statistical sample

8/13/2019 Statistical Sample

1/11

CF/7 CRD 23ORIGINAL LANGUAGE ONLY

JOINT FAO/WHO FOOD STANDARDS PROGRAMME

CODEX COMMITTEE ON CONTAMINANTS IN FOODS

Seventh Se!"nM"#"$% R&!'n Fe(e)'t!"n% * + ,2 A-)!. 2,3

C"00ent &10!tte( 1 J'-'n "n 'en(' !te0 ,4

PRELIMINARY REPORT ONINTERNATIONAL 5ALIDATION OFANALTICAL METHOD TO DETERMINE INORGANIC ARSENIC IN RICE6P)e-')e( 1 J'-'n

1 BACKGROUND..............................................................................................................................................................2

2 SCOPE OF THE STUDY...............................................................................................................................................2

3 OUTLINE OF VALIDATION.......................................................................................................................................3

1.1PRINCIPLEOFTHEMETHODS.......................................................................................................................................3

1.2TIMEFRAME..................................................................................................................................................................3

1.3ARSENICSPECIESANALYZED.......................................................................................................................................3

1.4PRELIMINARYANALYSIS..............................................................................................................................................3

4 TEST MATERIALS........................................................................................................................................................3

1.5PREPARATIONOFTESTMATERIALS.............................................................................................................................31.6HOMOGENEITY.............................................................................................................................................................4

1.7STABILITY....................................................................................................................................................................4

5 DISPATCH OF TEST MATERIALS............................................................................................................................4

1.8LABORATORYCODE.....................................................................................................................................................4

1.9DISPATCHOFTESTMATERIALS....................................................................................................................................4

6 RESULTS AND STATISTICAL ANALYSIS..............................................................................................................4

1.10RESLTSRETRNED..................................................................................................................................................4

1.11STATISTICALE!ALATION........................................................................................................................................5

1.12SMMARYOFRESLTSFOR"INASINRICEHPLC#ICP#MS 1$ METHOD................................................................5

1.12.1LIMITOFDETECTIONANDLIMITOF%ANTIFICATION..........................................................................................7

1.12.2RECO!ERY..............................................................................................................................................................7

1.12.3REPEATABILITY......................................................................................................................................................7

1.12.4REPRODCIBILITY...................................................................................................................................................7

1.12.5APPLICABLERANGE................................................................................................................................................7

7 FUTURE PLAN...............................................................................................................................................................8

ANNE! FLO" OF #IN AS IN RICE HPLC$ICP$MS 1% METHOD......................................................................1&


2/11

CRD 23 2

Summary

An analytical method employing separation by HPLC and detection by ICP-S !inducti"ely coupled plasma-mass spectrometry#de"eloped and nationally "alidated in $apan %as internationally "alidated through a collaborati"e study &or determining inorganicarsenic in rice' both indica and (aponica types)*he method determines inorganic arsenic in rice as the sum o& inorganic arsenicspecies !As!III# and As!+##)

*he inistry o& Agriculture' ,orestry and ,isheries !A,,#' $apan' organied the study in accordance %ith theIS./I0PAC/A.AC

International Harmonied Protocol) A total o& 1 laboratories &rom &our countries participated in this "alidation !one in Indonesia' t%oin Singapore' &i"e in *hailand and eight &rom $apan#)

*he study started in $anuary 212 and the &inal analytical results %ere recei"ed in December 212) It consisted o&4 preparation o&5nglish S.P and in"itation &or participation6 analysis o& three preliminary materials !indica and (aponica rice#6 re"ie% o& the S.P'ad(ustment thereo& and suggestions to participating laboratories as necessary6 analysis o& t%o series o& test materials' eachconsisting o& ten test materials !hus7ed and polished indica and (aponica rice#6 and statistical e"aluation o& recei"ed analyticalresults)

Analytical results o& 2 test materials %ere obtained &rom 12 laboratories and %ere statistically e"aluated) *he method per&ormancecharacteristics %ere then e"aluated against the Code8 criteria and &ound to be su&&icient &or determination o& inorganic arsenic at theconcentrations at or higher than )3 mg/7g)

