Proficiency testing has improved the
quality of data of total vitamin B2 analysis
in liquid dietary supplement
Analytical and Bioanalytical
Chemistry
ISSN 1618-2642
Volume 400
Number 1
Anal Bioanal Chem (2011)
400:305-310
DOI 10.1007/
s00216-011-4726-8
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Author's personal copy
Anal Bioanal Chem (2011) 400:305–310
DOI 10.1007/s00216-011-4726-8
ORIGINAL PAPER
Proficiency testing has improved the quality of data of total
vitamin B2 analysis in liquid dietary supplement
Mark Sykes & Joanne Croucher & Rosemary Ann Smith
Received: 4 November 2010 / Revised: 24 January 2011 / Accepted: 25 January 2011 / Published online: 12 February 2011
# British Crown Copyright 2011
Abstract A previously reported proficiency test for the
analysis of vitamin B2 in liquid dietary supplement
demonstrated bimodality. The same trend has now been
observed in four subsequent tests of this type. The trend
would not so easily have been observed without applying a
fit-for-purpose standard deviation that is more generous
than that predicted by the Horwitz equation. Since
originally reporting the bimodal problem and hypothesising
its cause by incomplete enzymic digestion of riboflavin-5phosphate, there has been a general improvement in the
reporting of the higher mode. This is thought to correspond
to free riboflavin following complete digestion of the
sample. Several individual participants appear to have
learned from the experience and have changed their
reporting of the lower mode to the higher mode.
Keywords Proficiency testing . Vitamin analysis .
Enzymic digestion
Introduction
Vitamin analysis is widely acknowledged as being difficult,
for a variety of reasons. One reason was highlighted by
FAPAS® [1] due to the likely difference in chemical form
of vitamin B2 in different matrices. Proficiency test (PT)
results for liquid supplement matrix have a bimodal
distribution, whereas for breakfast cereal matrix they are
unimodal. The hypothesis for the difference is that the liquid
supplement generally contains riboflavin-5-phosphate, which
M. Sykes (*) : J. Croucher : R. A. Smith
The Food and Environment Research Agency,
Sand Hutton, York YO41 1LZ, UK
e-mail: [email protected]
requires an enzymic dephosphorylation to the determinand,
riboflavin. Breakfast cereal, by contrast, is fortified with
riboflavin in which the extraction is not dependent on the
enzymic step. Laboratories that neglect the enzymic step or
fail to apply the necessary care with which enzymes must be
treated may have incomplete digestion of the sample. The
use of acid digestion is a necessary first step in digesting
protein-bound riboflavin. However, the acid digestion will
not hydrolyse riboflavin-5-phosphate to riboflavin. The
activity of enzymes will depend on batch-to-batch variation,
age, storage, and pH, temperature and duration of incubation
with the sample. The mixture of enzymes may also have an
effect on the digestion of some B group vitamins [2].
Vitamin B2 may be present in different forms, depending
on the matrix [3–6]. In foods, this may be as free riboflavin
or in combination with its nucleotide forms, the co-enzymes
flavin adenine dinucleotide and flavin mononucleotide. The
latter is more commonly referred to as riboflavin-5phosphate. EU legislation [7] allows for riboflavin and
riboflavin-5-phosphate to be used in the manufacture of
food supplements. Fortified foods or dietary supplements will
tend to contain only one form of the vitamin. In solid matrices,
such as breakfast cereals or vitamin tablets, this is likely to be
as free riboflavin. However, since riboflavin has a limited
solubility in water at fortified levels (about 10 mg/100 mL)
[6], liquid matrices (liquid dietary supplements and energy
drinks) will more likely contain the more soluble riboflavin5-phosphate (about 50 g/L).
Since the data relating to reference [1] were collated,
there have been a further four FAPAS® PTs of vitamin
analysis in liquid supplement matrix [8]. Although each
dataset for vitamin B2 shows the same bimodality, certain
trends are beginning to become apparent. In this follow-up
paper to the original report [1], we describe the PT data
collated over a 7-year period and discuss the supporting
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306
evidence that long-term participation in these PTs has been
beneficial to some laboratories.
