New reference material for analysis of elements in glutinous rice ...

Accred Qual Assur 

DOI 10.1007/s00769-009-0621-9 

PRACTITIONER’S REPORT 

New reference material for analysis of elements in glutinous rice 

produced at the National Institute of Metrology (Thailand) 

Charun Yafa • Sirinapha Srithongtim • Pranee Phukphatthanachai • Araya Thiparuk • Nittaya Sudsiri • 

Laddawan Rojanapantip • Mayuree Uraroongroj • Panawan Kluengklangdon • Preeyaporn Jaengkarnkit • 

Kittipong Sirisuthanant • Chamoy Thonglue • Thanida Pimma • Paramee Pengpreecha • 

Tippaya Julwee Fortune • Ratirot Zwicker • Vorapot Permnamtip • Sirinart Laoharojanaphnad • 

Boonlert Suanmamuang • Juwadee Shiowatana • Weerawan Waiyawat • Atitaya Siripinyanond • 

Kunchit Judprasong • Benjawan Boonsong • Suwaluck Talaluck • Chainarong Cherdchu 

Received: 1 September 2009 / Accepted: 16 November 2009 

Ó Springer-Verlag 2009 

Abstract Reference materials play an important role for 

evaluating the accuracy of analytical results, and are 

essential parts of good laboratory practice. They represent a 

key tool for quality control of chemical analyses. In Thailand, 

the demand of food and environmental reference 

materials is constantly increasing, and the National Institute 

of Metrology (NIMT, Thailand) is responding to the urgent 

Presented at BERM-12, July 2009, Oxford, UK. 

