Appendix Q - Blood urine analysis methods NI Report NDNS 2019-2023
Source: data.gov.uk (2025-10-10)

National Diet and Nutrition Survey 2017 to 2023 Report for Northern Ireland. This report presents results from 2017-2023 of the National Diet and Nutrition Survey (NDNS) for Northern Ireland. NDNS is a continuous cross-sectional survey, designed to assess the diet, nutrient intake and nutritional status of the general population aged 18 months and over living in private households in the UK.

1

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Appendix Q Methods of blood and urinary
analysis, quality control (QC) and quality
assessment (QA) in the National Diet and
Nutrition Survey 2019 to 2023

Q.1 Introduction

This appendix details the analytical methods, internal quality control (QC) and external
quality assessment (EQA) performance for each analyte measured as part of NDNS 2019
to 2023. The majority of assays were performed at the Nutritional Biomarker Laboratory
(NBL), MRC Epidemiology Unit, University of Cambridge. Other assays were performed
either at other laboratories on the Cambridge Biomedical Campus (Addenbrooke`s
Laboratory Research Services (LRS) and Core Biochemical Assay Laboratory (CBAL)) or
at specialist external laboratories (Trace Element Laboratory, University Hospitals
Southampton, UK (UHS) and Centers for Disease Control and Prevention, Atlanta, Georgia,
USA (CDC). Details are provided in table Q.1.
Table Q.1 Summary of analytes and methods of analysis during NDNS years 12 to 15
Analyte
Appendix
section
Analytical method or
platform
Responsible
laboratory
Full blood count
Q.3.1
Siemens ADVIA 2120
LRS
C-reactive protein
Q.3.2
Siemens Dimension
EXL200
CBAL
Ferritin
Q.3.3
Siemens Dimension
EXL200
CBAL
Serum cholesterol (total,
HDL, LDL)
Q.3.4
Siemens Dimension
EXL200
CBAL
Triglycerides
Q.3.5
Siemens Dimension
EXL200
CBAL
ETKAC (thiamin)
Q.3.6
Enzymatic method
NBL
EGRAC (riboflavin)
Q.3.7
Enzymatic method
NBL
Vitamin B6 (PLP and 4PA)
Q.3.8
HPLC
NBL
Total folate (serum)
Q.3.9
LC-MS/MS
NBL
Whole blood folate (red blood
cell folate)
Q.3.10
Microbiological method
CDC
Vitamin B12
Q.3.11
Siemens ADVIA Centaur LRS
Holotranscobalamin
Q.3.12
ELISA
NBL
Vitamin C
Q.3.13
Fluorescence method
NBL
Retinol (vitamin A), - and -
tocopherol (vitamin E) and
carotenoids
Q.3.14
HPLC
NBL
25-hydroxyvitamin D2/3
Q.3.15
LC-MS/MS
NBL
Selenium and zinc
Q.3.16
ICP-MS
UHS

2

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Iodine
Q.3.17
ICP-MS
UHS
Abbreviations: EGRAC, Erythrocyte glutathione reductase activation coefficient; ELISA,
enzyme-linked immunosorbent assay; ETKAC, Erythrocyte transketolase activation
coefficient; HPLC, high performance liquid chromatography; ICP-MS, inductively coupled
plasma mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry;
PA, pyridoxic acid; PLP, pyridoxal-5-phosphate.

The collection, transport and processing of blood and urine samples are described in
appendices M and P. A complete list of analytes measured and the priority order of
analyses are provided in the tables that accompany appendix P. There were no changes to
the laboratory analytical methods from the previous NDNS report for years 9 to 11 (2016 to
2019) and the methods described in this appendix are identical to those described
previously. As detailed elsewhere in the report, fieldwork years 12 to 15 were disrupted by
the COVID-19 pandemic. Consequently, field blood collections and laboratory analysis
were not performed continuously as during earlier years of NDNS. Year 12 blood samples
were collected between October 2019 and March 2020 and analysed during 2020. Blood
samples for years 13 to 15 were collected between November 2021 and June 2023 with
overlap between fieldwork years, and samples were analysed between November 2021
and September 2023. Therefore, rather than reporting quality control data for separate
NDNS fieldwork years as in earlier reports (where fieldwork years were distinct), data is
instead presented for year 12 and for years 13 to 15 combined.
Q.2 Notes on interpretation of external quality assessment
(EQA) schemes

Different schemes within the UK National External Quality Assessment Services (NEQAS)
and other EQA schemes (described relevant to analyte in the respective sections) have
varying approaches to reporting and presenting performance data. In this report, the results
of the performance in EQA schemes are presented as the per cent bias and SD of per cent
bias from the method mean or the all-laboratory trimmed mean (ALTM) calculated in-house
or using the scheme-specific scores; the notes below provide a summary of information to
interpret the scheme-specific scores.

EQA data points are missing for EQA schemes and EQA scores undertaken in 2020
(fieldwork year 12) due to the COVID-19 pandemic and closure of the NBL. In contrast,
CBAL and LRS remained open and enrolled in EQA schemes throughout ‘lock-down’ periods
and have EQA data available continuously from 2020 to 2023. Consequently, CBAL EQA
data are available for and are presented by calendar year rather than NDNS fieldwork
year(s). Details are provided under each analyte in the sections below.

Q.2.1 Performance scores for fat-soluble vitamins, vitamin D, vitamin B12,
holoTC, serum folate and ferritin

NEQAS scores for these schemes are reported as A, B and C scores:
Accuracy (A) score has been transformed and uses a ‘degree of difficulty’ factor. A scores
are broadly comparable across analytes and provide an overall performance accuracy
score which assesses bias, consistency of bias and clinical acceptability of performance
over approximately 6 months. A score of 100 indicates median performance for that assay.

3

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Bias (B) score has been calculated as the mean bias over last 6 months (per cent). Bias is
calculated as specimen per cent bias = [(result - target) ÷ target] × 100
Consistency (C) score of bias represented by the SD of bias over last 6 months (per cent)

Q.3 Methods

Details of the analysis method and the associated QC procedures for each analyte or set of
analytes are provided in section Q.3.1 onwards. For all analytes, internal QC samples were
run in every batch to assess assay imprecision. Accuracy was assessed by comparison
with certified reference materials and target values from external QC materials.
Laboratories participated in EQA schemes where such schemes were available. Analyses
were performed at NBL unless otherwise stated.

Q.3.1 Full blood count including haemoglobin and haematocrit
Full blood count was measured at LRS using a Siemens Advia 2120, which uses the
Coulter Principle to count red blood cells (RBC), mean cell volume (MCV), white blood
cells (WBC) and platelet counts. Haemoglobin was measured by photometric
measurement. Other parameters such as the mean cell haemoglobin (MCH), haematocrit
and red cell distribution width (RDW) were calculated from the above measured
parameters.

Q.3.1.1 Internal QC for full blood count
QC was performed as part of LRS standard practice. The QC results of the instruments
measuring full blood count are monitored continually, and analysis is stopped if the results
are not satisfactory, ensuring that results are only reported if the analysis is within the QC
parameters set by the relevant Pathology Department Laboratory Manager. However, it is
not possible to extract these results for reporting for each specific time period relevant to
the NDNS analyses. Typical LRS assay per cent CVs are 3.1% (RBC), 1.8% (MCV), 2.8%
(WBC), 3.9% (platelet count), 2.2% (MCH), 2.4% (RDW) 1.8% (haemoglobin) and 1.9%
(haematocrit).
Q.3.1.2 EQA for full blood count
Quality of results was assessed externally through NEQAS. In figure Q.1 NEQAS results
over the year to April 2023 are presented as a performance score. This score shows how
the laboratory and specific instrument performed against a trimmed (excluding a proportion
of the highest and lowest values) average for each analyte. The lower the performance
score, the closer the laboratory or instrument value is to the consensus value. A
performance score of between 0 and 75 is considered satisfactory.

4

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Figure Q.1 EQA data for full blood count

Q.3.2 C-reactive protein (CRP)

At CBAL, serum C-reactive protein (CRP) was assayed using a high-sensitivity (extended
range) assay (RCRP, DF34) on a Siemens Dimension EXL200 system clinical chemistry
analyser. The CRP method is based on a particle enhanced turbidimetric immunoassay
(PETIA) technique, giving high sensitivity by extending the detection range from 0.5mg/L
to 250mg/L. Latex particles coated with anti-CRP antibody aggregate in the presence of
CRP in the sample. The increase in turbidity that accompanies aggregation is proportional
to the CRP concentration.

5

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Consistent with reporting in previous NDNS RP years, values less than 1.1mg/mL were
given an assigned value of 0.7 mg/L (calculated as LOQ ÷ √2) to reflect the high
imprecision at values less than 1.1mg/L.

Q 3.2.1 Internal QC for CRP

Internal QC material for CRP (Bio-Rad Laboratories Ltd, Hertfordshire, UK) consisted of
Liquichek Immunology Control, levels 1 and 3 (product code 594) and Liquichek Cardiac
Markers Plus Control, level 2 (product code 180). Tables Q.3 and Q.4 show imprecision
data for year 12 and years 13 to 15, respectively.

