Certification and uncertainty evaluation of flavonoids certified reference materials

doi:10.4236/as.2013.49B016

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Vol.4, No.9B, 89-96 (2013) Agricultu ral Sciences

http://dx.doi.org/10.4236/as.2013.49B016

Certification and uncertainty evaluation of flavonoids

certified reference materials

Can Quan*, Huanfang Yao, Caixia Hou

Division of Chemistry, National Institute of Metrology, Beijing 100013, China;

*Corresponding Author: [email protected]

Received August 2013

ABSTRACT

Flavonoids are the most widely used in the field

of the food, medicine, and cosmetic due to their

pharmacological functions and biological activi-

ties, such as anti-tumor, anti-inflammatory and

antioxidation. This work described the assign-

ment of purity values to 3 flavonoids certified

reference materials (CRMs) including baicalein,

5,7-dihydoxy flavones and naringenine which

we re developed in this study according to the

ISO Guides 34 and 35. The qualitative analysis

was performed by liquid chromatography-mass

spectrometry (LC-MS) and infrared spectrosco-

py (IR). The CRMs’ purity values were assigned

based on the weighted average of quantitative

nuclear magnetic resonance method and mass

balance approach with high resolution liquid

chromatography-VWD. All the three CRMs with

following value mass fractions: baicalein at a

certified purity P ± U (k = 2) of 98.8% ± 0.8%;

5,7-dihydoxy flavones of 99.1% ± 0.7% and na-

ringenine of 99.5% ± 1.0% respectively. The

homogeneity of the CRMs was determined by an

in-house validated liquid chromatographic me-

thod. Potential degradation during storage was

also investigated and a shelf-life based on this

value was established.

Keywords: Flavonoids; Certified Refe rence

Material (CRM ); Quantitative Nucl ear Magnetic

Resonance (QNM R); M ass balance approa ch;

Uncertainty

1. INTRODUCTION

A certified reference material (CRM) is a material or

substance whose one or more property values are suffi-

ciently homogeneous, stable, and well established to be

used for the calibration of an apparatus, the assessment

of a measurement method, or for assigning values to ma-

terials [1].

Flavonoids are increasingly in demand in the field of

the food, medicine, and cosmetic [2] due to their phar-

macological functions and biological activities, such as

anti-tumor, anti-inflammatory and antioxidation [3 -5].

The qualities of flavonoids products will have analytical

challenges. The flavonoids CRMs have been determined

by at least two independent analytical methods. The fla-

vonoids CRMs could be used to ensure the accuracy,

traceability and comparability of the related products test

results.

This paper describes the development of three flavo-

noids CRMs including baicalein, 5,7-dihydoxy flavones

and naringenine. Qualitative analysis was carried out by

infrared spectrometry (IR) and liquid chromatography-

mass spectrometry (LC-MS). The moisture content and

inorganic e lements conten t were de termined by Kar l Fisch-

er titration, inductively coupled plasma mass spectrome-

try (ICP-MS), respectively. Homogeneity study was eva-

luated by F-test and t-test both for between vials and

within vial. The uncertainties of CRMs were evaluated

extensively.

2. EXPERIMENTAL

2.1. Materials

The chemical structure of flavonoid compounds stu-

died including baicalein, 5,7-dihydoxy flavones and na-

ringenine were shown in Figu re 1, all were purchased

from Sigma-Aldrich (USA). Benzoic acid certified ref-

erence material (99.9% ± 0.1% purity) used as QNMR

internal standard was obtained from NIST, China.

HPLC grade Acetonitrile and methanol were bought

from Merck (HPLC, Germany). Dimethyl sulfoxide-d6

solvent was purchased from Cambridge Laboratoties

(USA). Distilled deionized water (18.2 MΩ∙cm) was ob-

tained with Milli-Q water system and filtered through

0.22 μm membrane.

2.2. Instrument

Infrared spectr a were collected w ith FT-IR spectrometer

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Figure 1. Structure of the studied flavonoids CRMs.