C. Yafa (&) P. Phukphatthanachai N. Sudsiri C. Cherdchu 

National Institute of Metrology (Thailand) (NIMT), 

Ministry of Science and Technology, Technopolis, 

3/4-5 Moo 3, Klong 5, Klongluang, 

Pathumthani 12120, Thailand 

e-mail: Charun@nimt.or.th; charun_yafa@yahoo.com 

S. Srithongtim A. Thiparuk 

Environmental Research Training Center (ERTC), 

Department of Environmental Quality Promotion, 

Ministry of National Resources and Environment, 

Technopolis, Klongha, Klongluang, 

Pathumthani 12120, Thailand 

L. Rojanapantip M. Uraroongroj P. Kluengklangdon 

Bureau of Quality and Safety of Food, Department of Medical 

Sciences, The Ministry of Public Health, Tiwanon Road, 

Amphur Muang Nonthaburi 11000, Thailand 

P. Jaengkarnkit 

National Food Institute, 2008 Soi Arun Ammarin 36, 

Arun Ammarin Road, Bangyeekhan, Bangphlad, 

Bangkok 10700, Thailand 

K. Sirisuthanant 

Department of Livestock Development (DLD), 91 Moo 4 

Tiwanont Rd., Bangkadi Muang, Pathumthani 12000, Thailand 

C. Thonglue T. Pimma 

Central Laboratory (Thailand) Company Limited (CLT), 

50 Phaholyothin Rd., Ladyao, Jatujak, Bangkok 10900, Thailand 

needs for affordable materials, which require collaborative 

efforts at the national level. This paper describes the preparation 

of a new glutinous rice reference material, along with 

homogeneity and stability studies and the analytical work 

carried out for the certification of the contents of inorganic 

elements. The incurred material was collected from an 

actual rice paddy field. Material preparation along with 

homogeneity and stability testing were carried out at the 

Environmental Research Training Centre (ERTC). The 

P. Pengpreecha T. J. Fortune 

Thailand Industrial Metrology and Testing Service Centre, 

Thailand Institute of Scientific and Technological Research 

(TISTR), Klongluang, Pathumthani, Thailand 

R. Zwicker V. Permnamtip S. Laoharojanaphnad 

B. Suanmamuang 

Thailand Institute of Nuclear Technology (TINT), 

16 Vibhavadi Rangsit Rd, Ladyao, Chatuchak, 


J. Shiowatana W. Waiyawat A. Siripinyanond 

Department of Chemistry, Faculty of Science, 

Mahidol University, Rama 6 Road, Ratchthewee, 


K. Judprasong B. Boonsong 

Institute of Nutrition, Mahidol University, 

Phutthamonthon 4 Rd., Salaya, Phutthamonthon, 

Nakhon Pathom 73170, Thailand 

S. Talaluck 

Padaeng Industry Public Company Limited, 

13 Moo 4 Phratad Padaeng Subdistrict, Mae Sot, 

Tak 63110, Thailand 

123

homogeneity study was designed to have three experimental 

conditions; (A) 10 bottles of candidate materials being 

analyzed each with 2 replicates, (B) 20 bottles with 2 replicates, 

(C) 10 bottles with 7 replicates, in order to study the 

suitable treatments for homogeneity testing in the reference 

material production. It was shown that a minimum number 

of 10 bottles with duplicate analyses are enough to demonstrate 

the homogeneity of candidate reference material. 

Certification of a candidate reference material in a single 

laboratory using reference method was confirmed with an 

interlaboratory comparison participated by a certain number 

of well recognized testing laboratories in Thailand. Further 

elaborative results will be discussed. 

Keywords Inorganic analysis Elements Rice 

Reference material 

Introduction 

The world’s anthropogenic sources of inorganic elements 

deposition have been increasing following the industrial revolution 

when mining, smelting, electroplating, and other 

industrial activities are producing undesirable concentrations 

of metals, such as Cd, Cr, Cu, Ni, Pb, and Zn, which are not 

environmental friendly. Although trace metals are an important 

part of the soil ecosystem, accumulation of these metals is 

harmful to people, animals, plants and other organisms coming 

in contact with the soil and groundwater. It becomes more 

dangerous to human when the contaminated soil is cultivated 

for agricultural purposes. The metal mining industry in 

Thailand’s northern areas released a vast amount of cadmium 

(Cd), which contaminated the lands and river nearby. The 

lands are rice farms, which are the traditional sources of food 

for Thais and peoples in Asia. Determination of trace elements 

i.e. Cd, which is one of the main elemental pollutants in the 

environment, in rice samples is therefore very important from 

both a toxicological and nutritional point of views. Cadmium 

toxicity is hazardous for aquatic animal and plant life. Food is 

a pathway of the contamination. In man or animal, the metal is 

mainly accumulated in kidney and liver with long biological 

half life. Japanese scientists reported a serious health problem 

caused by consuming large amount of Cd in contaminated rice 

and water. Extensive researches later identified a symptom as 

‘Itai-itai’ disease. It has to be completely eradicated in Japan 

and yet their incident is increasing worldwide. 

The accurate determination of Cd in rice is vital. There 

has been a growing demand for rice flour national reference 

materials certified for both toxic and essential metals. 

Reference materials play an essential role for evaluating 

the accuracy of analytical results, and are important parts 

of good laboratory practice [1]. Quality control of analytical 

results obtained utilizing rice flour reference materials 

123 

is fundamental to the accurate analysis of rice. At present, 

several organizations provide rice reference materials certified 

for trace elements. The Japan National Institute for 

Environmental Studies (NIES) developed rice flour reference 

materials containing three levels of Cd. The National 

Institute of Standards and Technology (NIST) issued 

Standard Reference Material 1568 Rice Flour since January 

1978, though it provides certified values for toxic 

elements at only the normal level. The Korea Research 

Institute of Standards and Science (KRISS) and the 

National Measurement Institute Japan (NMIJ) have initiated 

the development of rice flour reference materials for 

Cd at both normal and elevated levels [2]. 

Each National Measurement Institute (NMI) of these 

countries has developed its own measurement standard for 

the measurement of inorganic elements in rice. In Thailand, 

the demand for food and environmental reference materials 

is rapidly increasing, NIMT together with ERTC respond 

to the urgent needs for affordable materials which require 

collaborative efforts at the national level. In this research 

paper, we describe the preparation of a new glutinous rice 

reference material. Glutinous rice is the major food of 

peoples from northern Thailand and some Southeast Asia 

countries. No reference material certified for Cd content in 

glutinous rice has commercially made available. This reference 

material will help improving the quality of 

analytical results generated from relevant chemical testing 

laboratories. It will provide the testing laboratories a reference 

material with the same matrix and concentration 

range with the field samples they always analyze. 