Table Q.2
Internal QC for CRP (NDNS year 12)
CRP QC
Year 12 QC1
Year 12 QC2
Year 12 QC3
Lot no.
664021
68911
660403
68913
29882
Mean (mg/L)
10.1
9.4
47.2
46.6
2.9
SD (mg/L)
0.15
0.24
1.38
1.13
0.22
Per cent CV
1.49
2.56
2.93
2.42
7.57
n
9
17
9
17
26

Table Q.3
Internal QC for CRP (NDNS years 13 to 15)
CRP QC
Years 13 to 15 QC1
Years 13 to 15 QC2
Years 13 to 15 QC3
Lot no.
68911 68951 68991 68913 68953 68993 29882 87812 87842
Mean (mg/L)
8.8
8.4
9.8
44.1
42.7
46.1
2.3
1.8
1.9
SD (mg/L)
0.26
0.35
0.23
1.05
1.78
1.68
0.26
0.28
0.13
Per cent CV
2.95
4.19
2.34
2.38
4.17
3.64
11.25
16.00
6.98
n
6
46
16
6
47
17
16
32
17

Q 3.2.2 External QA for CRP

External QA was achieved through the NEQAS CRP and ultra-sensitive CRP (usCRP)
schemes (table Q.4). Performance criteria for NEQAS are based on the following scores:
MRBIS (mean running bias) and MRVIS (mean running variance index score).
Performance of a laboratory over the given time period is characterised as follows:
Ideal: MRVIS < 50 Good:
MRVIS 50 to 100
Adequate: MRVIS 101 to 200
Poor: MRVIS > 200 or SDBIS > 200

Target values are based on all laboratory trimmed means and include a large number of
different analysers. The population of analysers and numbers of laboratories enrolled in the
scheme has changed dramatically over the course of years 12 to 15. In 2020, 276
participants were enrolled in the scheme for CRP and 44 in the usCRP scheme. In 2023, this
had risen to 606 and 102, respectively. It should also be noted that the method used in
NDNS is not designed as a high-sensitivity assay as generally used for the usCRP scheme.
This, together with the changes in scheme participants over the NDNS fieldwork year will
partly explain the variation observed in the external QC scores.

6

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.4
EQA for CRP (NDNS years 12 to 15)
CRP
NEQAS
Year 12
CRP
Year 12
usCRP
2021
CRP
2021
usCRP
2022
CRP
2022
usCRP
2023
CRP
2023
usCRP
MRVIS
+34
+158
+60
+124
+72
+125
+107
+177
MRBIS
+5
+49
+6
-11
+52
+15
+106
+155
SDBIS
43
202
79
167
70
165
69
143

Q.3.3 Ferritin

Serum ferritin was measured on a Siemens Dimension EXL200 analyser at CBAL. The
assay uses chromium dioxide particles coated with specific antibodies to human ferritin.
The assay is coupled to a colour reaction and the concentration of ferritin in the sample is
proportional to the intensity of colour produced in the reaction. Ferritin is quantified by
comparison to calibrants of known concentration.

Q.3.3.1 Internal QC for ferritin

QC material was Bio-Rad Lyphochek Immunoassay Plus Control - level 1 and 3 (product
code 370) and Bio-Rad Lyphochek Anemia Control (product code 500). Summaries of
internal QC data are shown in tables Q.5 and Q.6 for NDNS year 12 and years 13 to 15,
respectively.

Table Q.5
Internal QC for ferritin (NDNS year 12)
Ferritin QC
Year 12 QC1 Year 12 QC2 Year 12 QC3
Lot no.
40361
40363
43280
Mean (µg/L)
66.8
381
6.4
SD (µg/L)
1.19
8.5
0.51
Per cent CV
1.78
2.23
7.99
n
14
14
14

Table Q.6
Internal QC for ferritin (NDNS years 13 to 15)
Ferritin QC
Year 13 to 15 QC1
Year 13 to 15 QC2
Year 13 to 15 QC3
Lot no.
40361
40391
40411
40363
40393
40413
43280
43320
Mean (µg/L)
67.5
81.9
80.7
404
385
479
6.7
7.6
SD (µg/L)
1.22
3.56
2.02
24.0
14.3
11.9
0.66
0.71
Per cent CV
1.81
4.35
2.50
5.95
3.71
2.48
9.81
9.34
n
6
34
15
6
34
15
20
30

Q.3.3.2 External QA for ferritin

External QA was through the NEQAS Haematinics scheme. Table Q.7 shows the per cent
bias relative to the target concentration in ferritin results during years 12 to 15.

7

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.7
External QA for ferritin (NDNS years 12 to 15)
Ferritin
NEQAS
Year 12
2021
2022
2023
Mean per cent
bias
2.1
6.5
13.9
11.3
SD of per cent
bias
4.78
10.46
6.19
7.08
n
30
33
33
24

Q.3.4 Total, high density lipoprotein (HDL) and low density lipoprotein
(LDL) cholesterol

Total cholesterol and high-density lipoprotein (HDL) cholesterol were measured in serum
by Siemens Dimension EXL200 analyser at CBAL.

The total cholesterol method (Siemens CHOL DF27) uses cholesterol esterase to
catalyse the hydrolysis of cholesterol esters to produce free cholesterol which, along with
pre-existing free cholesterol, is oxidised in a reaction catalysed by cholesterol oxidase to
form cholest-4-ene-3-one and hydrogen peroxide. In the presence of horseradish
peroxidase, the hydrogen peroxide thus formed is used to oxidize N,N-diethylaniline-
HCl/4-aminoantipyrine to produce a chromophore that absorbs at 540nm.

The AHDL cholesterol assay (Siemens DF48B) is a method for directly measuring HDL
cholesterol concentrations. The method is based on accelerating the reaction of
cholesterol oxidase with non-HDL unesterified cholesterol and dissolving HDL selectively
using a specific detergent. In the first reaction, non-HDL unesterified cholesterol is subject
to a cholesterol oxidase reaction and the peroxide generated is consumed by a
peroxidase reaction with DSBmT yielding a colourless product. The second reagent
consists of a detergent capable of solubilising HDL specifically, cholesterol esterase and
chromagenic coupler to develop colour for the quantitative determination of HDL-C.
Low-density lipoprotein (LDL) concentration was calculated by CBAL using the
Friedmann calculation: LDL = (total cholesterol – triglycerides) ÷ (2.2 - HDL cholesterol). If
a triglyceride result was more than 4.5mmol/L then LDL cholesterol was not calculated
and no LDL cholesterol result was recorded.

Q.3.4.1 Internal QC for total cholesterol and HDL cholesterol

Internal QC material was Bio-Rad Liquid Unassayed Multiqual Levels 1 (QC1) and 3 (QC2)
(product code 697). Results are shown in tables Q.8 to Q.9 for total and HDL cholesterol
for Years 12 to 15, respectively.

Table Q.8
Internal QC for total cholesterol (NDNS years 12 to 15)
Total
cholesterol QC
Year 12
QC1
Year 12
QC2
Years 13 to 15
QC1
Years 13 to 15
QC2
Lot no.
47991
47993
xx-xxxx-xxxx
56701 47993 56673
56703

8

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Mean (mmol/L)
2.8
7.1
2.8
2.7
2.7
7.3
6.9
6.9
SD (mmol/L)
0.10
0.15
0.08
0.09
0.13
0.17
0.11
0.14
Per cent CV
3.42
2.13
2.86
3.44
4.82
2.34
1.60
2.07
n
69
69
30
93
69
28
91
68

Table Q.9
Internal QC for HDL cholesterol (NDNS years 12 to 15)
HDL cholesterol
QC
Year 12
QC1
Year 12
QC2
Years 13 to 15
QC1
Years 13 to 15
QC2
Lot no.
47991
47993 47991 56671
56701
xx-xxxx-xxxx
56703
Mean (mmol/L)
0.6
1.4
0.6
0.7
0.7
1.4
1.5
1.5
SD (mmol/L)
0.03
0.05
0.02
0.02
0.03
0.05
0.07
0.06
Per cent CV
4.38
3.44
2.55
3.59
3.56
3.79
4.43
3.73
n
46
48
18
47
41
17
46
41

Q.3.4.2 External QA for total and HDL cholesterol

External QA was achieved through NEQAS (for lipid investigations) and the Randox
International QA Scheme (RIQAS) (lipid programme); NEQAS pooled samples are
unsuitable for the total cholesterol method used by the Siemens instruments. Tables Q.10
and Q.11 provide EQA results from RIQAS for total and HDL cholesterol, respectively, and
table Q.12 NEQAS results for HDL cholesterol.
Table Q.10 External RIQAS QA results for total cholesterol (NDNS years 12 to 15)
Total
cholesterol
RIQAS
Year 12
Cycle 42
Year 12
Cycle 43
2021
Cycle 44
2021
Cycle 45
2022
Cycle 46
2022
Cycle 47
2023
Cycle 48
Mean per cent
bias
-1.6
-0.5
0.1
-1.3
-1.0
-2.5
-1.6
SD of per cent
bias
1.73
1.49
2.75
1.23
1.59
0.91
2.76
n
12
12
11
11
12
12
12

Table Q.11 External RIQAS QA results for HDL cholesterol (NDNS years 12 to 15)
HDL
cholesterol
RIQAS
Year 12
Cycle 42
Year 12
Cycle 43
2021
Cycle 44
2021
Cycle 45
2022
Cycle 46
2022
Cycle 47
2023
Cycle 48

9

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Mean per cent
bias
0.7
0.2
-0.6
-0.8
-1.0
-0.8
-1.4
SD of per cent
bias
1.83
2.46
2.98
1.66
2.30
1.21
1.33
n
12
12
11
11
12
12
12

Table Q.12
External NEQAS QA results for HDL cholesterol (NDNS years 12 to 15)
HDL cholesterol
NEQAS
Year 12
2021
2022
2023
Mean per cent bias
-12.0
-4.0
+1.1
-1.9
SD of per cent bias
12.54
12.51
8.74
7.15
n
33
30
33
24

Q.3.5 Triglycerides (triacylglycerols)
Serum triglycerides were measured on a Siemens Dimension EXL200 analyser at CBAL.
The triglycerides (triacylglycerols) method is based on an enzymatic procedure in which a
combination of enzymes are employed for the measurement of serum or plasma
triglycerides. The sample is incubated with lipoprotein lipase (LPL) enzyme reagent that
converts triglycerides into free glycerol and fatty acids. Glycerol kinase (GK) catalyses the
phosphorylation of glycerol by adenosine-5-triphosphate (ATP) to glycerol-3-phosphate.
Glycerol-3-phosphate-oxidase oxidises glycerol-3-phosphate to dihydroxyacetone
phosphate and hydrogen peroxide (H2O2). The catalytic action of peroxidase (POD) forms
quinoneimine from H2O2, aminoantipyrine and 4-chlorophenol. The change in absorbance
due to the formation of quinoneimine is directly proportional to the total amount of glycerol
and its precursors in the sample and is measured using a bichromatic (510nm, 700nm)
endpoint technique.