Limit o& detection !L.D# and limit o& 9uanti&ication !L.:# %ere estimated to be )2 mg/7g and )2 mg/7g' respecti"ely) Reco"eryo& the method %as in a range o& ;-11 < at the concentrations o& L.: !)2 mg/7g# and 1=L.: !)2 mg/7g#)

Repeatability relati"e standard de"iation !RSDr# and reproducibility relati"e standard de"iation !RSDR# %ere calculated at the di&&erentconcentrations o& inorganic arsenic bet%een )3-)> mg/7g)*he RSDr%as in a range o& 3)2-?) < and the RSDR%as in a range o&>);-3


3/11

CRD 23 3

3 O&t.!ne ": v'.!('t!"n

,;, P)!n#!-.e ": the 0eth"(

ethod %as de"eloped &or selecti"e determination o& inorganic arsenic in rice) Inorganic arsenic &rom rice po%der is e8tracted %ithnitric acid under a heating condition) +arieties o& inorganic arsenic species !As!III# and As!+## and organic arsenic compounds!monomethylarsonic acid !A#' dimethylarsinic acid !DA# and others# in the e8traction solution are separated by highper&ormance li9uid chromatography !HPLC# and detected by single ion monitoring using inducti"ely coupled plasma-massspectrometry !ICP-S#)

*he scheme o& method is described in the Anne8)

,;2 T!0e:)'0e

*he study started in $anuary 212 and the &inal analytical results %ere recei"ed in December 212)

,;3 A)en!# -e#!e 'n'.


4/11

CRD 23 B

*%o sets o& ten types o& rice samples totaling 2 test materials %ere dispatched to participating laboratories) Randomly selected t%o"ials &rom the abo"e mentioned 1 "ials &or each type o& rice sample %ere randomly labeled &rom A1 to A2 &or each laboratory) *heali9uots %ere stored at BFC)

,;? H"0"ene!t

*o test &or homogeneity' randomly selected ten samples o& each test material %ere analyed in duplicate and con&irmed to beGsu&&icient homogeneity according to the I0PAC G*he International Harmonied Protocol &or the Pro&iciency *esting o& Analytical

Chemistry Laboratories !2#)

*otal arsenic and inorganic arsenic !sum o& As!III# and As!+## concentrations %ere used as homogeneity parameters) *otal arsenic%asdetermined using inhouse-method coupling the A.AC >3)21 and A.AC >;)1' Atomic Absorption Spectrometry a&ter (eldahl,las7 digestion) Inorganic arsenic %as determined using the method used in this study)

,urthermore' t%o organic arsenic compounds' A and DA' %ere analyed using the methodused in this study' and moisturecontent %as analyed using IS. ?1242> method %ith slight modi&ication 1) Analytical results o& A' DA and moisture in the testmaterials %ere used as re&erence data &or homogeneity and stability)

,;7 St'1!.!t

Stability o& inorganic arsenic in the po%dered rice sample stored at BFC &or 3 months had been tested prior to starting this study byusing materials prepared &or the pre"iously conducted national "alidation)

In order to chec7 stability and inter con"ersion o& arsenic species and loss or gain o& moisture in the test materials ' participatinglaboratories %ere re9uired to analye A and DA concentrations and moisture content in the distributed test materials andreported results %ere assessed to e"aluate the stability o& the samples and to determine necessity o& data correction by moisturecontent)

*he reported analytical results pro"ide e"idence o& the stability o& the samples &or up to 3 months)

= D!-'t#h ": tet 0'te)!'.

,;* L'1")'t") #"(e

.ne o& lo%er case alphabetical codes Ga to Gp %as assigned to each laboratory and sho%n as blanch code on the label o& thedispatched test materials' e)g) GA1-8)

,;@ D!-'t#h ": tet 0'te)!'.

In August 212' test materials A1-A2 %ith a certi&ication o& radio cesium concentrations and a certi&ication o& plant protection %eredispatched to 1 laboratories) All materials %ere recei"ed by mid September) .ne laboratory recei"ed the materials in .ctober asthe dispatch %as pendingthe optimiation o& analytical condition o& that laboratory)

? Re&.t 'n( t't!t!#'. 'n'.!

