vitamin D receptor Arg274Leu

Vitamin D₃ analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1α-OH of 1α,25-dihydroxyvitamin D₃ to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2α-(3-hydroxypropyl)-1α-methyl-25-hydroxyvitamin D₃ (4), from d-galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1α,25-dihydroxyvitamin D₃ (1).     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization

The determination of the urinary vitamin D₃ metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D₃ metabolites, 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, ²H₄-DAPTAD (d-DAPTAD). The urine samples were treated with β-glucuronidase, purified with an Oasis® hydrophilic–lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n?=?5) and inter-assay (n?=?3) relative standard deviations did not exceed 9.5 %]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D₃ and 24,25(OH)₂D₃ levels due to vitamin D₃ administration. Graphical Abstract

Scheme of procedure for urinary vitamin D₃ metabolite analysis based on LC/MS/MS with ESI-enhancing and isotope-coded derivatization.     

Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography–tandem mass spectrometry

Milk is an important source of nutrients for various risk populations, including infants. The accurate measurement of vitamin D in milk is necessary to provide adequate supplementation advice for risk groups and to monitor regulatory compliance. Currently used liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods are capable of measuring only four analogues of vitamin D in unfortified milk. We report here an accurate quantitative analytical method for eight analogues of vitamin D: Vitamin D₂ and D₃ (D₂ and D₃), 25-hydroxy D₂ and D₃, 24,25-dihydroxy D₂ and D₃, and 1,25-dihydroxyD₂ and D₃. In this study, we compared saponification and protein precipitation for the extraction of vitamin D from milk and found the latter to be more effective. We also optimised the pre-column derivatisation using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), to achieve the highest sensitivity and accuracy for all major vitamin D forms in milk. Chromatography was optimised to reduce matrix effects such as ion-suppression, and the matrix effects were eliminated using co-eluting stable isotope labelled internal standards for the calibration of each analogue. The analogues, 25-hydroxyD₃ (25(OH)D₃) and its epimer (3-epi-25(OH)D₃) were chromatographically resolved, to prevent over-estimation of 25(OH)D₃. The method was validated and subsequently applied for the measurement of total vitamin D levels in human, cow, mare, goat and sheep milk samples. The detection limits, repeatability standard deviations, and recovery ranges were from 0.2 to 0.4 femtomols, 6.30–13.5%, and 88.2–105%, respectively.     

A gas chromatographic flow method for preconcentration and determination of vitamins D2 and D3 in pharmaceutical preparations

Summary A continuous-flow method that combines on-line preconcentration and isolation with gas chromatography for the direct determination of vitamins D₂ and D₃ in oily solutions is reported. A silica gel column permits preconcentration and isolation of analytes from the vitamin D matrix, although some triglyceride (ca. 25%) is also retained. To overcome problems associated with the low volatility of triglycerides, their retained fraction is further transesterified with potassium methylate to fatty acid methyl esters after elution. The proposed method was applied to the determination of vitamin D in pharmaceutical preparations.     

Automated method for the determination of vitamin D3 hydroxymetabolites in serum

An almost automated method for the determination of hydroxymetabolites of vitamin D₃ (cholecalciferol) in human serum is reported. The method consists of three steps: 1) a batch liquid–liquid extraction step with 2-propanol and hexane, and drying of the extract and reconstitution with phosphate buffer. 2) A cleanup and preconcentration step based on solid-phase extraction using Prospekt equipment, with CN group cartridges and elution with the chromatographic mobile phase. 3) A chromatographic step for individual separation of the target analytes starting with a 90:10 methanol–water mixture, then a linear gradient to obtain 100% methanol; followed by photometric detection. The method provides a linear range between 1.0 and 100 ng mL^–1 for 24,25-(OH)₂ vitamin D₃ and for 25-(OH)₂ vitamin D₃, and between 1.5 and 100 ng mL^–1 for 1,25-(OH) vitamin D₃, with correlation coefficients ranging between 0.993 and 0.987, repeatability between 1.9% and 4.8% and within-laboratory reproducibility between 2.8% and 8.8%.     

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 24,24-difluoro-25-hydroxyvitamin D3

24,24-Difluoro-25-hydroxyvitamin D₃ ($\text{13}$) has been prepared from commercially available lithocholic acid derivative to study the role of 24-hydroxylation in the metabolism of vitamin D₃.     

Evaluation of preliminary causes for vitamin D series degradation via DSC and HPLC analyses

Scientific evidence suggests that 25-(OH) vitamin D serum levels should be above 75 nmol L^?1, which is the level required for the beneficial effects of vitamin D on long-latency diseases. Supplementing vitamin D in a daily diet is advantageous for the health of elderly individuals. The focus of this study was on the causes of vitamin D₂ and D₃ degradation, such as vitamin D₂ being left in the open for days. The differences in the analysis results for fresh vitamin D and vitamin D stored in air for several days were compared. Comparisons were also made of the differences in vitamins D₂ and D₃ and their origins, i.e., oxidation, thermal decomposition, and photolysis, using high-performance liquid chromatography analyses and differential scanning calorimetry tests. We also developed a novel approach to examine the effect of heat on vitamin D, including the heat reactivity properties of vitamins D₂ and D₃, such as the kinetics of endothermic and exothermic reactions, the natural logarithm of the pre-exponential factor (lnk ₀), reaction order (n), activation energy (E _a), enthalpy of melting (?H), heat of decomposition (?H _d), isothermal conditions for the time to maximum rate, total energy release, and time until 10% conversion limited. Overall, the parameters, the reactivity properties, and the degradation results can be applied to determine the optimal conditions for vitamins D₂ and D₃ during cooking, storage, heat treatment, and food processing that will be beneficial to human health.