Methods 

Reagents 

All water used in this project is deionized water produced from 

the Milli-Q Element system with resistivity of 18.2 MX.Nitric 

acid used throughout the project is 65% Merck, ‘‘Suprapur’’ 

(\1 lg L -1 Pb) Merck catalogue number 100441. Stock 

standard solutions of the elements determined were prepared 

from its corresponded NIST SRM, i.e. NIST SRM3108 for Cd, 

NIST SRM3114 for Cu, NIST SRM3132 for Mn, and standard 

NIST SRM3168a for Zn. Working standard solutions were 

made by serial dilution of the stock solutions gravimetrically. 

Their corresponding spike isotopic standards, 111 Cd, 65 Cu, and 

67 Zn were purchased from Oak Ridge, USA. 

Preparation of candidate reference material 


A batch (100 kg) of incurred Cd contaminated, polished 

glutinous rice was collected from an actual rice paddy field. 

It was first air dried at room temperature to achieve less


than 10% moisture content before being pulverized with a 

rotary mill. About 15 kg of fine glutinous rice powder was 

obtained. The powder was then sieved through 90 lm sieve 

and later mixed in a V-shape blender for almost a week. 

Ten subsamples were taken from the bulk material for the 

preliminary homogeneity study. After satisfactory homogeneity 

level was achieved, the rice flour was then 

packaged in 30 g polypropylene bottles. Four hundred 

bottles of candidate rice reference material were finally 

obtained. They were irradiated by c radiation in order to 

minimize microbial contamination. The remaining material 

was discarded as it was considered to be less homogeneous. 

Homogeneity testing 

Homogeneity testing for mass fractions of elements in the 

candidate glutinous rice reference material was mainly carried 

out using the F test and ANOVA statistical test. Twenty bottles 

of candidate rice reference material were randomly selected 

from the batch. Samples were digested in HNO3 using hotplate 

digestion. Approximately 1 g of sample was accurately 

weighed into a beaker. Two portions of 5 ml HNO3 were 

added. They were covered with watch glass and then heated on 

the hotplate until nearly dry. The remaining was then re-dissolved 

with deionized water, before being filtered through 

filter paper and made up to 50 ml. The solution was then 

analyzed for inorganic elements content by ICP-OES. 

Stability study 

A literature review shows the good stability of inorganic 

elements in rice material [2]. It is expected that this glutinous 

rice reference material will be stable for at least 

12 months (especially after c radiation). The long-term 

stability of the candidate glutinous rice reference material 

was tested by storing three bottles of the candidate reference 

material at ?4 °C for a period of 6 months. After 1, 3, 

and 6 months, mass fraction of elements were determined 

in two replicates for each bottle. The procedures were the 

same as those used in the homogeneity study. Using linear 

regression as recommended in the ISO Guide 35: 2006 [3], 

instability would be detected if the slope of the regression 

line deviates from zero. The short-term stability study of 

the candidate glutinous rice reference material was tested 

by storing bottles of the candidate reference material at ?4, 

?20, and ?40 °C for a period of 8 weeks. After 4 and 

8 weeks, the mass fractions of elements were determined 

(in triplicate). The samples stored at ?4 °C were used as 

reference for the samples stored at ?20 and ?40 °C, 

respectively. Instability would be detected by comparing 

the measured mass fraction of elements of samples stored 

at ?20 and ?40 °C with those of samples stored at ?4 °C 

after 4 and 8 weeks. 

Certification of concentration with one laboratory 

Due to the low level of measurement uncertainty given for the 

certified reference materials produced by all reputable and 

highly experienced NMIs [4], it was decided to certify this 

glutinous rice reference material using the first approach given 

in the ISO Guide 34 [5]. A certification with measurement by 

one or more (reference) method in one laboratory has been 

employed. A certification of Cd, Cu, and Zn mass fractions in 

candidate glutinous rice reference material was initially performed 

by Inductively Coupled Plasma-Isotope Dilution 

Mass Spectrometry (ICP-IDMS) carried out by NIMT. Where 

the IDMS technique is not applicable for Mn analysis, the 

mass fraction of Mn is certified based on consensus value 

obtained from interlaboratory comparison. 

National interlaboratory comparison 

A series of national interlaboratory comparison exercise was 

conducted for confirmation of IDMS values. However, interlaboratory 

comparison is meant for certification of Mn content. 