,;, Re&.t )et&)ne(

Participants %ere re9uired to report the concentrations o& inorganic arsenic !As!III#' As!+# and the sum o& As!III# and As!+#' A'DA in mg/7g# obtained using the HPLC/ICP-S method in single analysisand moisture content &rom modi&ied IS. ?1242> !seeB)2' &ootnote 1#)

Results %ere submitted &rom 12 laboratories)

Some laboratories reported results o& duplicate analysis o& A1-A2'%hile the test materials A1-A2 %ere composed o& t%o sets o&rice samples to obtain duplicate data sets in single analysis) In this case' as IS. ?2-241>>B states' random selection %as made&rom the reported results to choose one analytical result &or each test material)

Some laboratories reported results corrected &or moisture content %hile the S.P does not re9uire such correction) hen thecorrected results %ere reported' inorganic arsenic concentrations %ithoutmoisture content correction %ere used &or statisticale"aluation)

1 Although in IS. ?1242> g o& sample is used &or moisture measurement' 2 g o& rice po%der %ere used in this studydepending

on%eight &or ali9uots)


5/11

CRD 23

,;,, St't!t!#'. Ev'.&'t!"n

*he 9uantitati"e results o& inorganic arsenic in ricesubmitted by participating laboratories %ere statistically e"aluated in order topro"ide per&ormance characteristics)

Analytical results o& moisture and organic arsenic species !A and DA# %ere used to e"aluate %hether signi&icant 9uality changein relation to moisture content and/or inter-con"ersion bet%een organic and inorganic arsenic species in test materials occurred) *he

mean moisture and A and DA concentrations o& 1 test materials != 2 series#calculated &or each laboratory' a&ter e8cludingoutliers by Cochran test and rubbs test' %ere "ery similar to those obtained by homogeneity testing) It suggests that moisturecontent and organic arsenic species in test materials %ere stable during transportation and storage)

Analytical results o& inorganic arsenic !sum o& As!III# and As!+## &or each sample material!12 data sets' t%o indi"idual results &romeach o& 12 laboratories# %ere sub(ected to Cochran test and rubbs test to detect outliers according to the IS./I0PAC/A.ACInternational Harmonied Protocol) +alues identi&ied as outliers %ere e8cluded &rom the statistical e"aluation)

,;,2 S&00') ": Re&.t :") In A !n )!#e HPLCBICPBMS , 0eth"(

*able 2 summaries the statistical "alues relating to the method coded GIn As in rice HPLC-ICP-S 1)

Although standard de"iations &or hus7ed rice %ere calculated to be slightly higher than those &or polished rice'relati"e reproducibilitystandard de"iations &or all test materials met the Code8 precision re9uirements sho%n in the Guidelines &or establishing numeric

"alues &or method criteria and/or assessing method &or compliance thereo& in the Procedural anual)

*hese data demonstratesu&&icient per&ormance o& the method and that it is applicable &or determination o& inorganic arsenic in rice ata concentration range o& )3 mg/7g to )? mg/7g)

*he "ariability o& repeatability and reproducibility &or indi"idual inorganic arsenic species !e)g) As!III## %asbigger than that &or totalinorganic arsenic !sum o& As!III# and As!+##' especially &or As!+# at lo% concentrations)

It must be noted that the method is applicable to determine total inorganic arsenicbut not As!III#or As!+# indi"idually) Inter-con"ersionbet%een As!III# and As!+# may occur during transportation' storage' e8traction step and e"en in a standby time &or insertion o& testportion to HPLC/ICP-S instrument)


6/11

T'1.e 2> St't!t!#'. v'.&e ": !nte)B.'1")'t") tet :") (ete)0!n't!"n ": !n")'n!# ')en!#

Tet M'te)!'.