Thermo Nicolet IS10 spectrophotometers. All the flavo-

noids reference material solutions, diluted to concentra-

tion of 10 μg∙mL−1 in methanol for LC/MS Analysis

were quantified at flow injection analysis mode with an

Agilent 6410 triple quad system with electro-spray ioni-

zation source (ESI). The QNMR measurements were

performed with a Bruker AV600 spectrometer (Bruker,

Billerica, MA). The Agilent 7500CE ICP-MS was used

for the analysis of trace metal residues in all the samples.

A Mettler Tolerdo DL39 Karl Fischer titrator was used

for water measurement of all the samples.

2.3. Qualitative Analysis Methods

2.3.1. LC-MS Method

The flavonoids CRMs in methanol with concentration

of 10 μg∙mL−1 were used to LC-MS analysis. The ion

spray voltage was set at 4.0 kV. Drying gas temperature

was maintained at 350˚C with flow rate of 9.0 mL∙min−1.

Nebulizer pressure was set at 40 psi. The fragmentor

energy was set at 135 V. The collision energy respectiv e-

ly were optimized at 30 V for baicalein and 5,7-dihydoxy

flavones, and 16 V for naringenine. The analysis the

mass spectrum peak molecular weight in an ESI positive

ion mode at specified MS scan range of 60 - 300.

2.3.2. IR Method

Infrared spectra were collected with FT-IR spectrome-

ter Thermo Nicolet IS10 spectrophotometers via KBr

pellets technique. Specimens were ground with dried

spectroscopic grade KBr powder and the mixture was

compressed to pellets for FT-IR measurements. The

sample to KBr mass ratio was optimized as 1:100. All the

spectra were collected in the 4000 - 400 cm−1 range at 4

cm−1 resolution.

2.4. Quantitative Analysis Methods

2.4.1. Mass Balance A pproach

The flavonoids were quantified by HPLC equipped

with an ZORBAX Eclipse XDB-C18(460 mm × 250 mm

× 5 μm, Agilent ) column with VWD detector. The

chromatography conditions are detailed in Ta b l e 1 . The

purity values of flavonoids CRMs were calculated using

the peak area normalization method. Following subtract

the moisture and inorganic elements impurities which not

measured by HPLC from 100%, as follows [6,7]:

( )

m aHPLC

Purity1 PPP=−− ∗

(1)

Where, PHPLC is the percentage area of main com-

pound acquired by HPLC-VWD, Pm is the moisture con-

tent measured by Karl Fisher method, and Pa is the inor-

ganic elements content measured by ICP-MS.

2.4.2. QNMR Method

The QNMR method is based on the directly propor-

tional relationship between the signal response (inte-

grated signal area, Ix) and the number of nuclei generat-

ing the corresponding resonance line, which is represented

by Eq.1 as [8].

std

P⋅⋅⋅⋅==

(2)

Where Px is purity of analyte; Ix and Istd are the inte-

grated signal areas of analyte and internal standard, re-

spectively; Nx and Nstd are the spin numbers of the ana-

lyte and internal standard, respectively; Mx and Mstd are

molar mass of analyte and internal standard, respectively;

m and mstd are the mass of analyte and internal standard,

respectively; Pstd is the purity of the internal stand ard.

The QNMR measurements were performed with a

Bruker AV600 spe ctrometer (Bruker, Billerica, MA). Spec-

tra were run with the following optimized parameters:

probe size, 5 mm; probe temperature, was 23.0˚C; exci-

tation pulse angle, 45˚; 32 K time domain points; 16 K

spectral data points; pulse delay, 4.15 μs; relaxation delay,

32 s and number of scans, 32. A sample solution con-

taining about 10 mg benzoic acid, 70 mg of carbohydrate

samples diluted in 0.5 mL DMSO was analyzed by

QNMR. The total mass of analyte in the sa mple solution

was determined usi ng Eq.1.