Laboratories invited to take part in the exercise were reputable, 

and highly experienced in this field of measurement throughout 

Thailand, considered to be well-equipped and employing 

quality control and quality assurance procedures. Eleven laboratories 

taking part were requested to verify the quality of their 

measurements, in particular, the validity of calibration 

(including calibration of balances, volumetric glass wares and 

other tools of relevance). They are the Department of Medical 

Sciences (DMSc), the National Food Institute (NFI), Department 

of Livestock Development (DLD), Central Laboratory 

Thailand (CLT), Thailand Institute of Scientific and Technological 

Research (TISTR), Thailand Institute of Nuclear 

Technology (TINT), Department of Chemistry, Mahidol University 

(MU), Institute of Nutrition, Mahidol University 

(INMU), Padaeng Industry Public Company Limited, Environmental 

Research and Training Centre (ERTC), and the 

National Institute of Metrology (Thailand) (NIMT). Participants 

were free to choose analytical methods of which they had 

previous experience and could therefore be expected to give 

valid results when applied by an experienced analyst. They 

were also asked to make a minimum of five independent replicate 

determinations of each element in the candidate reference 

material, each laboratory being supplied with one bottle of 

prepared candidate rice material. 

Results and discussion 

Homogeneity testing 

In order to study the suitable treatments for homogeneity 

testing in the reference material production, this 

123

experiment was designed to have three experimental conditions. 

In the first set of experiment (A), 10 bottles of 

candidate materials were analyzed, each with 2 replicates. 

In the second set of experiment (B), 20 bottles of candidate 

materials were analyzed, each also with 2 replicates. The 

third experiment (C) was carried out by analyzing 10 

bottles of candidate materials, each with 7 replicates. The 

three set of experiments were tested for homogeneity using 

ANOVA. To evaluate the level of work needed to carry out 

for homogeneity testing, the uncertainty due to homogeneity 

study arising from each condition was compared. 

Table 1 displays analytical results of Cd, expressed in 

mg kg -1 on a dry-mass basis, for treatment A (10 bottles, 2 

replicates), B (20 bottles, 2 replicates), and C (10 bottles, 7 

replicates). Their corresponding parameters calculated 

from ANOVA, i.e. mean squares within bottle (MSwithin), 

mean squares between bottles (MS between), F calculation 

(Fcal), F critical (Fcrit), P value are summarized in Table 2. 

Cochran’s test is employed to test for outlier due to variation 

of analytical method in each bottle. This test 

indicated the good performance of analyst who performed 

the analyses. From Tables 1 and 2, where it can be seen 

that Fcal does not exceed Fcrit with P value is greater than 0.05, 

there is strong evidence, at the 95% confidence level, that from 

these three sets of experiments the candidate glutinous rice 

reference material is homogeneous for Cd content. Uncertainty 

due to inhomogeneity (ubb) was calculated using the 

expression given in the ISO Guide 35: 2006. 

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 

MSbetween MSwithin 

ubb ¼ 

n0 

In case where MSbetween is close to MSwithin, the 

following calculation was employed: 

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 

sffiffiffiffiffiffiffiffiffiffiffiffiffi 

4 2 

ubb ¼ 

MSwithin 

n 

mMSwithin 

The same treatment was applied to other elements of 

interest, in each case demonstrating the homogeneity of the 

candidate material. Table 3 summarizes uncertainty due to 

inhomogeneity arising from those three sets of experiments 

for all four elements. On the basis of these results, the 

material was considered to be homogeneous at the level of 

1.0 g. From Table 3, it can be seen that all three sets of 

experiments yielded the relative standard uncertainty due 

Table 1 Data for mass fraction of Cd (mg kg -1 ) (dry-mass basis, i.e. corrected for moisture content) for the three treatments of homogeneity 

study of the candidate glutinous rice reference material 

No. Mass fraction of Cd (mg kg -1 ) 