Literature
Numerous papers have been published on vitamin analysis
in a variety of matrices. Some of these are pertinent to the
problem highlighted here, in terms of relevance to the
observed PT results. The preferred method of analysis for
vitamin B2 is now extraction followed by high performance
liquid chromatography (HPLC) determination [9, 10]. This
has largely superseded the microbiological method, which
is reflected in the method details returned by FAPAS® PT
participants. Several interlaboratory studies have been
conducted to establish performance characteristics for
different methods [2, 3, 5, 11, 12].
An early European interlaboratory comparison of methods for water-soluble vitamins asked participants to apply
their routine method to certified reference materials [2]. The
results highlighted the problem of vitamin B2 analysis with
observed relative standard deviation of reproducibility
(RSDR) of 28–74% and Horwitz ratio (HorRat) values of
2.6 and 5.9. HorRat values are frequently used in
collaborative trials as a measure of the observed precision
to the predicted precision using the Horwitz equation.
HorRat values are generally considered to demonstrate
acceptable precision if they are within the range 0.5–2.0.
Most laboratories used a similar extraction method of
autoclaving or boiling with acid, followed by dephosphorylation with takadiastase (for laboratories using HPLC
determination). Differences between laboratories largely
amounted to the detail of time or temperature for extraction.
Laboratories using microbiological determination did not
use the dephosphorylation step, since the microorganism
responds equally to free riboflavin and its phosphate forms.
The acid hydrolysis is still required, however, for all
determinations to free the vitamin from complexation with
proteins. The hydrolysis protocol needs to be rigorous,
whether acid or acid and enzymic, and tailored to the
foodstuff being analysed.
An HPLC-fluorescence method for thiamine and riboflavin [11] evolved the enzymic step by a limited
interlaboratory comparison, prior to a full collaborative
trial [3]. After some 8 years of routine use in French food
control laboratories, the official 1987 method for the
determination of vitamins B1 and B2 in foods was subjected
to collaborative study [3]. This was justified by the
observation that recovery rates were sometimes unsatisfactory in practice. The sample types used in the study
included “food complement” (i.e. supplement, not described further). Results were recovery-corrected and RSDR
M. Sykes et al.
ranged from 4% to 16% (7% for food complement).
Chocolate powders, previously shown to be a difficult
matrix for recovery, were treated in a separate study.
Recovery rates for vitamin B2 in chocolate powder were
lower (75%) than for other foodstuffs (89–94%).
It should be noted that the authors of [3] did not include
HorRat values but they calculate to a range of 0.53–1.25,
except “meal with fruits” (0.49) and “tube-feeding solution” (0.28).
A number of studies commented on the efficiency of the
enzyme used and variations between different suppliers of
enzyme. One laboratory in [2] reported different activity of
takadiastase from different suppliers. The authors of [11]
indicated that takadiastase is insufficient by itself for
complete dephosphorylation. During the development of
the method, it had been found that the use of takadiastase
on its own gave poor reproducibility, especially for foods
containing high levels of naturally phosphorylated vitamins. This may be due to differences in enzymic activity,
depending on the enzyme commercial product. The final
method [3] used a mixture of takadiastase and β-amylase
(10:1) for total dephosphorylation. Possibly, it is the
impurities in the amylase that are additionally responsible
for the efficiency of the process. A later EU-wide
interlaboratory comparison [12] provided an aliquot of
takadiastase enzyme to all laboratories from the same batch,
which had been characterised for its efficiency.
The prescribed optimised method described by van der
Berg et al. [12] commenced with hydrochloric acid
treatment, followed by incubation with takadiastase for
18 h. The in-house methods for all laboratories in the
interlaboratory comparison were similar with principal
differences being in the time for acid or enzymic treatment.
No raw data were provided but CVs were quoted as being
12–40% for in-house methods and 12–34% for the
optimised method. There was generally good agreement in
results between using the in-house and the optimised
method.
