PARAMEE PENGPRECHA

THAILAND INSTITUTE OF SCIENTIFIC AND TECHNOLOGICAL RESEARCH

Preparation of matrix sample for Food Testing Laboratory

INTRODUCTION

No Traceability

NIMT (IDMS method)

Traceability to SI unit via

NIMT

TISTR has been designated by NIMT

Be able to assign true value using

IDMS

Preservative is widely used in food and beverageIts use has to be controlled and labeled “Preservatives added” .Its amounts has to be declared when submitted to Authority by TEST REPORT.In fact, most of laboratories can provide additives testing in food. BUT in the condition of ISO/IEC 17025 accredited.

Paramee CP 2009 สถาบนัวิจัยวิทยาศาสตรและเทคโนโลยีแหงประเทศไทย(วว.)ti t th

สถาบนัวิจัยวิทยาศาสตรและเทคโนโลยีแหงประเทศไทยสถาบนัวิจัยวิทยาศาสตรและเทคโนโลยีแหงประเทศไทย((วววว.).)i hti t th

Spiked benzoic acid in (500mg/kg) and sorbic acid (250mg/kg) in

70%EtOH

CRM Production : Preservatives in Food

2kg(40 bags)

Preparation of Matrix food sample containing preservatives content for food testing Laboratory (PT sample and CRM Production)

Characteristic study

Test Items Sample blank Spiked sample

Appearance Red curry smooth paste

pH 5.07-5.10 5.06-5.07

Water activity (%) 0.891-0.917 0.881-0.876

Total plate count (CFU/g)

3.6x106 12.0x106

Homogeneity : Feasibility Study

No of Prep

SA %RSDSA BA %RSDBA

Lot 1 (2kg)

273 5.3 549 8.1

Lot 2 (10kg)

257 2.2 500 2.5

Lot 3 (10kg)

237 0.7 482 1.2

Lot 1 : Blending No 1

Lot 2 : Blending No 2Random Sampling : N=10-12 (n=2) by HPLC-UV

Method : Modified Determination of benzoic acid, sorbic acid and methyl, ethyl, propyl and butyl parabens by HPLC, SOP No. CLG-BSO.01,

US Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science

Lot 3 :Blending and kneading

ISO 13528 : Statistical methods for use in proficiency testing by inter laboratory comparisons)

Sampling N = 12(Between Sample)

n1 n2

Test portion n=2(Within sample)

Lot No.3

N2

n1 n2

N3

n1 n2

N4

n1 n2

N5

n1 n2

N6

n1 n2

N1

Homogeneity Study

HPLC UV-VISModified Determination of benzoic acid, sorbic acid and methyl, ethyl, propyl and

butyl parabens by HPLC, SOP No. CLG-BSO.01, US Department of Agriculture, Food Safety and Inspection Service

Measurement results for homogeneity check, Sorbic acid in curry paste (mg/kg)

Test portionAvera

ge (A-B)(A-

B)^2%RP

DSample No A B

mg/kg mg/kg

mg/kg

mg/kg

1026 237.5 241.5 239.5 4.0 16.0 1.671029 238.2 238.5 238.4 0.3 0.1 0.131036 236.2 234.8 235.5 1.4 2.0 0.591065 237.4 235.7 236.6 1.7 2.9 0.721067 239.7 238.1 238.9 1.6 2.6 0.671071 235.5 241.5 238.5 6.0 36.0 2.521072 238.8 240.1 239.5 1.3 1.7 0.541079 233.4 237.9 235.7 4.5 20.3 1.911088 239.3 237.4 238.4 1.9 3.6 0.801099 237.4 237.1 237.3 0.3 0.1 0.131121 232.0 235.9 234.0 3.9 15.2 1.671122 237.8 238.6 238.2 0.8 0.6 0.34

Mean (N=12)237.