Treatment A Treatment B Treatment C 

10 bottles, 2 replicates 20 bottles, 2 replicates 10 bottles, 7 replicates 

Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep3 Rep4 Rep5 Rep6 Rep7 

1 0.65 0.65 0.65 0.65 0.60 0.61 0.58 0.62 0.59 0.56 0.58 

2 0.67 0.63 0.65 0.67 0.61 0.58 0.59 0.58 0.58 0.60 0.60 

3 0.67 0.68 0.69 0.66 0.58 0.60 0.58 0.58 0.58 0.59 0.59 

4 0.67 0.66 0.68 0.68 0.58 0.59 0.61 0.62 0.59 0.61 0.60 

5 0.68 0.65 0.67 0.65 0.59 0.59 0.60 0.60 0.61 0.62 0.58 

6 0.68 0.66 0.67 0.68 0.61 0.59 0.59 0.59 0.59 0.59 0.61 

7 0.61 0.64 0.68 0.68 0.59 0.57 0.61 0.61 0.63 0.61 0.59 

8 0.63 0.62 0.67 0.64 0.62 0.60 0.59 0.57 0.60 0.54 0.59 

9 0.64 0.69 0.68 0.65 0.58 0.61 0.60 0.58 0.60 0.58 0.59 

10 0.66 0.67 0.69 0.64 0.58 0.59 0.61 0.58 0.60 0.60 0.59 

11 0.66 0.66 

12 0.67 0.66 

13 0.66 0.67 

14 0.69 0.66 

15 0.66 0.65 

16 0.68 0.68 

17 0.67 0.64 

18 0.66 0.68 

19 0.69 0.66 

20 0.65 0.66 

Rep replicate 

123 

Accred Qual Assur


Table 2 Analysis of variance (ANOVA) from three treatments of the homogeneity testing of mass fraction of Cd (mg kg -1 ) in the candidate 

glutinous rice reference material 

to inhomogeneity (ubb) of approximately 1%. It pointed out 

that the number of bottles taken for analysis and number of 

replicate analysis does not affect results of homogeneity 

study. Ten sub-samples with two replicates analysis are 

sufficient to demonstrate the homogeneity of the candidate 

material. This finding corresponds with many literatures [4] 

previously written on the homogeneity study of reference 

material. In this certification exercise, it was decided to 

employ information from 10 bottles of candidate material 

with 2 replicate analyses. 

Stability study 

Treatment A Treatment B Treatment C 

10 bottles, 2 replicates 20 bottles, 2 replicates 10 bottles, 7 replicates 

MSwithin 0.000334 0.000245 0.000255 

MSbetween 0.000589 0.000180 0.000166 

Fcal 1.764 0.737 0.652 

Fcrit 3.020 2.137 2.040 

P value 0.194 0.745 0.748 

MS within mean squares within bottle, MS between mean squares between bottles, F cal F calculation, F crit F critical 

Table 3 Relative standard uncertainty due to between-bottle inhomogeneity (u bb) arising from those three set of experiments for all four 

elements 

Experimental conditions Relative standard uncertainty due to between-bottle inhomogeneity (u bb) 

This candidate glutinous reference material is subjected to 

long term stability study over a period of 6 months. There 

is no apparent physical/chemical damage observed during 

this period of time. The experimental data are given in 

Table 4. As described in the ISO Guide 35:2006, there is 

no physical/chemical model that would realistically 

describe a degradation mechanism for this kind of material. 

The straight line model is employed as empirical model 

Cd (%) Cu (%) Mn (%) Zn (%) 

A (10 bottles, 2 replicates) 1.3 1.4 0.5 0.6 

B (20 bottles, 2 replicates) 0.9 1.1 0.4 0.4 

C (10 bottles, 7 replicates) 0.7 1.1 0.4 0.4 

Table 4 Long-term stability 

study of candidate glutinous rice 

reference material over a period 

of 6 months 

Time (months) Mass fraction of elements (mg kg -1 ) 

Cd Cu Mn Zn 

0 0.62 1.4 7.4 19.1 

1 0.62 1.4 7.4 19.2 

3 0.61 1.4 7.4 19.1 

6 0.62 1.4 7.4 19.2 

Relative standard uncertainty due to instability (ustab) 3% 2% 2% 1% 

(y = b1x ? b0). For this kind of material, it is expected that 

the intercept is (within uncertainty) equal to the values on 

the day of zero. It is also expected that the slope does not 

significantly differ from zero. Figure 1 shows information 

obtained from stability testing. Using Cd as example, the 

regression line is obtained as: y =-0.000048x ? 0.618. 

The slope (b1) is equal to -0.000048, with its associated 

uncertainty [s(b1)] of 0.00186. The intercept (b0) is 0.618. 