Q.3.5.1 Internal QC for triglycerides
Internal QC material was Bio-Rad Liquid Unassayed Multiqual Levels 1 (QC 1) and 3 (QC
2) (product code 697). Data are reported for years 12 to 15 (table Q.13).

Table Q.13 Internal QC for triglycerides (NDNS years 12 to 15)
Triglycerides QC
Year 12
QC1
Year 12
QC2
Years 13 to 15 QC1
Years 13 to 15 QC2
Lot no.
47991
47993 47991 56671
56701
xx-xxxx-xxxx
56703
Mean (mmol/L)
1.0
2.4
1.0
0.96
0.94
2.4
2.4
2.4
SD (mmol/L)
0.04
0.08
0.02
0.04
0.05
0.10
0.07
0.09
Per cent CV
3.69
3.33
2.24
3.78
4.91
4.30
2.98
3.79
n
76
76
33
94
66
32
93
68

Q.3.5.2 External QA for triglycerides

External QA was achieved through RIQAS (lipids programme) and NEQAS (for lipid

10

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

investigations). Table Q.14 indicates the percentage deviation of results obtained from the
target concentration.

Table Q.14 External RIQAS QA results for HDL cholesterol (NDNS years 12 to 15)
Triglycerides
RIQAS
Year 12
Cycle 42
Year 12
Cycle 43
2021
Cycle 44
2021
Cycle 45
2022
Cycle 46
2022
Cycle 47
2023
Cycle 48
Mean per cent bias
0.2
0.6
2.7
-0.9
-2.3
-1.1
0.0
SD of per cent bias
1.52
1.89
6.55
2.47
1.99
2.09
1.88
n
12
12
11
11
12
12
12

Table Q.15 External NEQAS QA results for HDL cholesterol (NDNS years 12 to 15)
Triglycerides
NEQAS
Year 12
2021
2022
2023
Mean per cent bias
-3.9
-4.2
0.5
-5.3
SD of per cent bias
3.85
3.78
17.03
10.33
n
33
33
36
24

Q.3.6 Erythrocyte transketolase activation coefficient (ETKAC) for thiamin
(vitamin B1) status

Thiamin diphosphate (ThDP) is a cofactor for the erythrocyte transketolase enzyme.
Measurement of transketolase activity in washed red blood cells before and after the
addition of exogenous ThDP, and calculation of the activity coefficient, provides a
measure of the saturation of the transketolase enzyme and consequently, thiamine
status. Erythrocyte transketolase activation coefficient (ETKAC) is a sensitive and specific
marker of thiamin status. Analysis was performed on a Thermo Multiskan FC plate reader.
The method details as performed for NDNS are published (Jones and others, 2021).

In thiamin sufficiency, addition of exogenous ThDP will make little difference to the
enzyme activity and an ETKAC close to 1 will be obtained. In thiamin insufficiency or
deficiency, the addition of exogenous ThDP has a progressively greater effect on ETKAC,
with higher values indicating a higher risk of thiamin deficiency. Although there is no
international consensus on cutoffs, the commonly used threshold for risk of deficiency is
an ETKAC of >1.25 (EFSA, 2016).
There are no available sources of erythrocytes with known ETKAC; therefore, QC material
was prepared in-house. Erythrocytes obtained from the National Blood Transfusion
Service (NBTS) or commercial sources were washed to remove the buffy coat and lysed
by threefold dilution with normal saline. This lysate was stored at -70°C in single-use
aliquots. The lysate was stored and assayed both neat and further diluted 2 times with
water. No source of thiamin deficient erythrocytes has been identified in sufficient quantity
with which to prepare a lysate giving high ETKAC.
QC material `S` and LR160223 are dilute lysates in which the reaction rates are very low
and are included to assess assay performance in similarly dilute samples.

11

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.6.1 QC for ETKAC

Descriptive statistics in table Q.16 for internal QCs indicate good batch-to-batch
consistency of ETKAC results during years 12 to 15.

There are no external QA schemes available for ETKAC.

Table Q.16
Internal QC for ETKAC (NDNS years 12 to 15)
ETKAC
QC

Year 12
QC C
Year 12
QC S
Year 12
QC Q
Year 13
to 15
QC C
Years
13 to 15
QC S
Year 13
to 15
QC Q
Year 13 to
15
LR160223
Year 13 to
15
Q160223
Mean
1.08
1.18
1.22
1.08
1.18
1.19
1.13
1.16
SD
0.03
0.05
0.02
0.03
0.04
0.03
0.02
0.02
Per cent
CV
2.9
4.1
1.6
2.4
3.7
2.8
2.1
2.1
n
8
8
8
49
28
37
19
19

Q.3.7 Erythrocyte glutathione reductase activation coefficient (EGRAC)
for riboflavin (vitamin B2) status

Riboflavin (vitamin B2) is a component of the co-enzyme, flavin adenine dinucleotide
(FAD). As for thiamin and ETKAC in section Q.3.6 the activity coefficient of erythrocyte
glutathione reductase (EGRAC), a FAD-dependent enzyme, is a biomarker of riboflavin
status. The EGRAC is calculated from the ratio of unstimulated (‘basal’) EGR to
stimulated (‘activated’) EGR with exogenous FAD. The preincubation with FAD is carried
out for a relatively long period, 30 minutes at 37°C, in order to ensure full reactivation of
apo-enzyme, and, at a low final concentration of FAD (1.5μM), to eliminate activation
coefficients (ratios) <1.0; this can result from enzyme inhibition by FAD, or its breakdown
products, which may occur if the final concentration of FAD is too high. The generally
accepted threshold for riboflavin adequacy is EGRAC below 1.30 (EEFSA, 2017).
Analysis was performed on a Thermo Multiskan FC plate reader following a published
method (Parkington and others, 2023).

Q.3.7.1 QC for EGRAC

There is no QC material available with known EGRAC, therefore washed erythrocytes
were prepared in-house. Erythrocytes obtained from the National Blood Transfusion
Service (NBTS) or commercial sources were washed to remove the buffy coat and lysed
by threefold dilution with normal saline. This lysate was stored at -70°C in single-use
aliquots. In addition to the native samples a saturated control was made by incubation
with FAD before aliquoting (Control X). These 3 controls were run on each assay plate.
There is no external QA scheme available for EGRAC.

Descriptive statistics in table Q.17 for internal QCs indicate good batch-to-batch
consistency of EGRAC results during years 12 to 15.

12

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

There are no external QA schemes available for EGRAC.

Table Q.17
Internal QC for EGRAC (NDNS years 12 to 15)
EGRAC QC Year 12
QC X
Year 12
QC C
Year 12
QC A
Year 13 to 15
QC X
Year 13 to 15
QC C
Year 13 to 15
QC A
Mean (ratio)
0.99
1.49
2.17
0.98
1.57
2.34
SD
0.01
0.06
0.10
0.02
0.05
0.10
Per cent CV
0.7
4.1
4.4
2.3
3.2
4.1
n
9
9
9
66
66
65

Q.3.8
Plasma vitamin B6 (Pyridoxal-5-phosphate and 4-pyridoxic acid)

The assay for vitamin B6, based on that published by Rybak and Pfeiffer (2004), uses a
metaphosphoric acid protein precipitation followed by filtration of the supernatant to
prepare plasma samples for analysis. Detection of pyridoxal-5-phosphate (PLP) and 4-
pyridoxic acid (PA) is accomplished by HPLC with fluorescence detection employing a
post-column derivatisation step with sodium chlorite.
Analytes are resolved using reversed phase HPLC on a BDS hypersil, C18 3 × 150 mm 5
µm column at 35°C with a gradient mobile phase combining 50mM sodium dihydrogen
phosphate buffer at pH 2.7 plus 0.2% acetonitrile and methanol at 0.7mL/min.

Q.3.8.1
Internal QC for vitamin B6

In years 12 to 15 three in-house plasma controls containing endogenous vitamin B6
across the assay range for both PLP and PA and a spiked plasma sample with a
50nmol/L nominal concentration were analysed at the beginning and at the end of each
batch of samples. Years 12 to 15 QC data are summarised for PLP and PA in tables Q.18
and Q.19.