5SD

1.750

% CV0.73

7Within-sample standard deviation

2.051

Between-sample standard deviation0.97

9SD robust statistic from Proficiency assessment Robust statistic (3 outliers excluded)

19.06

0.3xSD (robust statistic)5.74

8One-way ANOVA between-sample (F) 1.46One-way ANOVA between-sample (F critical) 2.71

Measurement results for homogeneity check, Sodium benzoate in curry paste (mg/kg)

Sample NoTest portion

Average (A-B)

(A-B)^2 %RPD

A B mg/kg mg/kg mg/kg mg/kg1026 480.0 490.3 485.2 10.3106.09 2.121029 480.4 482.2 481.3 1.8 3.24 0.371036 478.7 475.1 476.9 3.6 12.96 0.751065 472.3 482.5 477.4 10.2104.04 2.141067 491.8 485.6 488.7 6.2 38.44 1.271071 482.1 489.1 485.6 7 49 1.441072 486.7 492.8 489.8 6.1 37.21 1.251079 484.9 481.8 483.4 3.1 9.61 0.641088 464.4 482.3 473.4 17.9320.41 3.781099 483.4 483.6 483.5 0.2 0.04 0.041121 473.9 480.7 477.3 6.8 46.24 1.421122 490.7 491.7 491.2 1 1 0.20

Mean (N=12) 482.8SD 5.670% CV 1.174Within-sample standard deviation 5.509Between-sample standard deviation 4.120SD robust statistic from Proficiency assessment Robust statistic (4 outliers excluded) 41.4300.3xSD (robust statistic) 12.429One-way ANOVA between-sample (F) 2.11One-way ANOVA between-sample (F critical) 2.71

ISO Guide 35 -Reference materials-General and statistical principles for certification

HOMOGENEITY (☺)

Short term Stability Study

Storage condition : in Nylon transparent plastic bag : Dark color were observed after 3 months at 25OC.

ySod.BA@4C = -0.0625x + 503.78�R² = 0.0672 y Sod.BA@25C= -0.2285x + 494.93�R² = 0.20

ySA@4C = -0.3473x + 254.96�R² = 0.7725

ySA@25C = -0.9157x + 244.88�R² = 0.8918

Instability of sorbic acid

PT Food Sample : Stability Study

Stability study and shelf life for short term study (3months)

Data (Na.benzoate)

25C 4C Remark

Slope (b1) -0.2285 -0.0625

Intercept(b0) 494.931 503.779

S2 (sd of the point among the line)

662.56 822.16

S(b1) (uncertainty associated with the slope)

0.175 0.195

Significant of slope (t0.95, n-2=26 ,=2.056)

0.228<0.360 0.062<0.401

2

)(1

2110

2

−

−−

=∑=

n

xXbbY

s

n

ii

∑=

−

=n

ii XX

ss

1

2)(

)( 12,95.01 bxstb n−<

No instability was observed at 25C and 4C of storage condition for 90days

ISO Guide 35 – Reference materials-General and statistical principles for certification (Annex B5)

PT(Local Labs) : Sodium benzoate in curry paste

MTC-PT-1000-2550

-assigned value from 6 Labs (ISO/IEC 17025 accredited), - Evaluation of the PT is according to ISO 13528 : Statistical methods for use in proficiency testing by inter laboratorycomparisons)

-Reference Standards were from NIST, RMIPO, ACROSS, BDH, Carlo Erbra, Fluka, MERCK, Riedel-dehaenAG, SIGMA Aldich, Supelco, UNILAB,and USP

Oct 2007-Feb 2008

77.11175.0553.9509.5120.4 2222 =+++=PTu

2222stabPTwbbPT uuuuu +++=

Sodium benzoate PT Sample =474+24 mg/kgU= 4.9%

where k = 2 is the coverage factor for a 95 % confidence level and uCRM is the combined standard

uncertainty calculated according to ISO Guide 35

22hom* assignoPT uuu +=

APMP –P14 : BENZOIC ACID IN CURRY PASTE

CRM Production : Benzoic acid in curry paste

Material No of bags (50g)