According to the ISO Guide 35:2006, the slope insignificantly 

differs from zero, and hence no instability is 

observed if |b 1| \ t 0.95,n-2 s(b 1). In this case, the student 

factor for n - 2 degree of freedom and P = 0.95 (95% 

confidence level) is equal to 4.30. It is proven that the slope 

is insignificant, hence no instability was observed. By 

applying the same treatment with Cu, Mn, and Zn, no 

instability was detected over a period of study. The 

uncertainty due to instability (u stab) is calculated using the 

following expression: ustab = s(b1) t. Table 4 shows 

uncertainty due to instability over a shelf life of 12 months. 

As for the short-term stability, the ratios (R T) of the 

mean values (xT) of triplicate measurements made for 

123

Mass fraction of Cd (mg/kg) 

Mass fraction of Mn (mg/kg) 

0.65 

0.63 

0.61 

0.59 

0.57 

0.55 

7.7 

7.6 

7.5 

7.4 

7.3 

7.2 

7.1 

7.0 

samples stored at ?20 and ?40 °C and the mean value 

(xþ4 C) from the three determinations at ?4 °C were calculated 

as: 

RT ¼ x T =xþ4 C: 

The combined uncertainty at each temperature (uc,T) 

was obtained from the coefficient of variation (CV) of three 

measurements: 

uc;T ¼ RT 

0 2 4 6 8 

Time (months) 

0 1 2 3 4 5 6 7 

Time (months) 

Fig. 1 Stability study for Cd, Cu, Mn, and Zn in candidate rice reference material 

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 

: 

ðs=xÞ 2 

T 

ðs=xÞ 2 

þ4 C 

The RT ratio should be 1 in the case of ideal stability. 

But as slight instability might be expected at the high 

temperature, the value 1 should lie between RT - uc,T and 

RT ? uc,T. From this experiment, more than 98% of the 

measurements made for samples stored at ?20 and ?40 °C 

conditions, the values fell within RT ± uc,T. It was 

concluded that there was no instability during transport. 

As a result of the stability testing, the candidate material 

was considered to be suitable for certification, provided 

that the material is stored at typical room temperature or 

under refrigeration. 

Certification of concentration in single laboratory 

Analytical data obtained at NIMT were quantified by the 

Isotope Dilution (ID), which is an especially useful method 

when a sample has to undergo some chemical pre-treatment 

123 

Mass fraction of Cu (mg/kg) 

Mass fraction of Zn (mg/kg) 

1.50 

1.45 

1.40 

1.35 

1.30 

19.5 

19.3 

19.1 

18.9 

18.7 

0 2 4 6 8 

Time (months) 

18.5 

0 2 4 6 8 

Time (months) 


before analysis, because any loss of analyte during the pretreatment 

does not affect the final results. The ID method is 

based on the spiking of a known amount of a material of 

same element with different isotopic composition. After 

equilibration, the isotope ratio of two selected isotopes in the 

blend is measured using a mass spectrometer. The IDMS 

technique [6] carried out at NIMT is called ‘‘Double IDMS’’. 

The double IDMS analysis can be carried out in such a way 

as to minimize the need for correction factors due to mass 

discrimination and linearity effects by using a technique 

known as exact matching. In this technique, the sample and 

calibration standard are both spiked with the same amount of 

the same spike solution; and the amount of analyte in the 

calibration standard is matched to be the same as in the 

sample. This will remove the effect of any systematic error 

associated with measurement of these ratios when they are 

measured consecutively over a short period of time. 

Using the IDMS exact matching technique, the glutinous 

rice flour samples were decomposed with an AntonParr 

Multiwave 3000 microwave digestion system. About 

500 mg of the rice flour sample placed in a Teflon vessel, 

5 ml of HNO3 and 1 ml of H2O2 were added into the vessel 

together with appropriate amounts of their corresponding 

spike isotopes. The samples were digested inside a 

microwave oven with a digestion program of 1000 W for 

35 min. After cooling, they were diluted to about 50 ml. 

They were then analyzed for Cd, Cu, and Zn mass fractions


Table 5 Digestion methods 

and instrumental analytical 

techniques used by participants 

in the interlaboratory 

comparison exercise for 

elemental concentrations in 

candidate rice reference 

material 

by ICP-QMS using IDMS equation. The ICP-MS instrument 

employed in this work was a PerkinElmer SCIEX 

ELAN DRC II ICP-QMS. Details of the instrument operating 

conditions are as follows: RF power 1125 W, plasma gas 

flow rate 15 L min -1 , nebulizer gas flow rate 0.91 L min -1 , 

and auxiliary gas flow rate 1.2 L min -1 . 