2.5. Homogeneity Study and Stability Study

According to the Technical Norm of Primary Refer-

ence Material of China [9], the homogeneity study and

the stability study were carried out by HPLC-VWD me-

thod. 15 bottles, were randomly selected and assayed for

OOH

HO 7

6510 43

81' 4'

2'3'

Baicalein CRM

OOH

HO7

6510 43

81'

5,7-dihydoxy flavones CRM

OOH

HO7

651043

81'

Naringenine CRM

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Table 1. The chromatography conditions of the studied flavonoids CRMs.

CRMs Mobile phase

(Acetonitrile:0.1% phosphoric acid) Wavelength (nm) Injection volume (μL) Flow (mL/min) Concentration (mg/mL)

Baicalein 35:65 275 5 1 0.5

5,7-dihydoxy fl avones 43:57 268 5 1 0.3

Naringenine 35:65 288 2 1 1

homogeneity between bottles, and 7 portions from each

bottle were assayed for homogeneity within bottle. The

results were examined via F-test and t-test. In F-test,

standard deviations were used to examine whether the

deviation between bottles was significantly greater than

the deviation within bottle. In t-test, means were used to

examine whether the mean between bottles was signifi-

cantly different from that of within bottle [10].

A stability study monitored a significant change in

value of the flavonoids CRMs was performed over one

year.

A study of long-term stability for storage at 4˚C was

carried out by HPLC-VWD method [11 ] in which ma-

terial was determined at 1, 3, 6 and 12 months. For each

condition, two solutions were prepared and analyzed in

duplicate. A comparison was made between the purity

determined by HPLC-VWD for different storage time. A

freshly prepared control sample was analyzed at each

time. All the samples were randomized for analysis.

2.6. Uncertainty Evaluation

The uncertainty evaluation for the flavonoids CRMs

derived from the certified value, homogeneity and stabil-

ity study. The expanded uncertainty was calculated as

follows:

222

c vhs

uuuU kUk=⋅=⋅ ++

( 3)

where, k is the coverage factor, Uc is the combined un-

certainty of the sample, uv, uh, us are the uncertainty

components of the assigned value, homogeneity and sta-

bility study.

3. RESULTS AND DISCUSSION

3.1. Qualitative Analysis Methods

3.1.1. LC-MS Method

The experiment obtained the molecular weight of the

flavonoids CRMs by full scan mass spectra. The results

indicted the molecular ion peak (M + H)+ of mass charge

ratio m/z of baicalein and 5,7-dihydoxy flavones was the

molecular weight plus the atomic weight of hydrogen

(1.0), the molecular ion peak (M − H)− of mass charge

ratio m/z of naringenine was the molecular weight sub-

tract the atomic weight of hydrogen (1.0). Ion fragments

by product ion scan are detailed in Figu re 2. The results

showed the flavonoids mainly fractured in 1,2 C-Ochem-

ical bond and 3,4 C-C chemical bond.

3.1.2. IR Method

The Infrared spectrum of the flavonoids CRMs were

shown in Figu re 3. The results revealed the peaks at

3090, 3097, and 3114 cm−1 belong to hydroxide peaks,

1660, 1653, and 1630 cm−1 belong to associat ed car bon yl

peaks, 1449 ~ 1618 cm−1 belong to benzene ring skeleto n

of C = C peaks of the flavonoids CRMs. The Infrared

spectrums of the flavonoids CRMs were consistent with

those spectrums from literature [13-15] and can be used

for quantification.

3.2. Quantitative Analysis Methods

3.2.1. Mass Balance A pproach

The HPLC chromatogram of the flavonoids CRMs

were shown in Figure 4. The results of the moisture

content and the inorganic elements contents were listed

in Table 2, as indicated, The water content for Baicalein,

5,7-dihydoxy flavones, and naringenine were 0.538%,

0.150%, 0.098% and 0.115%, respectively, all were less

than 0.1%, while the inorganic content all warless than

0.00002% at the studied conditions.