The results of a collaborative trial [5] for vitamins B1
and B2 in animal feed had RSDR values ranging from
4.283% to 9.006% (HorRat values 0.423–0.747) for mean
values of 5.55–20.8 μg/g for vitamin B2, expressed as
riboflavin. Variations in the methodology in relation to the
need for the enzymic step have been tested [2–4, 10]. In
particular, Viñas et al. [4] compared acid hydrolysis alone
to acid plus enzymic hydrolysis. Of the eight B group
vitamins under investigation, thiamine (B1) and riboflavin
(B2) both required the enzymic step as well as the acid step.
The recoveries from spiked samples for acid hydrolysis
only were 66.5% and 70.3% for B1 and B2, respectively,
which improved to 99.4% and 99.0% with the additional
enzymic step. Blake [10] suggests that dephosphorylation is
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Proficiency testing has improved the quality of data
307
preferable to attempting to determine riboflavin 5-phosphate
separately and to analyse the total as free riboflavin.
A variation for the type of enzyme was developed by
interlaboratory trial [5]. Although the method variations
were aimed mainly at improving the performance of the
method for vitamin B1, the procedure was adopted for B2 as
well. The initial method hydrolysed with hydrochloric acid
in an autoclave (121 °C for 30 min), followed by
takadiastase enzyme incubation at 50 °C for 2 h. The first
variation in extraction protocol was to substitute takadiastase for clara-diastase enzyme. The second variation was
hydrolysed with hydrochloric acid on a steam bath for
60 min and incubated with clara-diastase at 37 °C for 16 h.
The contrast of analysing dietary supplements was
highlighted in the context of multi-vitamin analysis in a
single method [6]. Since dietary supplements tend to have
the vitamins present in a single form, and without the
presence of complex matrix, this made a good test material
for a multi-analyte approach. The standard reference
material (NIST SRM 1849) used was a milk-based powder
with vitamin B2 present as free riboflavin. Hence, the
sample preparation methodology was simplified to a low
pH extraction only, with no dephosphorylation step. Free
riboflavin, although a water-soluble vitamin, has limited
solubility at dietary supplement concentrations. A solid
matrix for the supplement is therefore suitable for riboflavin, which does not have to be in the form of (more
expensive) riboflavin-5-phosphate. The vitamin standard
solutions were tested for long-term stability, and riboflavin
was stable for over a year at 4 °C. However, the solution
was prepared in pH 2.1 phosphate buffer plus 0.1% βmercaptoethanol preservative. The purity of vitamin standards was thought to be an important factor in the
measurement uncertainty (although probably at negligible
levels compared to the 20% RSDR used for the FAPAS®
standard deviation for proficiency).
FAPAS® PT results and discussion
Standard deviation for proficiency
PTs are quantitatively assessed by the use of z scores. A
result equal to the assigned value corresponds to a z
score of zero. Results within two standard deviations of
the assigned value correspond to z scores within ±2 or a
95% probability of the result falling within the normal
distribution.
The percentage of PT z scores within ±2 for breakfast
cereal matrix is generally in excess of 80%. By contrast, the
liquid supplement matrix PTs only achieve <60% z scores
within ±2. Table 1 summarises the PT statistics for the
liquid supplement tests being reported here. The previous
report [1] was based on the discussion of PT 2139 (June
2006). This PT contained the first use of the mode to set the
assigned value, and also the fit-for-purpose standard
deviation for proficiency based on an RSDR of 20%, rather
than Horwitz. Hence, z scores for PTs 2126 (July 2004) and
2133 (June 2005) were not issued because the statistics
based on the Horwitz standard deviation were clearly not
suitable. (Other B group vitamins in these tests were
evaluated, however.) This is compounded by the u/σp
(observed uncertainty/standard deviation for proficiency)
test, which provides a measure of the observed variability
on the z scores. Ideally, this should be <0.4 for a negligible
effect. PTs 2126 and 2133 evidently demonstrate that the
Horwitz standard deviation is not fit-for-purpose. Breakfast
cereal PTs, by comparison, continue to use a tighter
Horwitz-derived standard deviation, at similar levels of
vitamin B2.