Table Q.18
Internal QC for PLP (NDNS years 12 to 15)
PLP QC

Year 12
A02
Year 12
A07
Year 12
PA Old
Year 12
Spike QC
Years 13
to 15
A02
Years 13
to 15
A07
Years 13
to 15
PA Old
Years 13
to 15
Spike
Mean
(nmol/L)
48.0
26.6
14.0
52.5
48.5
25.0
11.7
53.2

13

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

SD
(nmol/L)
2.6
1.5
1.3
2.5
2.1
1.7
1.5
3.1
Per cent
CV
5.3
5.5
9.5
4.7
4.4
6.9
12.5
5.8
n
10
10
10
10
51
51
49
51

Table Q.19
Internal QC for PA (NDNS years 12 to 15)
PA QC
Year 12
A02
Year 12
A07
Year 12
PA Old
Year 12
Spike QC
Years 13
to 15
A02
Years 13
to 15
A07
Years 13
to 15
PA Old
Years 13
to 15
Spike
Mean
(nmol/L)
120.1
12.6
36.6
51.8
128.2
13.4
37.4
53.5
SD
(nmol/L)
8.8
0.7
2.2
2.9
5.0
1.2
1.1
2.7
Per cent CV
7.3
5.7
6.0
5.5
3.9
9.2
3.0
5.1
n
10
10
10
10
51
51
49
51

Q.3.8.2
Accuracy for vitamin B6

Standard Reference Material (SRM) 1950 from the National Institute of Standards and
Technology (NIST) was assayed once with every run to assess the accuracy of the assay
(table Q.20).

Table Q.20
Accuracy for PLP and PA (NDNS years 12 to 15)
Vitamin B6
SRM 1950
Year 12 PLP
Year 12 PA
Years 13 to
15 PLP
Years 13 to
15 PA
Mean (nmol/L)
34.2
26.9
34.9
28.2
SD (nmol/L)
2.2
0.3
2.7
1.3
Per cent CV
6.4
1.1
7.8
4.8
n
3
3
12
12
Target value (nmol/L)
33.17 ± 1.86
28.71
33.17 ± 1.86
28.72

Q.3.8.3
External QA for PLP

The NBL participated in the Royal College of Pathologists of Australasia Quality Assurance
Program (RCPAQAP). There are only 6 participants in the scheme, the majority of whom use
the Chromsystems complete HPLC kit (Chromsystems, Germany) reporting PLP. There is no
external QA scheme for PA. Table Q.21 provides a summary of EQA data for years 12 to 15.

1 Values do not have an associated measurement uncertainty
2 Values do not have an associated measurement uncertainty

14

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Due to laboratory closures associated with the COVID-19 pandemic, no EQA material was
analysed for year 12, however accuracy of the method was validated through inclusion of
NIST SRMs as described above.

Table Q.21 External QA for vitamin B6 (RCPQAP) (NDNS years 12 to 15)
Vitamin B6 (PLP)
RCPAQAP
Year 12
Years 13 to 15
Mean bias (per cent ) 3
-
-10.8
SD of bias
-
9.9
Mean Z-score4
-
-0.72
n
-
8

Q.3.9
Serum folate

The assay for serum total folate uses solid phase extraction with phenyl columns to isolate
the folate vitamers from serum samples (Meadows. 2017). Analyte matched stable isotope
labelled internal standards are added during the extraction step and undergo processing
identical to the analytes thereby normalizing for sample preparation and instrument
variability. Highly specific detection of the 6 vitamers; methyltetrahydrofolate (MTHF),
tetrahydrofolate (THF), formyltetrahydrofolate (FTHF), folic acid, 5,10-
methenyltetrahydrofolate (CH+THF) and pyrazino-s-triazine derivative of 4α-hydroxy-5-
methyl tetrahydrofolate (MeFox) and internal standards is accomplished by UPLC-MS/MS
analysis and the ratio of analyte to internal standard signal is compared to that of a
calibration curve to determine analyte concentration.

Analytes are separated using reversed phase UPLC on a Waters ACQUITY UPLC® HSS
T3 C8 2.1 × 100 mm 1.7 µm column at 30°C with a 49.5:40:10:0.5
water:methanol:acetonitrile:acetic acid isocratic mobile phase prior to mass spectrometry
analysis. In NDNS, serum total folate is calculated from the sum of the 6 folate vitamers,
including MeFox.

Q.3.9.1
Internal QC for serum folate

In-house plasma controls containing endogenous folates across the assay range for
MTHF, folic acid and MeFox and a spiked serum sample with a 5 nmol/L nominal
concentration for FTHF, THF and CH+THF with endogenous levels of MTHF, folic acid
and MeFox were analysed at the beginning and at the end of each batch of samples for
assessment of assay precision. Tables Q.22 to Q.25 provide a summary of QC data for
years 12 to 15.
Table Q.22 Internal QC for MTHF (NDNS years 12 to 15)
MTHF QC
Year 12
QC 1
100118
Year 12
QC 3
100118
Year 12
Spike QC
170418
Year 12
QC 4
070220
Year 13 to
15 Spike
QC 170418
Year 13 to
15 QC 4
070220
Year 13 to
15 QC 6
151020

3 Z score = (lab result - all results mean) ÷ all results SD
4 Z score = (lab result - all results mean) ÷ all results SD

15

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Mean
(nmol/L)
27.2
17.2
62.0
8.9
62.7
9.4
26.3
SD (nmol/L)
1.38
1.24
4.85
0.44
3.38
0.50
1.33
Per cent CV
5.1
7.2
7.8
4.1
5.38
5.31
5.06
n
4
4
6
6
27
27
27

Table Q.23
Internal QC for folic acid (NDNS years 12 to 15)
Folic acid QC Year 12
QC 1
100118
Year 12
QC 3
100118
Year 12
Spike QC
170418
Year 12
QC 4
070220
Year 13 to
15 Spike
QC 170418
Year 13 to
15 QC 4
070220
Year 13 to
15 QC 6
151020
Mean
(nmol/L)
7.6
0.6
5.0
1.6
5.1
1.6
0.75
SD (nmol/L)
0.20
0.14
0.28
0.23
0.38
0.16
0.11
Per cent CV
2.6
23.5
5.7
14.3
7.5
9.9
15.0
n
4
4
6
6
27
27
27

Table Q.24
Internal QC for MeFox (NDNS years 12 to 15)
MeFox QC
Year 12
QC 1
100118
Year 12
QC 3
100118
Year 12
Spike QC
170418
Year 12
QC 4
070220
Year 13 to
15 Spike
QC 170418
Year 13 to
15 QC 4
070220
Year 13 to
15 QC 6
151020
Mean
(nmol/L)
6.3
1.2
8.2
6.4
8.3
6.5
6.4
SD (nmol/L)
0.65
0.09
0.37
0.47
0.46
0.29
0.29
Per cent CV
10.4
7.7
4.5
7.3
5.5
4.4
4.5
n
4
4
6
6
27
27
27

Table Q.25
Internal QC for other folates (NDNS years 12 to 15)
Other folates
QC
Year 12 QC
170418
CH+THF
Year 12 QC
170418
THF
Year 12 QC
170418
FTHF
Year 13 to
15 QC
170418
CH+THF
Year 13 to
15 QC
170418
THF
Year 13 to
15 QC
170418
FTHF
Mean
(nmol/L)
5.2
5.5
5.7
5.3
5.4
6.2
SD (nmol/L)
0.30
0.43
0.30
0.44
0.45
0.58
Per cent CV
5.7
7.9
5.3
8.4
9.32
0.58
n
6
6
6
27
27
27

Q.3.9.2
Accuracy of serum folate

NIST SRM 1950 was assayed with every run to assess and monitor the accuracy of the
assay for MTHF and folic acid. Results are presented in table Q.26.
Table Q.26
Accuracy for serum folate (NDNS years 12 to 15)
Serum folate
SRM 1950
Year 12
MTHF
Years 13 to 15
MTHF
Year 12
Folic acid
Years 13 to 15
Folic acid

16

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Mean (nmol/L)
27.1
27.6
3.1
3.0
SD (nmol/L)
0.93
1.22
0.19
0.19
Per cent CV
3.4
4.4
6.2
6.5
n
3
14
3
14
SRM target values
(nmol/L)
26.91 ± 0.7
26.91 ± 0.7
3.42 ± 1.02
3.42 ± 1.02

Q.3.9.3
External QA of serum folate

The NBL participated in NEQAS and VITAL quality assurance schemes. Table Q.27 shows
the NEQAS scores for year 12 and years 13 to 15. Due to laboratory closures associated
with the COVID-19 pandemic, no VITAL EQA material was analysed for year 12. However,
accuracy of the method was validated through inclusion of NIST SRMs as described
above. VITAL-EQA samples were received twice a year and had total folate target
concentrations assigned using the ‘gold standard’ microbiological method run by CDC.
Rounds 38, 39 and 40 were run during years 13 to 15 (table Q.28).
Table Q.27
External QA (NEQAS) for serum folate (NDNS years 12 to 15)
Total folate
NEQAS
Year 12
Year 13 to 15
Limits
A
71
100
200
B (per cent)
-5.4
-3.8
±25
C (per cent)
19.6
26.1
25

Table Q.28 External QA (VITAL) for serum folate (NDNS years 13 to 15)
Total folate
VITAL
Round
38 S1
Round
38 S2
Round
38 S3
Round
39 S1
Round
39 S2
Round
39 S3
Round
40 S1
Round
40 S2
Round
40 S3
Mean
(ng/mL)
6.8
8.3
22.6
5.5
6.4
8.0
3.0
9.3
12.3
Imprecision
(per cent)
5.7
6.3
3.8
5.1
7.8
1.8
9.4
5.4
1.1
Bias (per
cent)
-9.8
-17.4
5.7
-15.6
-17.4
-12.4
-1.4
1.9
4.1
Target value
(ng/mL)
7.5
10.0
21.4
6.5
7.7
9.1
3.0
9.1
11.8