PT Sample 40

CRM needed 50

Feasibility study 20

Characteristic study 20

Homogeneity study 10

Stability study 60

10kg

Gamma radiationEliminate bacteria*

*Curry paste samples were sterilized by gamma ray irradiation at Thailand Institute of Nuclear and Technology (TINT)

•Decreasing of sorbic acid was observed and inhomogeneity was found.•Study on stability was found that 39.5% of sorbic acid was decreased @ 1000 mg/l) after 1h at 105C.•Therefore sorbic acid was not considered for the APMP.QM-P14

*the homogeneity were carried on before and after radiation

Analysis Blank sample PT sampleBenzoic acid Not detected

(less than 3 mg/kg)See homogeneity study

pH (10%w/v) 5.09 5.02Water activity 0.89% 0.88%Total plate count 1.0x106 CFU/g 4.2x106 CFU/g (before irradiation)

8.3x103 CFU/g (after irradiation)Solubility 80% w/w Non soluble matter 4% w/w on 1 micron membrane filter

16% w/w on 22 micron membrane filter

Characterization Study : Benzoic acid in curry paste

Result is similar to the previous study

CRM Homogeneity Study : Benzoic acid in Curry Paste

Homogeneity: Benzoic acid(mg/kg)n 1 2 3 Average SD

1 381.6 387.7 388.6 386.0 3.82 390.7 387.1 390.9 389.6 2.1

3 377.3 388.9 390.0 385.4 7.0

4 386.6 388.9 389.7 388.4 1.6

5 388.2 389.5 391.1 389.6 1.4

6 389.9 388.3 391.0 389.7 1.3

7 388.6 390.3 392.9 390.6 2.2

8 389.8 392.7 392.8 391.8 1.7

9 391.2 393.9 389.3 391.5 2.3

10 388.0 382.3 384.9 385.1 2.8

Figure 1: Participants’ results for benzoic acid content in curry paste sample. The horizontal blue lines represent the proposed reference value (median) and its associated uncertainty. The horizontal brown line is the arithmetic mean value and its associated uncertainty.

assigned Value for CRM =378 mg/kgU=12 mg/kg

CRM Stability Study : Benzoic Acid in Curry Paste

BA -22C

Slope (b1) 0.262

Intercept(b0) 379.334

S2

(sd of the point among the line) 9.162

S(b1) (unc associated with the slope)

0.143

Significant of slope

(t0.95, n-2=10 ,=2.776)

0.261<0.396)( 12,95.01 bxstb n−<

No instability was observed at -22C for 2 years

CRM : Benzoic acid in curry paste

22222ltsstsassignedwbbCRM uuuuuu ++++=

Sources of unc ui ui2

usts 0.175 0.031

ults 0.143 0.020

u(assigned) 6.500 42.250

Sbb 2.928 8.572

S within gr 3.097 9.591SQRT(ui

2

combined) 7.776

U (k=2) 15.6

%U 3.2

Benzoic acid = 378 +15.6 mg/kg

•The certified value is based on isotope dilution mass spectrometry method which is analysed by National Metrology Institute of Thailand•Benzoic acid SRM is used for matrix matching standard curve

CRMCRM kxuU

•uncertainty is the combined standard uncertainty multiply by k calculated according to ISO Guide 35,•where k = 2 is the coverage factor for a 95 % confidence level

=

ACKNOWLWDGEMENT

Dr. Chainarong Cherdchu NIMTDr. Charun Yafa NIMTMs.Pranee Phukphatthanachai NIMTDr. Luxsamee Plungsangmas TISTRMr. Cheumsak Sinchaisri TISTRMs.Vilaval Pongpitak et.al. TISTR (BCL)Ms.Thippaya Junvee Fortune et.al. TISTR (ACL)

ACL team fromTISTR