A further consequence of the adoption of the fit-forpurpose RSDR is the comparison with collaborative trial
data. The collaborative trials referenced above [3, 5] all
demonstrate reproducibility precision in keeping with that
Table 1 Summary of the results statistics for seven FAPAS® vitamin B2 PTs in liquid supplement matrix
PT
Date
n
AV
Units
RSDR%
σp
u
2126
2133
2139
2146
2152
2158
2164
July 2004
June 2005
June 2006
June 2007
June 2008
June 2009
June 2010
40
34
42
37
39
34
41
46.8 R
46.4 R
88.6 M
87.6 M
93.9 M
10.2 M
9.98 M
μg/g
μg/g
μg/g
μg/g
μg/g
mg/100 mL
mg/100 mL
8.97 H
8.98 H
20 F
20 F
20 F
20 F
20 F
4.20
4.17
17.7
17.5
18.8
2.04
2.00
7.37
9.08
4.25
1.87
3.85
0.436
0.184
u/σp test
1.76
2.18
0.240
0.106
0.205
0.214
0.092
|z|≤2
a
a
51%
57%
53%
59%
56%
n number of valid results used in the calculation of the assigned value (AV), R robust mean, M mode, H Horwitz standard deviation as RSDR,
F fit-for-purpose RSDR (expert advice [1]), σp standard deviation for proficiency, u uncertainty of reported results, |z| z scores
a
2126 and 2133, observation of results only, no z scores issued
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308
M. Sykes et al.
predicted by the Horwitz equation. In RSDR terms, this is in
the range of about 5–14% (depending on concentration and
matrix). Collaborative trials, however, concentrate the
laboratories’ efforts on a single well-defined method.
Interlaboratory comparisons [2] and PT, by contrast, may
use a range of methodology as used routinely by each
individual laboratory. In most cases, the Horwitz standard
deviation is entirely fit for the purpose of prescribing the
acceptable precision. The experiences of [2] and of the PTs
described here demonstrate an interesting deviation from
this rule of thumb.
Bimodality of vitamin B2 PT data
PT 2139 set the new standard for this test. By adopting a
fit-for-purpose standard deviation for proficiency based on
20% RSDR, the multi-modality of the results distribution
a
2146 Vit B2 Horwitz
0.035
0.030
Density
0.025
0.020
0.015
0.010
0.005
0.000
-100
b
0
100
Analytical result
200
300
200
300
2146 Vit B2 FFP
0.020
0.015
Density
Fig. 1 a PT 2146 kernel density
plot using Horwitz standard
deviation. Analytical result units
are μg/g. b PT 2146 kernel
density plot using standard
deviation based on 20% RSDR.
Analytical result units are μg/g
could now be resolved into a distinct bimodal distribution.
The two modes could be regarded as corresponding to free
riboflavin and riboflavin-5-phosphate. The subsequent PTs
have continued with this approach to issue z scores based
on the higher mode of a bimodal distribution.
The method of bump-hunting [13] is now well established to determine the modal distribution of PT results.
The kernel density plot produced depends on the σp value,
the standard deviation for proficiency. Figure 1a shows the
kernel density plot of the PT 2146 dataset using σp
derived using the Horwitz equation (σp =5.118). Figure 1b,
by contrast, shows the kernel density plot of the same
dataset but using σp based on the fit-for-purpose RSDR of
20% (σp =11.82). There is a clear visual improvement in
simplifying the modal distribution. The interpretation of
the dataset, based on the hypothesis of free riboflavin vs.
riboflavin-5-phosphate, appears to be justified.
