Table 1a.
Methodology Used by Participating Laboratories to Analyze Vitamin D Content in Foods and 25(OH)D Content in Foods and a Dietary Supplement
| Laboratory A | Laboratory B | Laboratory C | Laboratory D | Laboratory E | |
|---|---|---|---|---|---|
| Method reference | Huang, M.; LaLuzerne, P.; Winters, D.; Sullivan, D., Measurement of vitamin D in foods and nutritional supplements by liquid chromatography/tandem mass spectrometry. J AOAC Int 2009, 92, 1327–1335. | Not available | Not available | Burild, A.; Frandsen, H. L.; Poulsen, M.; Jakobsen, J., Quantification of physiological levels of vitamin D(3) and 25-hydroxyvitamin D(3) in porcine fat and liver in subgram sample sizes. J. Sep. Sci. 2014, 37, 2659–2663. | Not available |
| Sample size | 3 g to 10 g depending on moisture content | 0.8 g to 3 g depending on moisture content | 0.6 g to 2 g | 1 g | 2 g to 3 g |
| Internal standard | (2H3)vitamin D3 and (2H3)25(OH)D3 (IsoSciences, King of Prussia, PA) | (2H6)vitamin D3 and (2H6)25(OH)D3 (Chemaphor, Ottawa, Canada) | (3H)vitamin D3 (in-house) and (2H3)25(OH)D3 (Sigma-Aldrich) | (2H6)vitamin D3 and (2H6)25(OH)D3 (Chemaphor, Ottawa, Canada) | (13C5)vitamin D3 and (13C5)25(OH)D3 (IsoSciences, King of Prussia, PA) |
| Initial treatment | reagent-grade alcohol with 2 % pyrogallic acid, 50 % KOH added | 10 mL pyrogallol 1% in EtOH and 8 mL KOH 60 % | methanoic KOH | sodium ascorbate, KOH, and ethanol | lipase and ethanol |
| Lipid treatment | saponification under N2 overnight | saponification under N2 at room temperature overnight | saponification for 2 hours at 60°C | saponification under N2 at room temperature for 16–18 hours | enzymatic digestion for 2 hours at 37° C |
| Extraction step | solution of 30 mL 20 % ether and 80 % hexane | 3× petroleum ether/diethyl ether (80/20) | 3× hexane/ethyl acetate (85/15) | ethyl acetate/n-heptane (20/80) | 2× hexane extracted, pooled, with addition of ~1g MgSO4 to remove extra water; centrifuged; and dried |
| Clean-up step | dried and redissolved in 1 mL 70 % acetonitrile–H2O [not done for 25(OH)D] | organic phase washed with water, taken to dryness, and dissolved in 800 μL 1-chlorobutane | dried and redissolved in 1 mL hexane/methylene chloride | dried and redissolved in 2-propanol in n-heptane | |
| Additional clean-up | purified using HPLC normal phase (silica column) and fractions collection | loaded into an SPE cartridge, eluted with methylene chloride/isopropanol, two cleanings through HPLC columns, final solution methylene chloride and isopropanol | loaded onto a silica SPE cartridge, rinsed with 0.5 % 2-propanol in n-heptane, eluted with 6% and 10 % 2-propanol in n-heptane | ||
| Derivatization | evaporated to dryness and 2 mg/mL PTAD in 100 % ACN added. | fractions evaporated to dryness, PTAD reagent in ethyl acetate added, evaporated to dryness, reconstituted in 0.2 mL of methanol | not derivatized | dried under N2 and derivatized with PTAD in anhydrous acetonitrile, solvent evaporated, sample reconstituted in 60% methanol | derivatized with PTAD in acetonitrile, dried, reconstituted in 500 uL 70:30 methanol:water. |
| HPLC column | Thermo, UHPLC column, Hypersil GOLD aQ (1.9μm) 100 mm × 2.1 mm | Phenomenex LUNA-C18 (3.0 um) 150 mm × 2.1 mm | Supelco-Sil LC-18 ODS column (5μm) 3.3 cm × 4.6 mm | Ascentis Express C18 (2.1 mm × 10 cm, 2.7 μm particles) column and C18 (2.1 × 5 mm, 2.7 μm particles) guard column | YMC-Pack Pro C18 RS (5μm,8nm) 150 mm × 4.6 mm |
| HPLC separation | mobile phase: (1) 0.1% formic acid, 20% MeOH in ultrapure water; (2) 0.1 % formic acid in methanol | mobile phase: (1) 45 % ACN/55 % H2O; (2) methanol, both with 2 mM ammonium acetate and 0.04 % formic acid | mobile phase: methanol/acetonitrile/water (48.5/48.5/3) | mobile phase: (1) Milli-Q water, methylamine (5 mM), and formic acid (0.1 %); (2) methanol, methylamine (5 mM), and formic acid 0.1 % | mobile phase: (1) water, (2) methanol |
| Quantification | 4000 LC-MS/MS System (Sciex): triple quadrupole tandem mass spectrometer | LC-Agilent 1100 with binary pump coupled to a WATERS Quattro Premier XE MSMS system (ESi mode) | Agilent 1290 UHPLC system coupled to an Agilent 6460 triple quadrupole LC/MS/MS ESI operated in positive MRM mode | Agilent 1200 series HPLC coupled to an Agilent 6460 series triple quadrupole mass spectrometer operated in positive MRM mode | Agilent 6410B MS/MS system, APCI, positive ionization mode |
| Ion Massesa | D3 - 560 (383, 298, 280) 25(OH)D3 558 (298, 280, 247) D2 572 (298) 25(OH)D2 N/A |
D3 - 560 (298, 280) 25(OH)D3 593 (298, 280) D2 572 (298, 280) 25(OH)D2 605 (298, 280) |
D3 - 385 (367, 259) 25(OH)D3 401 (383, 365) D2 397 (379, 107) 25(OH)D2 413 (395, 355) |
D3 - 591 (298) 25(OH)D3 607 (298) D2 603 (298) 25(OH)D2 619 (298) |
D3 - 560 (298) 25(OH)D3 558 (298) D2 572 (298) 25(OH)D2 570 (298) |
| Limit of quantification | Vitamin D3/D2: 0.05/0.20 μg/100g; 25(OH)D3: 0.012 μg/100g | high moisture: 0.08 ng/g low moisture: 0.2 ng/g | 0.5 ng/g | <0.1 ng/g | vitamin D3/D2: 0.4/0.3 ng/g 25(OH)D3/D2: 0.2/0.7 ng/g |
a
Masses in parenthesis are the product ions.
Abbreviations: HPLC, high-performance liquid chromatography; SPE, solid-phase extraction; PTAD, 4-phenyl-1,2,4-triazole-3,5-dione; ACN, acetonitrile; UHPLC, ultra-high-performance liquid chromatography; MS/MS, tandem mass spectrometry; ESi, electrospray ionization; MRM, multiple reaction monitoring