Q.3.10 Whole blood (WB) folate

Whole blood (WB) haemolysate specimens (WB diluted and stabilised with ascorbic acid,
1/11) were analysed for total folate at CDC in years 12 to 15 using the Lactobacillus
rhamnosus microbiologic growth assay by an adaptation of O’Broin and Kelleher (1992)
and Molloy and Scott (1997) as described by Pfeiffer and others (2011).
Aliquots of whole blood (WB) samples were preserved with 1% ascorbic acid (appendix P)
and frozen at -70 °C before sending on dry ice to CDC.
Diluted specimen (4 replicates, duplicates at 2 dilutions) was added to an assay medium
containing the microorganism and all of the nutrients, except for folate, necessary for the

17

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

growth of the microorganism. Since the growth of L. rhamnosus is proportional to the
amount of total folate present in the specimen, the total folate level was assessed by
measuring the turbidity of the inoculated medium at 590nm in a microplate reader. The
assay was calibrated with 5-methyl-tetrahydrofolate (MTHF), using an 11-point calibration
curve (0–1.0 nmol/L; 8 replicates/point) with a 3rd degree polynomial curve fit.
Sample dilutions with a concentration below the lowest calibrator or above the highest
calibrator were repeated for confirmation, at lower or higher dilution. The standard
dilution used for WB haemolysate specimens in this study was 1/70.
Results from the 4 replicates were averaged to generate the final result and the CV from
the 4 replicates had to be ≤15% (≤10% if only 3 replicates were used). No result was
reported from less than 3 replicates. Assays were repeated where necessary.
Samples with a WB folate concentration <127nmol/L (corresponding to a RBC folate
concentration of <305nmol/L RBC if a haematocrit of 0.4L/L is assumed) were considered
to represent potential folate deficiency and samples were repeated for confirmation. This
is the standard practice in the CDC laboratory.
Accuracy has been established by spiked recovery and by periodic assaying of the
International Standard for Whole Blood Folate (NIBSC World Health Organization (WHO)
standard (95/528))

Q.3.10.1 Internal QC for WB folate

QC limits for WB folate were established by duplicate analysis of 3 in-house
quality control pools. The 3 WB bench QC pools were analysed in duplicate in
each run (n=5 in year 12 and n=10 in years 13 to 15). The between-run
imprecision for WB folate and the target concentration are shown in table Q.29.

18

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.29 Internal QC for WB folate (NDNS years 12 to 15)
WB folate QC

LB14810_MA
Year 12
LB14810_MA
Year 13 to 15 A
LB14810_MA
Year 13 to 15 B
MB14811_MA
Year 12
MB14811_MA
Year 13 to 15 A
MB14811_MA
Year 13 to 15 B
HB14812_MA
Year 12
HB14812_MA
Year 13 to 15 A
HB14812_MA
Year 13 to 15 B
Mean (nmol/L)
264
256
249
438
433
418
729
725
702
SD (nmol/L)
21
12
23
18
19
25
35
34
31
Per cent CV
7.9
4.7
9.3
4.1
4.5
5.9
4.8
4.6
4.4
Target
(nmol/L)
254
254
254
443
443
443
745
745
745
Difference
from target,
per cent
3.8
1.0
-2.1
-1.1
-2.2
-5.6
-2.2
-2.7
-5.8

Four additional WB QC pools were analysed `blind` (that is target concentration unknown
to analyst) as part of this study at a rate of 1 blind QC sample in every 20 unknown
samples. The between-run imprecision and target concentration are shown in table Q.30.

Table Q.30 WB folate blind QC pool analysis (NDNS years 12 to 15)
WB folate
blind QC
936 Year 12
936 Year 13 to 15
A
936 Year 13 to 15
B
937 Year 12
937 Year 13 to 15
A
937 Year 13 to 15
B
938 Year 12
938 Year 13 to 15
A
938 Year 13 to 15
B
939 Year 12
939 Year 13 to 15
A
939 Year 13 to 15
B
Mean
(nmol/L)
529
493
512
301
260
249
416
385
367
200
214
178
SD (nmol/L)
20
32
20
-
23
17
18
27
19
12
-
10
Per cent CV
3.7
6.5
4.0
-
8.9
6.7
4.3
6.9
5.2
6.0
-
5.5
n
4
7
5
1
4
4
4
5
3
3
1
8
Target
(nmol/L)
527
527
527
256
256
256
354
354
354
187
187
187
Difference
from target,
per cent
0.4
6.3
-2.7
17.5
1.7
-2.8
17.5
8.7
3.5
7.3
14.7
-4.5

Q.3.10.2 Calculation of red blood cell (RBC) folate

Red blood cell (RBC) folate is calculated from WB folate concentration, serum
folate concentration and haematocrit (quantified as part of the full blood count)
using the equation:
RBC folate = (WB folate - (serum folate × (1 - haematocrit))) ÷ haematocrit

19

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Where a serum folate concentration was not available a surrogate of 18nmol/L
was used in the calculation. Where haematocrit was not available, a surrogate of
0.4L/L was used. If neither serum folate or haematocrit were available then no
RBC folate was calculated.
For the calculation of RBC folate, if the WB folate concentration was less than the limit of
detection (44nmol/L) then a value of 31nmol/L (calculated as the LOD divided by the
sqrt(2)) was assigned to that sample for the calculation of RBC folate. Where there was
no result for WB folate then the calculation of RBC was not performed.

Q.3.11 Vitamin B12

Serum vitamin B12 was measured at LRS. The ADVIA Centaur B12 assay is a competitive
immunoassay using direct chemiluminescence. Vitamin B12 from a participant’s sample
competes with vitamin B12 labelled with acridinium ester for a limited amount of labelled
intrinsic factor. The intrinsic factor is covalently bound to paramagnetic particles. The
assay uses a releasing agent (sodium hydroxide) and dithiothreitol (DTT) to release the
B12 from the endogenous binding proteins in the sample.

Q.3.11.1 Internal QC for vitamin B12

The QC results of the instrument measuring Vitamin B12 at LRS are monitored continually,
and analysis is stopped if the results are not satisfactory, ensuring that results are only
reported if the analysis is within the QC parameters set by the Laboratory Manager.
However, it is not possible to extract these results for NDNS reporting. Therefore in order
to monitor between-batch precision, control material was aliquoted and included in the
regular delivery of NDNS samples to LRS. Control material was purchased from Bio-Rad
and was chosen to reflect the full range of vitamin B12 concentrations. Different batches of
control material (indicated by underscore) are used across NDNS years as batches expire
or run out which allows monitoring of continuity between NDNS years. QC data are
summarised in table Q.31.

Table Q.31
Internal QC for vitamin B12 (NDNS years 12 to 15)
Vitamin
B12
QC
QC1_1
Y12
QC1_1
Y13 to
15
QC2
Y12
QC2
Y13 to
15
QC3
Y12
QC3
Y13 to
15
QC4
Y12
QC4
Y13 to
15
QC5
Y12
QC5
Y13
to15
QC1_2
Y12
QC1_2
Y13to
15
Mean
(ng/L)
155
164
176
193
124
141
612
602
1017
1052
-
181
SD
(ng/L)
23
20
11
27
17
25
33
49
52
91
-
24
Per
cent
CV
15
12
6
14
13
18
5
8
5
9
-
13
n
7
19
7
38
7
39
7
39
7
39
-
25
Abbreviations: QC1: Lyphochek Anemia Control, single level QC2: Liquid Assayed
Multiqual, Level 2 QC3: Lyphochek Assayed Chemistry Control, Level 2 QC4: Lyphochek
Immunoassay Plus Control, Level 2 QC5: Lyphochek Immunoassay Plus Control, Level 3

20

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.11.2 External QA for vitamin B12

External QA was achieved through the UK NEQAS Haematinics scheme (table Q.32).

Table Q.32 Vitamin B12 NEQAS (NDNS years 12 to 15)
Vitamin B12
NEQAS
Year 12
Years 13 to 15
Limits
A
52
81
200
B (per cent)
+3.0
+4.0
± 20
C (per cent)
7.3
10.8
15.0

Q.3.12 Holotranscobalamin (holoTC); `active` B12`

Vitamin B12 (cobalamin) is transported in the circulation bound to transcobalamin (TC) (10
to 30%) and to haptocorrin (HC) (70 to 90%). When TC and HC bind vitamin B12 the
resulting complexes are known as holotranscobalamin (holoTC) and holohaptocorrin
(holoHC) to distinguish them from the proteins carrying no vitamin.
The serum holoTC assay is an enzyme-linked immunosorbent assay (ELISA)
manufactured by Axis Shield. It is conducted in 96-well microplates. HoloTC reacts with a
specific antibody immobilised on the plate surface; a second, labelled antibody then reacts
to form a `sandwich`. The enzyme label is quantitated using a coloured substrate and the
absorbance read in a microplate spectrophotometer.

Q.3.12.1 Internal QC for holoTC

Two sets of QC samples were measured. Those supplied by the manufacturer were
measured in duplicate with every assay. Measured values were always with the limits set by
the manufacturer for different lot numbers and per cent CV within lots was less than 5%.
Three serum QC samples spanning the normal range were prepared in-house were used to
monitor lot to lot variation. Additional serum QC samples (QC D, E and F) were introduced
in years 13 to 15 due to limited remaining aliquots of QC A, B and C. Results are provided
in Table Q.33. In year 12, analysis was performed on a single ELISA lot. In years 13 to 15,
QCs A, B and C were measured on 2 ELISA lots and QCs D, E and F on 4 ELISA lots.
Between lot variation was <10%.

