Development and validation of

SAGL Inhouse method

for the determination of

vitamin D2

in bread and yeast products

The Southern African Grain Laboratory NPCWe measure so that you know

Introduction

A Canadian company produce a new food ingredient – a vitamin D2-enriched, UV-treated baker’s yeast

The baker’s yeast is treated with UV irradiation to induce the conversion of ergosterol to vitamin D2

The enriched yeast can be used in breads, rolls and fine pastry, as well food supplements

Introduction

There was a need for a quantitative method for the determination of vitamin D2 in bread and yeast

SAGL developed and validated a method for the determination of vitamin D2 in bread and cream and dry yeast

Optimisation of Analytical method

Method development

It was an interesting exercise with a few unexpected challenges

The challenges were :

• Internal standard

• Pre-vitamin D

• The type of matrix and the concentration range

• Optimisation of saponification step

Vitamin D2 & Vitamin D3

Isomerization of Vitamin D2

• Vitamin D can undergo thermal isomerization to reversibly

change to pre-vitamin D

• It must be noted that there is no pure std for pre-vitamin D.

Products

Products tested

• Dry yeast with Vitamin D2

• Reporting unit IU/100g

• Concentration ± 2500000 IU/100g

• Creamed Yeast

• Reporting unit µg /L

• Concentration ± 2025 µg /L

• Bread with vitamin D2

• Reporting unit µg/100 g on 39% moisture base• Expected minimum concentration 2 µg/100 g

Optimisation of Analytical method

• Saponification:

o SAGL tested different temperatures and extraction times

on bread and yeast.

o The best saponification was achieved for 16 hours at room

temperature to avoid thermal isomerization to previtamin

D.

• KOH concentration increased to 8 N KOH

• Extraction / clean-up:

o The diethyl ether/ petroleum ether (1:1 v/v) extraction was

evaluated to optimise the volume and number of

extractions.

Analytical method

Method

• 16 hours at room temperature alcoholic KOH

saponification

• Diethyl ether/ petroleum ether (1:1 v/v)

extraction

• Vitamin D2 is determined by reverse-phase

HPLC with UV detection at 268 nm

• With vitamin D3 as internal standard

• Column: C18, 5µm, 4.6 x 150 mm

• Mobile Phase: Acetonitrile: Methanol 75:25

• Flow rate 1.2 mL/min

Chromatogram

D2 D 3

• Column: C18 4.6 x 150 mmStandard

Bread

Method validation

Method validation on optimized method

Bread, dry yeast and creamed yeast

• Specificity must be able to analyse both Vitamin D2 and D3

• Accuracy:

o Yeast samples supplied with COA

o Spiked samples of creamed yeast and bread with vitamin D2

addition @ three concentration levels in triplicate

• Precision - Repeatability and reproducibility on spiked samples

• Concentration ranges: 0.02 – 6 µg/mL 3 -33 µg/mL

• Homogeneity of bread and yeast – six replicate samples

• Stability of extracts – 24 h & 48 h

• Interlaboratory testing of the same samples

Validation resultsSpiked Bread samples: Reproducibility and Accuracy

Vitamin D2

Spiking

Levels,

µg/100g

Amount

measured

µg/mL

Accuracy Reproducibility

%

Recovery

% Mean

Recovery

%Mean

Recovery

of all

levels

Mean

Amount

measured,

µg/100g

Standard

deviation%RSD n

2 2.10 105

112

106

2.23 0.19 8.48 32 2.45 123

2 2.15 108

4 4.90 122

114 4.57 0.38 8.36 34 4.65 116

4 4.15 104

8 8.35 104

101 8.06 0.32 3.92 7

8 7.90 99

8 8.35 104

8 7.45 93

8 8.15 102

8 8.00 100

8 8.20 103

Validation resultsVitamin D2 spiked creamed yeast samples:

Reproducibility and Accuracy

Spiking

Levels,

µg/mL

(HPLC

value)

Amount

measured,

µg/mL

Accuracy Reproducibility

%

Recovery

% Mean

Recovery

%Mean

Recovery

of all

levels

Mean

Amount

measured,

ug/mL

Standard

deviation%RSD n

2 2.098 105

107

104

2.13 0.03 1.42 32 2.140 107

2 2.157 108

4 4.111 103

102 4.08 0.03 0.76 34 4.050 101

4 4.071 102

6 6.307 105

103 6.18 0.16 2.89 36 6.239 104

6 6.002 100

Validation resultsAccuracy of dry yeast samples,

Sample reference Vitamin D2

Lot 75876D0K1

(17/18) 410

COA 2500000 IU/100g

SAGL 2521647 IU/100g

% Recovery 101%

Lot 76097D0K1

(17/18) 917

COA 2590000 IU/100g

SAGL 2647765 IU/100g

% Recovery 102%

Analyses resultsCreamed yeast and bread samples

Lab No

Creamed yeast

expected value,

µg/L

Measured value, µg/L % Recovery

(17/18)2012 2025 1752 87%

Type of breadLabel value,

µg/100g

Measured value 39% moisture base,

µg/100g

White bread 10 7.52

Brown bread 11 7.01

White bread 2 2.50

Brown bread 2 3.84

White bread 2 1.56

Brown bread 2 1.78

Finalisation of the project

SANAS Accreditation December 2018

Thank You