3.2.2. QNMR Method

QNMR was also used to determinate purity values

flavonoids CRMs. The NMR spectrums were shown in

Figu re 5. The C3-H peak areas of baicalein, C8-H peak

areas of 5,7-dihydoxy flavones, C4,-OH peak areas of

naringenine, C2,2-2H peak areas of the benzoic acid in-

ternal standard were selected for integra tion .

3.2.3. Purity Values of the Flavonoids CRMs

The purity values of flavono ids CRMs were calculated

with the average of mass balance approach and QNMR

method. The results are listed in Table 2.

3.3. Homogeneity Study and Stability Study

The F-Test and t-test results of all the studied were

summarized in Table 3. No significant differences were

found for all the studied compounds in homogeneity test

as the calculated F-values were less than the critical

F-values (Table 3). Although a slight difference was ob-

served, this was attributed to the variation of the analyti-

cal method.

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Figure 2. Main fragmentation schemes of the flavonoids CRMs.

Figure 3. The infrared spectrums of the studied flavonoids CRMs.

The flavonoids CRMs stored at 4˚C were used to test

stability. Th e testing data of stability study are showed in Table 4. The results in different times were consistent

with the standard value through the t-test analysis. There

4000 3500 3000 2500 2000 1500 1000500

100

1470

640

827

899

1090

1160

1618

3090

3410

Wavenumbers

(

-1)

Transmitt ance

（

%）

1300

1586

1660

1505

1390

1020

Baicalein C RM

4000 3500 3000 2500 2000 1500 1000500

100

641

807

842

1168

1500

1577

1034

1449

1612

1653

2712 2631

Transmitt ance

（

%）

Wavenumbers

(

-1

)

3097

5,7-dihydoxy flavones CR M

4000 3500 3000 2500 2000 1500 1000500

100

1083

1065 532

731

832

1520

1499

1158

1602

1630

2833

3114

Transmitt ance

（

%）

Wavenumbers

(

-1

)

3286

Naringenin e CR M

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Figure 4. Purity determination HPLC chromatogram of the flavonoids CRMs.

Tab le 2. The purity values results of flavonoids CRMs.

CRMs HPLC (%) QNMR (%) Average (%)

Baicalein 98.78 98.88 98.8

5,7-dihydoxy fl avones 99.40 98.84 99.1

Naringenine 99.47 99.46 99.5

were no obvious changes in purity values monitored of

the flavonoids CRMs over one year. The results mean

that they were stable.

3.4. Uncertainty Evaluation

The uncertainty for the flavonoids CRMs is composed

of the uncertainty from the certified value, homogeneity

and stability study.

3.4.1. Uncertainty of Certified Value

1) Uncertainty from HPLC measurement [16].

a) u1 (type A uncertainty) is the uncertainty from re-

peatability of HPLC measurement.

b) u2 (type B uncertainty) is the uncertainty from dif-

ferent responses from flavonoids CRMs and impurities at

different wavelengths of HPLC measurement.

The com bined uncerta inty is as follow:

HPLC1 2

uuu= +

2) Uncertainty of the QNMR measurement

a) u3 (type A uncertainty) is the uncertainty from re-

peatability of QNMR measurement.

b) u4 is the uncertainty from relative molar masses of

flavonoids CRMs and benzoic acid internal standard

CRM. The value of u4 calculated is less than 0.1%. In

this case, u4 is ignored.

c) u5 is the uncertainty from benzoic acid CRM. The

uncertainty of benzoic acid CRM was 0.1%, according to

the uniform distribution, u5 = 0.1%/31/2 = 0.06%.

d) u6 is the uncertainty from balance weighing. The

balance (Max = 2.1 g, d = 0.1 μg) was used to weigh the

flavonoids CRMs and benzoic acid internal standard

CRM. Balance changing and test error estimate for the

0.5 μg, therefore u6 is estimated 0.01%.