21

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.33 Internal QC for holoTC (NDNS year 12 to 15).
HoloTC QC
Year
12
QC A
Year
12
QC B
Year
12
QC C
Year
13 to 15
QC A
Year
13 to 15
QC B
Year
13 to 15
QC C
Year
13 to 15
QC D
Year
13 to 15
QC E
Year
13 to 15
QC F
Mean (pmol/L)
33.7
55.0 78.6
47.8
60.1
101.1
42.2
66.1
93.5
SD (pmol/L)
1.3
1.5
1.8
3.4
6.0
6.5
7.9
7.6
9.1
Per cent CV
3.7
2.7
2.2
7.2
10.0
6.4
18.6
11.5
9.7
n
6
6
6
24
25
25
23
23
23

Q.3.12.2 External QA for holoTC

In each survey year, analysis of samples for holoTC was performed as batches over a short
time scale and therefore did not allow for `live` participation in the EQA scheme. Three
samples are received per NEQAS distribution (4 distributions per year). NEQAS samples
were stored at -70°C and assayed retrospectively alongside respective samples for each
year, the results being compared with the NEQAS ALTM for results obtained by all
participating laboratories (table Q.34), most of whom used automated methods.

Table Q.34 External QA for holoTC (NDNS years 12 to 15)
HoloTC
NEQAS5
Year 12
Years 13 to 15
Bias against ALTM (per cent)
-17 (10)
6 (27)
Bias against method mean (per cent) -24 (11)
15 (27)
n
18
65
Q.3.12.3 Accuracy for holoTC

In year 13, NIBSC World Health Organization (WHO) international standard (03-178) was
also run regularly. The standard 03/178 has an assigned consensus holoTC value of
107pmol/L. Performance of the assay against the consensus value for holoTC is reported in
table Q.35.

Table Q.35 Performance against WHO International Standard for holoTC (NDNS
years 13 to 15)
HoloTC
NIBSC (03-178) Accuracy6
Years 13 to 15
Consensus (target) value, pmol/L
107
Mean (SD), pmol/L
109 (6)
Per cent CV
6
n
19
Mean (SD) bias, per cent
2 (6)

Q.3.13 Vitamin C (ascorbic acid)

This assay is based on the procedure described by Vuilleumier and Keck (1989). Samples
are stabilised immediately after separation using an equal volume of 10%
metaphosphoric acid and plasma (see appendix P).

5 Bias was calculated against the target value as % bias = (result - target)/target) × 100
6 Bias was calculated against the target value as % bias = (result - target)/target) × 100

22

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Vitamin C (ascorbic acid) in the sample is converted to dehydroascorbic acid by
ascorbate oxidase, followed by coupling of the resulting dehydroascorbate with o-
phenylene diamine to form a fluorescent derivative quinoxaline. The formation of
quinoxaline is linearly related to the amount of vitamin C in the sample. The assay was
performed on the BMG Labtech FLUOstar Omega plate reader.

Q.3.13.1 Internal QC for vitamin C

QC samples were made in-house by spiking vitamin C-depleted plasma. Results are
provided in table Q.36.
Table Q.36 Internal QC for vitamin C (NDNS years 12 to 15)
Vitamin C
QC
Year
12
QC1
Year
12
QC2
Year
12
QC3
Years
13 to 15
QC1
Years
13 to 15
QC2
Years
13 to 15
QC3
Years
13 to 15
QC1
Years
13 to 15
QC2
Years
13 to 15
QC3
Mean
(µmol/L)
10.5 34.1 50.9
10.8
30.8
47.8
14.1
26.8
55.1
SD (µmol/L)
0.9
3.7
1.5
0.7
1.7
2.8
1.2
2.0
3.8
Per cent CV
8
11
3
6
6
6
9
7
7
n
12
12
12
7
77
7
64
64
64

Q.3.13.2 External QA for vitamin C

NBL participated in the Royal College of Pathologists of Australasia Quality Assurance
Program (RCPAQAP). The bias is calculated from the ALTM. The majority of the
participating laboratories use the Chromsystems HPLC method and calibrator, which are
different from the fluorescence method and calibrator used in the NDNS; results from NBL
show a bias relative to the Chromsystems method (table Q.37). The NBL calibrators were
prepared gravimetrically from the AnalaR ascorbic acid, the purest grade available. This
methodological difference shows as a deviation of NBL results from the Chromsystems
consensus. The consistency of the bias indicates consistency in NBL results, as does the
low ‘per cent imprecision’ result.

Table Q.37 External QA for vitamin C (NDNS years 12 to 15)
Vitamin C
RCPAQAP
Year 12
Cycle 41
Year 12
Cycle 42
Years 13 to 15
Cycle 22
Years 13 to 15
Cycle 23
Mean Bias (per
cent)7
-23
-19
-29
-27
Mean bias
imprecision (SD)
3.1
8.7
4.0
4.1
n
8
14
17
16

7 Bias was calculated against target value as % bias = (result - target)/target) × 100 when measured value
was greater than the assay limit of quantitation. The reported target values were obtained from the end of
cycle reports and represent the median of all assay results submitted to the scheme

23

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.14 Retinol, − and −tocopherol, and individual carotenoids (fat-
soluble vitamins (FSVs)
The assay for FSVs is based on that published by Sowell and others (1994) and uses a
methanol protein precipitation step followed by liquid-liquid extraction in hexane to
separate fat soluble vitamins from other analytes in plasma samples. Internal standards
are added during the extraction step and undergo processing identical to the analytes
thereby normalising for sample preparation and instrument variability. Detection of the 9
analytes; retinol, α-tocopherol, γ-tocopherol, lutein and zeaxanthin (combined due to co-
elution), lycopene, α-cryptoxanthin, β-cryptoxanthin, α-carotene, β-carotene and internal
standards; tocopherol acetate and apo-8’-carotenal is accomplished by HPLC analysis
with PDA (UV, photo diode array) detection. The ratio of analyte to internal standard
signal is compared to that of a calibration curve to determine analyte concentration.

Analytes are resolved using reversed phase HPLC on a YMC-pack pro C18 4.6 x 150 mm 3
µm column at 40°C with a 25:75 ethanol + 0.1% triethylamine: acetonitrile + 0.1%
triethylamine isocratic mobile phase at 1.2mL/min.

Q.3.14.1 Internal QC for plasma retinol, − and −tocopherol and
individual carotenoids

Serum internal quality controls contained physiologically relevant concentrations of each
analyte; these were aliquoted for use in each analytical run. Between-batch precision was
calculated from these values, as for all other analytes measured in NDNS. Some QC
material used in previous NDNS years had limited remaining aliquots and therefore
during a review of FSV QCs, the use of some QC materials was discontinued, whilst
new QCs were introduced prepared from commercial sources of plasma. In order to
provide sufficient concentration range for the analytes in this assay, a larger number of
QC materials are required than for standard assays. Assay LOD is 0.09µmol/L for all
analytes except α-cryptoxanthin (0.05µmol/L). Tables Q.38 and Q.39 provide a
summary of QC data for year 12 and years 13 to 15, respectively.

24

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.38 Internal QC for fat soluble vitamins (NDNS year 12)8
Fat-soluble
vitamins QC

µmol/L
QC1
QC068
QC071
QC636775 QC (high)
Retinol

Mean
SD
Per cent CV
2.64
(0.17)
3%
0.97
(0.04)
4%
1.60
(0.08)
3%
2.35
(0.10)
4%
2.73
(0.08)
3%
α-tocopherol

Mean
SD
Per cent CV
34.81
(1.33)
4%
18.26
(0.62)
3%
17.01
(0.55)
3%
21.76
(0.43)
2%
32.49
(1.11)
3%
γ-tocopherol

Mean
SD
Per cent CV
2.32
(0.13)
6%
5.87
(0.27)
5%
3.88
(0.17)
4%
6.93
(0.18)
3%
2.01
(0.09)
5%
Lutein &
zeaxanthin

Mean
SD
Per cent CV
0.50
(0.02)
4%
0.16
(0.01)
5%
0.19
(0.005)
2%
0.21
(0.01)
4%
0.40
(0.02)
6%
α-cryptoxanthin

Mean
SD
Per cent CV
0.07
(0.01)
9%
0.03
(0.004)
12%
0.03
(0.004)
14%
0.03
(0.002)
8%
0.03
(0.005)
18%
β-cryptoxanthin

Mean
SD
Per cent CV
0.31
(0.02)
6%
0.14
(0.01)
8%
0.04
(0.004)
10%
0.07
(0.01)
8%
0.16
(0.01)
9%
Lycopene

Mean
SD
Per cent CV
0.49
(0.03)
6%
1.26
(0.11)
9%
1.23
(0.10)
8%
1.02
(0.07)
7%
0.75
(0.05)
6%
α-carotene

Mean
SD
Per cent CV
0.13
(0.01)
6%
Not
detected
0.05
(0.02)
44%
Not
detected
0.17
(0.01)
4%
β-carotene

Mean
SD
Per cent CV
0.77
(0.04)
5%
0.23
(0.01)
6%
0.28
(0.02)
8.4%
0.15
(0.01)
6%
1.25
(0.04)
04%

8 Data generated from 6 replicates

25

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.39 Internal QC for retinol (NDNS years 13 to 15)
Fat-soluble
vitamins
QC

µmol/L
QC068
(n 27)
QC071
(n 27)
QC636775
(n 26)
QC (high)
(n 23)
QC11237645
(n 27)
QC11237830
(n 27)
Retinol

Mean
SD
Per cent CV
0.90
0.09
11
1.54
0.10
6
2.30
0.19
8
2.68
0.21
8
1.56
0.13
8
2.30
0.16
7
α-tocopherol

Mean
SD
Per cent CV
19.4
1.75
9
17.9
0.54
3
22.3
0.95
4
33.5
0.84
3
25.4
1.13
4
37.8
4.1
11
γ-tocopherol