0.010
0.005
0.000
-100
0
100
Analytical result
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Proficiency testing has improved the quality of data
PT
2126
2133
2139
2146
2152
2158
2164
Units
Lower mode
Lower mode density
Higher mode
Higher mode density
Ratio of modes as %
μg/g
7.48
0.025
92.0
0.016
8%
μg/g
6.03
0.030
83.8
0.013
7%
μg/g
10.4
0.016
90.7
0.014
12%
μg/g
9.43
0.015
87.7
0.018
11%
μg/g
14.8
0.013
93.9
0.013
16%
mg/100 mL
0.789
0.080
10.2
0.12
8%
mg/100 mL
0.822
0.12
9.95
0.14
8%
Although the datasets from the earlier PTs 2126 and
2133, based on the Horwitz standard deviation statistics,
could not issue evaluative z scores, retrospective bumphunting based on 20% RSDR shows the same bimodal trend
of PT 2139 and subsequent PTs. Table 2 summarises the
PTs and their two modes. It is observed that the lower mode
is some 10% of the higher mode. We can surmise two
possibilities from this. The first is that the riboflavin-5phosphate in the test material contains 10% free riboflavin.
The second is that the acid hydrolysis step by itself has
about 10% dephosphorylation efficiency. In reality, there
could well be a combination of these reasons.
Evolution of methodology by participants
Kernel density plots can be created for all the datasets
corresponding to PTs 2126 to 2164. Figure 2 shows the ratio
of lower mode density to higher mode density. There is a
trend towards a decreasing ratio with later PTs, i.e., there is a
general move towards reporting the higher mode in more
recent PTs, from reporting the lower mode in earlier PTs. It
seems that some participants are learning from the PT reports
that the method needs to incorporate dephosphorylation in
order to report total vitamin B2 expressed as riboflavin.
The identity of participants in FAPAS® PTs remains
confidential. However, by internally looking at the results
of participants who have returned results for more than four
of the seven PTs being examined, some observations can be
made. Twenty-two laboratories submitted results in four or
more PTs. Of these laboratories, ten fairly consistently
reported a result corresponding to the higher mode. Four
laboratories consistently reported a result corresponding to
the lower mode. One laboratory was completely inconsistent in this respect. Seven laboratories, however, showed a
general change from reporting the lower mode result in
early PTs to the higher mode result in later PTs. The change
in results reported by these laboratories coincides with the
PT 2139 report, i.e. the higher mode is reported from PT
2146 onwards. This finding is in keeping with the trend in
kernel density ratio observed above.
It is evident that some laboratories are still not
comfortable with the idea that the lower mode is an
incomplete result for vitamin B2 in liquid supplement
matrix. Four laboratories have consistently reported the
lower mode for at least four PTs, including the change of
standard deviation reported in PT 2139. There may still be
some confusion over the purpose of the two-step hydrolysis
procedure. Additional comments submitted by two participants support this idea. Two participants in recent PTs both
specified that their result was for riboflavin only, since
riboflavin-5-phosphate was not analysed in their laboratory.
The FAPAS® PTs ask participants for method details,
although the submission for this is optional. Unfortunately,
the method details returned for these PTs are too insubstantial to compare methodological trends.
It may be hypothesised that the same PT based on a solid
dietary supplement test material (tablet or powder) would
produce a unimodal dataset from the same participants.
This is because the solid supplement need not contain the
more expensive and more soluble riboflavin-5-phosphate
but free riboflavin which would not benefit from the
enzymic digestion step.
Conclusions
Standard deviation based on fit-for-purpose RSDR of 20%
permits the modes of vitamin B2 PT data to be calculated in
liquid vitamin supplement. This is not so easily possible
using the tighter Horwitz standard deviation. The HorRat
2.5
2
Density ratio
Table 2 Summary of the modes
for seven FAPAS® vitamin B2
PTs in liquid supplement matrix
309
1.5
1
0.5
0
PT 2126
PT 2133
PT 2139
PT 2146 PT 2152
PT 2158
PT 2164
PT number
Fig. 2 Ratio of lower mode density to higher mode density for seven
total vitamin B2 FAPAS® PTs in liquid supplement
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310
values are still reliable indicators of precision in single
method collaborative trials. The observation reported in PT
2139 and its hypothesis of incomplete digestion of
riboflavin-5-phosphate producing the modal data continues
to be supported by later PT data. Some laboratories have
learned from the PT exercises and appear to have modified
their methods to encompass the necessary enzymic dephosphorylation step.
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