BS ISO 20636:2018 pdf download – Infant formula and adult nutritionals — Determination of vitamin D by liquid chromatography-mass spectrometry

BS ISO 20636:2018 pdf download – Infant formula and adult nutritionals — Determination of vitamin D by liquid chromatography-mass spectrometry
5.3 Standard preparation
5.3.1 Vitamin D is sensitive to light. Perform all steps under low-level incandescent lighting. If exclusively vitamin D 3 is required for analysis, then standards pertaining to vitamin D 2 need not be used and vice versa. Calibration standards should be bracketed at the beginning and at the end of an analytical run.
5.3.2 Vitamin D 2 stable isotope labelled stock standard solution, ρ ≈ 10 μg/ml. Dispense the contents of a 1 mg vial of d6-vitamin D 2 (5.1.4) into a 100 ml volumetric flask. Dissolve in 90 ml of ethanol (5.1.10). To promote dissolution, sonicate if necessary. Mix thoroughly, make up to volume with ethanol (5.1.10). Measure the absorbance of an aliquot at 265 nm. The spectrophotometer should be zeroed against an ethanol (5.1.10) blank solution. Calculate and record concentration. Immediately dispense aliquots (~1,3 ml) into cryogenic vials and freeze at < –15 °C for up to 6 months.
5.3.3 Vitamin D 3 stable isotope labelled stock standard solution, ρ ≈ 10 μg/ml. Dispense the contents of a 1 mg vial of d6-vitamin D 3 (5.1.5) into a 100 ml volumetric flask. Dissolve in 90 ml of ethanol (5.1.10). To promote dissolution, sonicate if necessary. Mix thoroughly, make up to volume with ethanol (5.1.10). Measure the absorbance of an aliquot at 265 nm. The spectrophotometer should be zeroed against an ethanol (5.1.10) blank solution. Calculate and record concentration. Immediately dispense aliquots (~1,3 ml) into cryogenic vials and freeze at < –15 °C for up to 6 months.
5.3.4 Stable isotope labelled internal standard solution, ρ ≈ 1 μg/ml. Depending on the number of samples that need to be analysed in a run, more or less stable isotope labelled internal standard solution needs to be made up. For every 15 samples (or part thereof) in an analytical run, remove 1 vial of Vitamin D 2 stable isotope labelled stock standard solution (5.3.2) and/or 1 vial of Vitamin D 3 stable isotope labelled stock standard solution (5.3.3) from the freezer and allow to warm to room temperature. Pipette 1,0 ml of vitamin D 2 stable isotope labelled stock standard solution (5.3.2) and/or 1,0 ml of vitamin D 3 stable isotope labelled stock standard solution (5.3.3) into a 10 ml volumetric flask (use a separate 10 ml volumetric flask for each set of 15 samples). Make each 10 ml volumetric flask to volume with acetonitrile, pool together and mix thoroughly. Make fresh daily.
5.3.5 Vitamin D 2 non-labelled stock standard solution, ρ ≈ 1 mg/ml. Weigh accurately, approximately 50 mg of vitamin D 2 (5.1.2) into a 50 ml volumetric flask. Dissolve in 40 ml of ethanol (5.1.10). To promote dissolution, sonicate if necessary. Mix thoroughly, make up to volume with ethanol (5.1.10). Store in freezer at < –15 °C for up to 1 month.
5.3.6 Vitamin D 3 non-labelled stock standard solution, ρ ≈ 1 mg/ml. Weigh accurately, approximately 50 mg of vitamin D 3 (5.1.3) into a 50 ml volumetric flask. Dissolve in 40 ml of ethanol (5.1.10). To promote dissolution, sonicate if necessary. Mix thoroughly, make up to volume with ethanol (5.1.10). Store in freezer at < −15 °C for up to 1 month.
5.3.7 Vitamin D 2 non-labelled purity standard solution, ρ ≈ 10 μg/ml. Pipette 1,0 ml of vitamin D 2 non-labelled stock standard solution (5.3.5) into a 100 ml volumetric flask. Make to volume with ethanol (5.1.10). Measure the absorbance of an aliquot at 265 nm. The spectrophotometer should be zeroed against an ethanol (5.1.10) blank solution. Record absorbance and calculate concentration. Make fresh daily.
5.3.8 Vitamin D 3 non-labelled purity standard solution, ρ ≈ 10 μg/ml. Pipette 1,0 ml of vitamin D 3 non-labelled stock standard solution (5.3.6) into a 100 ml volumetric flask. Make to volume with ethanol (5.1.10). Measure the absorbance of an aliquot at 265 nm. The spectrophotometer should be zeroed against an ethanol (5.1.10) blank solution. Record absorbance and calculate concentration. Make fresh daily.
5.3.9 Non-labelled working standard solution, ρ ≈ 1 μg/ml. Pipette 1,0 ml of vitamin D 2 non-labelled purity standard solution (5.3.7) and/or 1,0 ml of vitamin D 3 non-labelled purity standard solution (5.3.8) into a 10 ml volumetric flask. Make to volume with acetonitrile (5.1.14) and mix thoroughly. Make fresh daily.