Mean
SD
Per cent CV
5.30
0.48
20
3.52
0.18
3
6.33
0.31
5
1.73
0.10
6
0.82
0.12
15
1.23
0.20
16
Lutein &
zeaxanthin

Mean
SD
Per cent CV
0.20
0.04
21
0.29
0.06
23
0.29
0.06
21
0.53
0.12
23
0.23
0.04
19
0.37
0.07
19
α-cryptoxanthin

Mean
SD
Per cent CV
0.05
0.01
21
0.04
0.01
23
0.04
0.01
23
0.04
0.01
27
0.04
0.01
22
0.08
0.01
12
β-cryptoxanthin

Mean
SD
Per cent CV
0.19
0.03
17.8
0.05
0.02
28
0.10
0.02
17
0.21
0.03
15
0.21
0.03
13
2.21
0.28
13
Lycopene

Mean
SD
Per cent CV
1.40
0.17
12
1.38
0.12
9
1.13
0.11
10
0.81
0.07
8
1.23
0.11
9
0.65
0.07
10
α-carotene

Mean
SD
Per cent CV
Not
detected
0.11
0.03
27
Not
detected
0.23
0.05
21
0.25
0.05
19
0.13
0.03
22
β-carotene

Mean
SD
Per cent CV
0.25
0.03
11
0.30
0.02
6
0.17
0.01
5
1.27
0.11
9
1.10
0.11
10
1.30
0.13
10

26

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.14.2
Accuracy for plasma retinol, − and −tocopherol and individual
carotenoids

NIST SRM 1950 was assayed with every run (n= 12) during years 13 to 15 to assess the
accuracy of the assay. Results are presented in table Q.40.
Table Q.40
Accuracy for FSVs assessed with NIST SRM 19509
Fat
soluble
vitamins
SRM 1950
Retinol
α-
tocopherol
γ-
tocopherol
Lutein and
zeaxanthin
Lycopene
β-
carotene
Mean
(µmol/L)
1.50
19.9
3.98
0.19
0.66
0.17
SD
(µmol/L)
0.09
0.74
0.21
0.2
0.08
0.01
Per cent CV
6
4
5
12
11
9
n
12
12
12
12
12
12
NIST
targets
(µmol/L)
1.41 ±
0.122
19.0 ±
0.511
4.1 ±
0.408
0.16 ±
0.049
0.615 ±
0.037
0.15 ±
0.07

Q.3.14.3 EQA for plasma retinol, − and −tocopherol and individual
carotenoids

NBL participated in the NEQAS and VITAL (retinol only) EQA schemes during NDNS
RP Years 12 to 15, although participation was affected by the break in laboratory work
during the COVID-19 period.

Table Q.41 shows NEQAS scores for year 12 and years 13 to 15. It should be noted that
there were 14 laboratories returning results for β-carotene and 4 laboratories returning
results for lutein + zeaxanthin and lycopene. NBL returned results for α-carotene and α-
and β-cryptoxanthins when possible but these were frequently below the limit of
detection so there are no cumulative scores consistently available for these assays.
Table Q.42 shows the VITAL EQA scheme results for retinol.

9 Lutein and zeaxanthin are not separated in the method used for NDNS and consequently the reported result
is for the combined concentration. For this comparison, we have therefore summed the target concentrations
for lutein and zeaxanthin reported for SRM 1950

27

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.41 External QA (NEQAS) for FSVs (NDNS years 12 to 15)
FSVs
NEQ
AS
Retin
ol
Y12
Retin
ol
Y13
to 15
α-
tocophe
rol Y12
α-
tocophe
rol Y13
to 15
Lutein
and
zeaxant
hin Y12
Lutein
and
zeaxant
hin Y13
to 15
Lycope
ne Y12
Lycope
ne Y13
to 15
β-
carote
ne
Y12
β-
carote
ne
Y13 to
15
Limi
ts
A
131
93
55
101
92
113
34
29
124
70
200
B
(per
9
-7.6
3.2
-4.6
-17.1
20.8
-1.3
9
20.9
8.4
±15
C
(per
10.9 5.5
3.5
6.7
5.3
10.4
17.8
10
13.2
11.3
20

Table Q.42 VITAL EQA performance for retinol (NDNS years 13 to 15)
VITAL EQA
- retinol
Round
38 S1
Round
38 S2
Round
38 S3
Round
39 S1
Round
39 S2
Round
39 S3
Round
40 S1
Round
40 S2
Round
40 S3
Mean (µg/dL)
39.3
45.4
52
29.5
43.9
45.4
43.6
49.9
48.8
Impression
(per cent)
13.5
10.3
0.3
6.7
5.6
1.2
16.7
3.3
2.9
Bias (per cent)
0.0
4.3
9.4
5.4
5.1
-2.8
1.7
6.4
0.0
Target (µg/L)
39.3
43.5
47.5
28
41.7
46.7
42.8
46.9
48.8

Q.3.15 25-hydroxyvitamin D (25OHD)

The assay for serum 25(OH)D uses a methanol protein precipitation step followed by
liquid-liquid extraction in hexane to separate 25OHD from other analytes in serum
samples. Stable isotope labelled internal standards are added during the extraction step
and undergo processing identical to the analytes thereby normalizing for sample
preparation and instrument variability. Highly specific detection of the analytes and internal
standards is accomplished by UPLC-MS/MS analysis using a Waters ACQUITY UPLC
system coupled to an AB Sciex QTrap mass spectrometer and the ratio of analyte to
internal standard signal is compared to that of a calibration curve to determine analyte
concentration.
Analytes are resolved using reversed phase UPLC on a Waters HSS PFP 2.1 × 100 mm
1.8 µm column at 40°C with a 72% methanol +0.1% formic acid isocratic mobile phase at
0.3L/min prior to mass spectrometry analysis.
Total 25(OH)D is calculated from the sum of 25(OH)D2 and 25(OH)D3. The epimer of 25
hydroxyvitamin D3 is not reported in NDNS but is chromatographically separated using this
method and so is not included in the total 25OHD.

Q.3.15.1 Internal QC for total 25(OH)D

28

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

QC material was Bio-Rad Lyphochek Specialist Immunoassay Control. The concentrations
of 25(OH)D2 and 25(OH)D3 controls covered the concentration range observed in NDNS;
QC material was analysed at the beginning and at the end of each batch of samples.
Tables Q.43 and Q.44 provide a summary of QC data for 25(OH)D2 and 25(OH)D3. An in-
house serum QC was also analysed and included C3-epimer 25(OH)D3 (table Q.45). In
NDNS, vitamin D status is reported as total 25(OH)D calculated from the sum of 25(OH)2
and 25(OH)D3. Where 25(OH)D2 concentration was below the assay LOQ (6nmol/L) and
above from the LOD, a concentration was recorded as LOQ/√2 (4.2nmol/L) to calculate
total 25(OH)D.

Table Q.43 Internal QC for 25OHD2 (NDNS years 12 to 15)
25(OH)D2
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Year
13 to
15
Bio-
Rad
QC
Year
13 to
15
Bio-
Rad
QC
Year
13 to
15
Bio-
Rad
QC
Year
13 to
15
Bio-
Rad
QC
Year
13 to
15
Bio-
Rad
QC
Year
13 to
15
Lot
25251
25252
25283
88701
88702
88703
88721
88722
88723
Mean
(nmol/L)
11.9
19.6
53.2
7.1
17.9
49.2
5.2
16.5
48.0
SD
(nmol/L)
1.10
0.71
2.20
0.91
1.30
2.31
0.94
2.36
4.86
Per cent
CV
9
4
4
13
7
5
18
14
10
n
8
8
7
20
20
18
14
16
16

Table Q.44 Internal QC for 25OHD3 (NDNS years 12 to 15)
25(OH)D3
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Year
12
Bio-
Rad
QC
Years
13 to
15
Bio-
Rad
QC
Years
13 to
15
Bio-
Rad
QC
Years
13 to
15
Bio-
Rad
QC
Years
13 to
15
Bio-
Rad
QC
Years
13 to
15
Bio-
Rad
QC
Years
13 to
15
Lot
25251
25252
25283
88701
88702
88703
88721
88722
88723
Mean
(nmol/L)
16.0
26.0
77.2
11.3
22.4
69.1
12.4
21.5
48.0
SD
(nmol/L)
0.71
0.61
2.7
0.87
1.39
3.85
1.49
1.67
4.86
Per cent
CV
5
2
4
8
6
6
12
8
10
n
8
8
7
20
20
18
16
16
16

29

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.45 In-house serum QC (NDNS years 12 to 15)
25(OH)D
QC
Year 12
25(OH)D2
Year 12
25(OH)D3
Year 12
C3-epimer
25(OH)D3
Years
13 to 15
25(OH)D2
Years
13 to 15
25(OH)D3
Years
13 to 15
C3-epimer
25(OH)D3
Mean
(nmol/L)
7.9
58.6
9.4
7.56
65.42
8.91
SD (nmol/L)
0.7
1.5
1.2
1.14
3.31
0.80
Per cent CV
9
3
13
15
5
9
n
8
8
8
33
34
33

Q.3.15.2 Accuracy for 25(OH)D

Samples from the Vitamin D EQA Scheme (DEQAS), which have concentrations assigned
by the reference measurement procedure at CDC, were analysed every quarter. Table
Q.46 summarises NBL performance in the DEQAS scheme.

