I need standard method for qualitative and quantitative detection of vitamins with HPLC-MS.
Dear doctor
About Vitamin E tocopherol I'll send you a method I used for oil
Tocopherols analysis
The tocopherol composition was determinate using HPLC according to standard ISO 9936. 0.5 g of oil was diluted with 5 ml hexane and 20 µl samples were injected into HPLC system (Waters 2690) consisting of a quaternary pum, auto sampler and a fluorescence detector set at 295 nm. A reverse phase C 18 column (150 mm × 4.6 mm × 5 µm) was used with isopropanol/hexane (0.5/99.5, v/v) as a mobile phase at an isocratic elution at a flow rate of 1,5 ml/min. The mixed tocopherol standards in hexane (2 mg/ml) solution were prepared from the standard compounds (α, δ and γ tocopherols from Sigma- Aldrich)
Tocopherol peaks identified by comparison with chromatogram of standard compounds obtained under similar analytical conditions.
about vit D
Colum: Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U)
mobile phase: [A] 5 mM Ammonium Formate; [B] Methanol : Water (75:25)
Flow rate: 0.4 mL/min
Pressure: 3553 psi (245 bar)
Column temperature: 20 °C
Detector: (UV, 265 nm)
Injection: 1 μL,
Sample: 500 mg/L in Water: Methanol (25:75)
SM
ESI(+), m/z 100 - 1000)
The VitD metabolites 25(OH)D3 and 25(OH)D2 were obtained from Fluka Chemicals. 25(OH)-epi-D3 was kindly supplied by Dr. G. Satyanarayann Reddy (Providence, RI). Laurophenone (99%), ACS reagent-grade acetonitrile (CH3CN), and ethyl acetate were obtained from Fisher. Methanol (HPLC grade) was obtained from Mallinckrodt Chemicals. Ultrapure water (18.2 MΩ/cm) was obtained from a MilliQ water purification system (Millipore). The precipitation reagent contained the internal standard laurophenone (400 μg/L) in CH3CN and was stored in an amber bottle. Strata-X (surface-modified styrene-divinylbenzene resin) 60-mg (1 mL) extraction cartridges were from Phenomenex. An automated extraction instrument, the Gilson ASPEC XL4 (Gilson Instruments), consisted of a 4-syringe pump module and a 4-needle sampler module with four 2-way solvent ports. Areas in the sampler racks were defined as the sample zone, reagent zone, result zone, and a disposable extraction column (DEC) zone. Acetonitrile was delivered via solvent ports. Acetonitrile–water (35:65 by volume) was stored and delivered from tubes within the reagent zone. The main reservoir contained water. The solvent evaporator was a Turbo Vap™ LV (Caliper Life Sciences). Temperature was set at 35 °C, nitrogen flow was adjusted to 10 psi on the instrument gauge, and the typical drying time setting was 25 min. The HPLC unit was an integrated system with a UV3000 detector set at 275 nm, a P4000 pump set at 1.2 mL/min, an AS2000 autosampler, and a SCM1000 solvent system, all from Thermo Separation Products. A silica-saturator column [250 × 4.6 mm (i.d.) stainless steel column; Alltech] packed with ICN silica gel (particle size, 63–100 μm; MP Biochemicals) was installed in the oven between the pump and injector and is necessary here to prevent deterioration of the analytical column (20). The guard column [(12.5 × 4.6 mm (i.d.)] and analytical column
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