Where the IDMS technique is not applicable for Mn 

analysis, the certified value is calculated from the consensus 

value obtained from interlaboratory comparison. 

This value was subsequently confirmed by analysis of Mn 

mass fraction in single laboratory. The candidate material 

was carefully analyzed for mass fraction by ICP-OES. The 

LAB ID Sample preparation Analytical techniques 

LAB1 AOAC 2005: 199.10 GF-AAS 

LAB2 1 g/HNO3, HClO4/Hotplate digestion ICP-OES 

LAB3 1.5 g/HNO3, HClO4/Block digester ICP-OES 

LAB4 AOAC 2005: 199.10 ICP-OES/ICP-MS 

LAB5 HNO3, HClO4/Hotplate AAS 

LAB6 0.2 g INAA 

LAB7 0.5 g/HNO3,H2O2/Microwave digestion ICP-OES 

LAB8 Microwave digestion ICP-MS/ICP-OES 

LAB9 10 g/HNO3, HClO4, HCl/Hotplate digestion ICP-OES 

LAB10 1 g/HNO3/Hotplate digestion ICP-OES 

LAB11 0.25 g/HNO3,H2O2/Microwave digestion ICP-MS 

LAB12 0.25 g/HNO3,H2O2/Microwave digestion ICP-MS 

ICP-OES instrument employed in this work was an 

ICP-OES PerkinElmer Optima 2000 DV. Details of the 

instrument operating conditions are as follows: RF power 

1500 W, plasma gas flow rate 15 L min -1 , nebulizer gas 

flow rate 0.7 L min -1 , auxiliary gas flow rate 0.2 L min -1 , 

with the wavelength of 257.610 nm. 

Uncertainty evaluation of Cd, Cu, Mn, and Zn analysis 

Measurement uncertainty for Cd, Cu, and Zn analysis 

carried out by IDMS are calculated according to the Guide 

on the Expression of Uncertainty in Measurement (GUM 

Fig. 2 The sets of results from interlaboratory comparison for Cd, Cu, Mn, and Zn accepted on technical and statistical grounds compared with 

values obtained from measurement in single laboratory (except Mn) 

123

1995), and EURACHEM (2000) expression. Uncertainty 

from characterization for Mn was estimated according to 

guide line given in the ISO Guide 35: 2006. 

National interlaboratory comparison 

As for the interlaboratory comparison, on receipt of data 

from participants, an identification number (Laboratory ID) 

was assigned to each laboratory. Nine laboratories reported 

data for Cd and Zn mass fractions, eight laboratories for Cu 

and Mn mass fraction, using a range of digestion conditions 

(HNO 3-based acid combination) and a variety of analytical 

techniques (AAS, GFAAS, ICP-OES, ICP-MS), including, 

in the case of one laboratory, INAA analysis of the solid 

phase. Table 5 lists the digestion methods and instrumental 

analytical techniques used by the participants. 

The sets of results submitted by participants were 

assumed to be normally distributed and analyzed statistically 

using Grubbs and Cochran’s tests to detect outlying 

values. The Grubbs test was used to detect outlying values 

in the population of individual results and in the population 

of laboratory means, while Cochran’s test was used to 

identify outlying values in the laboratory variances. The 

sets of results from interlaboratory comparison accepted on 

technical and statistical grounds are also presented in 

Fig. 2. Table 6 shows the mass fractions of Cd, Cu, and Zn 

carried out by IDMS analysis at NIMT, along with Mn 

result obtained from ICP-OES analysis at ERTC compared 

with results obtained from the interlaboratory comparison. 

It showed no significant differ between results obtained 

from the two approaches. 

Certification and calculation of uncertainty of reference 


Again, using Cd as the example, the uncertainty of the 

value assigned to the Cd concentration (0.69 mg kg -1 )of 

the glutinous rice certified reference material was calculated 

according to a modification of the GUM [7–11] and 

the recommends given in the ISO Guide 35: 2006, using 

the equation: 

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 

UCRM ¼ k u2 char þ u2 bb þ u2 q 

stab 

where U CRM denotes expanded uncertainty of the total Cd 

concentration of the glutinous rice certified reference 

material, k coverage factor, uchar uncertainty of the certified 

metal concentration in the candidate glutinous rice reference 

material, ubb uncertainty of the between-bottle 

inhomogeneity, ustab uncertainty of the instability. Table 7 

summarizes information used for certification of mass 

fraction of elements and their associated certified values. 