Table Q.46 DEQAS performance (NDNS years 12 to 15)
25(OH)D
DEQAS
Year 12
Years 13 to 15
Mean bias (per
cent )
-7.1
-6.8
SD of per cent bias
4.7
9.9
n
5
23

Q.3.15.3 External QA for 25(OH)D
NBL participated in NEQAS and VITAL EQA schemes for vitamin D. Table Q.47 shows the
NEQAS scores. NEQAS performance is assessed against the mass spectrometry mean.
Table Q.48 summarises performance in the CDC’s VITAL EQA scheme against their
reference method.
Table Q.47 External EQA (NEQAS) performance for 25(OH)D (NDNS years 12 to
15)10
25(OH)D
NEQAS
Year 12
Year 13 to 15
Limits
A
Not applicable
Not applicable
Not applicable
B (per cent)
-7
-2.2
±20
C (per cent)
3.7
8.9
25

10 No A-score is currently reported by NEQAS for this EQA scheme

30

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.48
VITAL EQA performance for 25(OH)D (NDNS years 12 to 15)
VITAL EQA –
vitamin D
Round
38 S1
Round
38 S2
Round
38 S3
Round
39 S1
Round
39 S2
Round
39 S3
Round
40 S1
Round
40 S2
Round
40 S3
Mean (µg/L)
19.5
39.3
107
27.6
71.0
81.1
26.9
24.6
27.6
Impression (per
cent)
0.4
4.1
3.3
1.5
16.8
4.7
1.8
2.9
1.5
Bias (per cent)
-11.5
6.2
-2.1
-7.9
11.2
3
0.8
-8.3
1.7
Target (µg/L)
22
37
108.8
30
63.8
78.8
26.6
26.8
27.1

Q.3.16 Serum selenium and serum zinc

Analysis of selenium and zinc concentrations was sub-contracted to the Trace Element
Laboratory, UHS.
Selenium (Se) and zinc (Zn) concentrations in serum were determined by inductively
coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NEXION 300D,
equipped with a dynamic reaction cell (DRC).

Prior to analysis the ICP-MS instrument was tuned for optimum signal sensitivity and
minimum oxide species and doubly charged ion formation.
Serum was diluted 1 in 50 in 0.01% nitric acid for Zn and 0.5% butanol for Se with
rhodium (Rh) as internal standard and run against matrix matched calibration solutions
prepared with bovine serum (Sigma-Aldrich). 66Zn and 78Se were determined and the
analyte isotope signals were compared against the internal standard, to compensate for
matrix related effects and any signal fluctuation due to instrument drift. Se analysis
employed dynamic reaction cell (DRC) suppression to overcome argon-based
interferences, with methane as the DRC gas.
Trial samples, blanks, calibration standards and QCs were analysed in each batch and the
signal data generated was converted to concentrations via the calibration plot.

Q.3.16.1 Internal QC for selenium and zinc

Internal QC material was analysed in conjunction with the blanks, calibration standards
and samples. Tables Q.49 and Q.50 summarise QC data for selenium and zinc,
respectively.

31

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Table Q.49 Internal QC for selenium analysis (NDNS years 12 to 15)
Selenium
Year 12
AS 1
Year 12
AS 2
Year 12
AS 3
Years 13
to 15 AS 1
Years 13
to 15 AS 2
Years 13
to 15 AS 3
Target (µmol/L)
0.65
1.15
2.10
0.30
0.90
2.10
Acceptable
range (µmol/L)
0.50 to
0.74
0.95 to
1.35
1.85 to
2.35
0.15 to
0.45
0.70 to
1.10
1.80 to
2.40
Mean (µmol/L)
0.60
1.11
1.97
0.29
1.02
2.20
SD (µmol/L)
0.04
0.06
0.14
0.03
0.13
0.26
Per cent CV
7
6
7
12
13
12
n
18
14
15
60
56
53

Table Q.50 Internal QC for zinc analysis (NDNS years 12 to 15)
Zinc
Year
12
171
Year
12
172
Year
12
173
Years
13 to 15
171
Years
13 to 15
172
Years
13 to 15
173
Years
13 to 15
181
Years
13 to 15
182
Years
13 to 15
183
Target
(µmol/L)
5.0
11.0 21.0
5.0
11.0
21.0
4.1
12.7
19.8
Acceptable
range
(µmol/L)
3.0 to
7.0
9.0 to
13.0
17.0
to
25.0
3.0 to
7.0
9.0 to
13.0
17.0 to
25.0
3.2 to
5.0
10.2 to
15.3
16.2 to
23.4
Mean
(µmol/L)
5.27 10.41 20.85
5.38
11.41
20.61
4.50
13.08
19.76
SD
xx-xxxx-xxxx
0.80
0.90
1.27
0.44
1.09
2.04
Per cent CV
11
9
8
15
8
6
10
8
10
n
19
16
15
12
11
10
51
46
46

Q.3.16.2 EQA for selenium and zinc in serum

Southampton Trace Elements participates in TEQAS (NEQAS for Trace Elements,
Guildford). Performance in TEQAS scheme is shown in figures Q.2 and Q.3 for selenium
and zinc, respectively. Closed circles indicate the rolling average score and open circles
observed results for each round.

32

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Figure Q.2 TEQAS serum selenium performance March 2022 to March 2023

Figure Q.3 TEQAS serum zinc performance March 2022 to March 2023

33

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.17 Urinary iodine

Analysis of urinary iodine concentration was sub-contracted to the Trace Element
Laboratory, UHS. Urine iodine concentrations were determined by inductively coupled
plasma mass spectrometry (ICP-MS), using a Perkin Elmer NEXION 300D.
Urine samples and QC materials were diluted 1 in 25 in 0.33% blood diluent (ammonia
dihydrogen orthophosphate, EDTA and ammonia solution) with rhodium (Rh) as
internal standard. 127I isotope signals were measured and compared against the
internal standard, to compensate for matrix related effects and any signal fluctuation
due to instrument drift. Matrix matched external calibration standards were prepared in
human urine for each analytical batch.
Prior to analysis the ICP-MS instrument was tuned for optimum signal sensitivity and
minimum oxide species and doubly charged ion formation. Trial samples, blanks,
calibration standards and QCs were analysed in each batch and the signal data
generated converted to concentrations via the calibration plot.
Q.3.17.1
Internal QC for urinary iodine

In order to establish quality assurance of each analytical batch and inter-batch
variation, QC material (Seronorm trace elements urine was analysed in conjunction
with blanks, calibration standards and samples (table Q.51).

Table Q.51 Internal QC analysis urinary iodine (NDNS years 12 to 15)
Urinary iodine
(Seronorm urine) QC
Year 12
Level 1
Year 12
Level 2
Years 13 to 15
Level 1
Years 13 to 15
Level 2
Target concentration /
range (µmol/L)
0.83
(0.66 to 0.99)
2.30
(1.9 to 2.8)
1.34
(1.07 to 1.61)
4.50
(3.6 to 5.4)
Mean measured
concentration
(µmol/L)
0.76
2.21
1.18
3.88
SD (µmol/L)
0.05
0.13
0.04
0.11
Per cent CV
7
6
3
3
n
54
51
32
33

34

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Q.3.17.2
External QC for urinary iodine

During year 12, the laboratory participated in the Interlaboratory Comparison Program
for Metals in Biological Matrices (PCI). Results are provided in table Q. 52. Participation
in the PCI scheme was later discontinued and EQA subsequently provided by
participation in the Multi-Element External Quality Assurance Scheme (QMEQAS)
operated by Centre de Toxicologie du Québec at the Institut national de santé publique
du Québec (INSPQ) (table Q.53).
Table Q.52 EQA performance (PCI EQA scheme) for urinary iodine analysis
(NDNS year 12)
Urinary iodine
TEQAS
Sample I-2013
Sample I-2014
Sample I-2015
Result (µmol/L)
0.91
1.55
0.69
Target (µmol/L)
0.86
1.69
0.71
Per cent bias
5.5
-8.3
-3.2
Z-score
0.8
-1.2
-0.5

Table Q. 53 EQA performance (QMEQAS) for urinary iodine analysis (NDNS
years 13 to 15)
Urinary
iodine
QMEQAS
2204 2205 2206 2213 2214 2215 2304 2305 2306 2313 2314 2315
Result
(µg/L)
104 246 192
75 240
155
83
121 245
96
327 123
Z-score
2.2
1.8
2.6 -1.1 -1.0 -0.8 -0.1 -xx-xxxx-xxxx

35

National Diet and Nutrition Survey: 2019 to 2023 (years 12 to 15)

Acknowledgements
We would like to thank personnel at the following institutions for their assistance in
local sample processing during NDNS years 12 and 13 prior to and during the
evaluation of the postal processing model:

Aberdeen Royal Infirmary, Aberystwyth University, Basildon Hospital, Bristol Royal
Infirmary, Countess of Chester Hospital, University Hospital Coventry , Great
Western Hospital (Swindon), Hull Royal Infirmary, James Cook Hospital
(Middleborough), Kings Mill Hospital (Mansfield), Leicester Royal Infirmary,
Middlesex University, Midland Pathology Services, Nationwide Laboratories
Poulton, Nuffield Hospital Glasgow, Nuffield Hospital Newcastle, Queen Elizabeth
Hospital Woolwich, Royal Bournemouth Hospital, Royal Cornwall Hospital, Royal
Devon & Exeter Hospital, Royal Oldham Hospital, Royal Shrewsbury Hospital,
Royal Sussex Hospital (Brighton), Sheffield Hallam University, St James Hospital
(Leeds), UK Biocentre (Milton Keynes), University of East Anglia (Norwich),
University of Dundee, University of North Midlands, University of Surrey, Wellcome
Trust Clinical Research Facility Edinburgh, Wellcome Trust Clinical Research
Facility Manchester

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