The combined uncertainty is as follow:

22 2 2

QNMR3 45 6

uu uu u=++ +

3.4.2. The Combined Uncertainty

The combined uncertainty for the flavonoids CRMs is

composed of the uncertainty from the certified value,

homogeneity and stability study. The results of homo-

geneity and stability study completely conformed to the

requirement of homogeneity and st ability in the regula-

tion of the CRMs. The homogeneity and stability of the

flavonoids CRMs both are good. Therefore the uncer-

tainty of homoge ne i ty (uh) and stab ility (us) is neglected.

2222222

Cvh sHPLCQNMRh s

22 22 2222

123 45 6hs

22 22 22

123 45 6

Uu uuuuuu

u uuuuuuu

u uuuuu

= ++=+++

=++ ++ +++

≈++ ++ +

3.4.3. The Expanded Uncertainty

The expanded uncertainty (U) for this flavonoids

CRMs was calculated by multiplying the combined un-

certainty by a coverage factor of k = 2. The uncertainty

evaluation for flavonoids CRMs were listed in Table 5.

4. CONCLUSION

The three flavonoids CRMs were developed with the

procedures of sample preparation, certified value, homo-

geneity study, and stability study and uncertainty evalua-

tion. The three flavonoids CRMs can not only meet the

requirement of quantitative detection of food, medicine

and related materials, but also provide a more quickly

and accurately experimental methods for the research

and devel o pment of related prod uc ts.

5. ACKNOWLEDGEMENTS

The authors acknowledge Special Program for Key Basic Research

of the Ministry of Science and Technology, China (Grant No.

2011FY130100) and the National Natural Science Fou ndation of C hina

(Grant No 21275134/ B 0505) for financing this work.

05 10 15 20 25 30 3540 45

A / mAu

t / m in

Baicalein C RM

05 10 15 20 25 30 35 40 45

t / m in

A / mAu

5,7-dihydoxy flavones CR M

05 10 15 20 25 30 35

A / mAu

t / m in

Naringenin e CR M

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Figure 5. The spectrums of the studied flavonoids CRMs.

C. Quan et al. / Agricultural Sciences 4 (2013) 89-96

Table 3. Homogeneity results for the contents, the within-bottle, and between-bottle and homogeneity test of flavonoids CRMs.

Homogeneity test Baicalein CRM 5,7-dihydoxy flavones CR M Naringenine CRM

X between-bottle (s1,%) 99.31 ± 0.018 99.58 ± 0.013 99.58 ± 0.015

within-bottle (s2,%) 99.31 ± 0.011 99.58 ± 0.010 99.58 ± 0.010

F-test

/Fss=

2.63 1.86 2.60

0.05

F (14,6)

3.96 3.96 3.96

0.05

FF<

Y Y Y

t-test

txx= −

0.0010% 0.0011% 0.0022%

t2 nn



 



= +

 



 



0.0064% 0.0049% 0.0053%

≤

Y Y Y

Table 4. The results of stability of flavonoids CRMs.

Time interval Baicalein 5,7-dihydoxy fl avones Naringenine

Zero month 99.27 99.50 99.53

One month 99.37 99.56 99.57

Three months 99.31 99.54 99.58

Six months 99.31 99.52 99.50

Twelve months 99.35 99.46 99.55

Average (%) 99.32 99.52 99.55

RSD (%) 0.04 0.04 0.03

Table 5. The results of uncertainty evaluation of flavonoids CRMs.

Uncertainty Baicalein CRM 5,7-dihydoxy flavones CRM Naringenine CRM

u1/% 0.01 0.02 0.03

u2/% 0.04 0.04 0.01

u3/% 0.36 0.33 0.50

u4/% 0 0 0

u5/% 0.06 0.06 0.06

u6/% 0.01 0.01 0.01

uc/% 0.37 0.34 0.50

k 2 2 2

U/% 0.8 0.7 1.0

Purity value/% 98.8 99.1 99.5

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