Future work 

In the future this candidate glutinous rice will be characterized 

for some more other elements i.e. As, Cr, Pb, and Hg. Stability 

of the reference material will be continuously monitored. 

Table 6 Analytical results of Cd, Cu, Mn, and Zn from interlaboratory comparison compare to values obtained by IDMS analysis performed at 

NIMT, along with values of Mn analysis taken from ICP-OES analysis using external calibration 

Elements Mass fraction of elements from 

interlaboratory comparison 

(mg kg -1 )(x ± 1s) 

Cd 0.63 ± 0.09 0.69 ± 0.02 

Cu 1.7 ± 0.6 1.5 ± 0.05 

Mn 7.8 ± 0.5 7.5 ± 0.2* 

Zn 22.0 ± 4.3 21.2 ± 0.4 

*Value for Mn analysis was taken from ICP-OES analysis using external calibration 

Mass fraction of elements 

from IDMS (mg kg -1 )(x ± u c) 

Table 7 Certified values with uncertainties (coverage factor of 2) for the mass fraction of Cd, Cu, Mn, and Zn in the glutinous rice reference 


Elements Mass fraction 

(mg kg -1 ) 

Standard uncertainty (mg kg -1 ) Combine uncertainty 

(mg kg -1 uchar ubb ustab ) 


U (k = 2) Certified values 

(mg kg -1 ) 

Cd 0.69 0.02 0.01 0.02 0.03 0.06 0.69 ± 0.06 

Cu 1.5 0.05 0.02 0.03 0.06 0.1 1.5 ± 0.1 

Mn 7.8 0.5 0.04 0.2 0.5 1.0 7.8 ± 1.0 

Zn 21.2 0.4 0.1 0.2 0.5 1.0 21.2 ± 1.0 

123


Applicability and availability 

Although this glutinous rice reference material has been 

developed specifically for use in the analysis of glutinous 

rice, it may also be of some value in the analysis of some 

other type of rice material. Enquiries concerning the 

availability of this material should be made to NIMT, from 

whom instructions for use can also be obtained. 

Conclusion 

This study confirmed that the number of samples or number 

of replicates measurements is not so critical for the 

homogeneity testing in the process for production of reference 

material. A minimum number of 10 samples with 2 

replicates measurements performed on each sample were 

sufficient to demonstrate homogeneity of the candidate 

reference material. Another conclusion that can be drawn 

from this study is the sample preparation is not a critical 

issue for determination of inorganic elements in rice sample. 

It was proven that an open digestion on the hotplate 

can easily give the analytical results relatively close (within 

its uncertainty) to the results obtained by microwave 

digestion. Microwave digestion of rice flour samples with 

HNO3 alone or in combination with other organic acids 

was adequate for the accurate determination of Cd, Cu, and 

Zn. The IDMS method employed in the present work 

enabled accurate and precise determination of the analytes 

in spite of non-quantitative recovery. 

Acknowledgments We gratefully acknowledge assistance from 

Dr. Pantip Klomjek of Naresuan University and Mr. Chaiwat 

Phadermrod of the Padaeng Industry Public Company Limited in the 

collection of the starting material. We are in debt to ERTC member 

of staffs, Sununtha Boonprakong, Watjarin Panyasa, Nuttaporn 

Sukhakasikorn, Phornjit Mungwongsa, Prakong Kotarapong, Wichai 

Rattanasriboonya, and Kamonchanok Tiyawan, for their hard work in 

the preparation of candidate material. Special thanks go to the 

Thailand Institute of Nuclear Technology (TINT) for facilitating the 

radiation of the sample. Finally, we would like to express our deep 

appreciation to the National Institute of Metrology, Thailand (NIMT) 

for sponsoring this project. 

References 

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Reference Materials–General and Statistical Principles (2006) 

ISO Guide 35-2006(E) (2006). ISO, Geneva 

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5. International Organisation for Standardisation (2009) General 

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