(aponica indica

hus7ed polished polished polished polished hus7ed polished polished polished polished

,A1 2A$ 3A$, 4A$2 =A$3 ?81 7C$ *D$ @E$ ,F$

*otal data sets!Jumber o& laboratories submitted results#

12 12 12 12 12 12 12 12 12 12

Jumber o& laboratoriesa&ter eliminating outliers

11 11 11 11 1 12 11 1 1 1

Jumber o& outliers 1 1 1 1 2 1 2 2 2

Jumber o& analysis &or each test material 2 2 2 2 2 2 2 2 2 2

ean "alue o& inorganic arsenicKmg/7g )21 )?; )1;B )1?B )B? )1BB )>2 )3 )2> );;

Repeatability standard de"iation !Sr# )1 ) )12 )? )21 )11 )B; )21 )1 )B3

Repeatability "alue' r!2); = Sr# )2; )1B )33 )1> ); )32 )13 ); )B )12

Repeatability relati"e standard de"iation!RSDr# K )2 ?) )? )3

Reproducibility standard de"iation !SR# )B? )13 )31 )2; )B )3 )2 )11 )2> )?

Reproducibility "alue' R!2); = SR# )13 )3? ); )?; )1; )>? ) )3 );2 )1>

Reproducibility relati"e standard de"iation!RSDR#K); 2B 22 3 1 >);

HorRat 1)2 ); ); ); ) 1)1 1) 1)? ) )


7/11

,;,2;, L!0!t ": Dete#t!"n 'n( L!0!t ": &'nt!:!#'t!"n

Limit o& detection and limit o& 9uanti&ication o& the method %ere estimated by a re&erence laboratory2)

,or the di&&iculty to obtain blan7 rice samples' a rice sample %ith inorganic arsenic &orti&iedat a concentration o& )2 mg/7g !)1 Ego& inorganic arsenic into ) g rice# %as analyed ; times in one day' and standard de"iation !s# o& the results %as calculated)

L.D %as estimated to be )2 mg/7g based on 3)3s!)1B M )2 Kmg/7g# and L.: %as estimated to be )2 mg/7g' the higher"alue o& calculated 1s!)BB Kmg/7g# and the &orti&ied concentrationo& )2 mg/7g)

,;,2;2 Re#"ve)

Percent reco"ery o& the method %as estimated by a re&erence laboratory)

*o assess reco"ery'rice samples %ith inorganic arsenic&orti&ied at t%o di&&erent concentrations' )2 mg/7g !L.:# and )2 mg/7g!1=L.:#' %ere analyed 1 times in di&&erent days' once in each day)Reco"ery %as calculated to be in a range ;-11 mg/7g)*he RSDr%as in arange o& 3)2-?)


8/11

CRD 23 ;

7 F&t&)e P.'n

e are planning to submit detailed report &or publication in a peer-re"ie%ed scienti&ic (ournal)

e also plan to conduct comparison o& more "ersatile and easier methods %ith the abo"e-mentioned HPLC/ICP-S method in "ie%o& selecting appropriate routine methods)


9/11

CRD 23 >

Re&erences

1) *hompson' )' et al)' *he International Harmonied Protocol &or the Pro&iciency *esting o& Analytical Chemistry Laboratories)Pure Appl) Chem)' ?;!1#' 1B-1> !2#

2) Hor%it' )' Protocol &or the Design' Conduct and Interpretation o& ethod-Per&ormance Studies) Pure Appl) Chem)' ?!2#'331-3B3 !1>>#

3) A.AC .&&icial ethods o& Analysis !22#) Interlaboratory Collaborati"e Study' Appendi8 D4 uidelines &or Collaborati"e StudyProcedures *o +alidate Characteristics o& a ethod o& Analysis

B) IS. ?2-241>>B' Accuracy !trueness and precision# o& measurement methods and results -- Part 24 @asic method &or thedetermination o& repeatability and reproducibility o& a standard measurement method

) IS. ?1242>'Cereals and cereal products -- Determination o& moisture content -- Re&erence method

) IS./I5C 1?2' eneral re9uirements &or the competence o& testing and calibration laboratories

?) Code8 Procedural anual 2thedition' Guidelines &or establishing numeric "alues &or method criteria and/or assessingmethods &or compliance thereo&

;) A.AC >3)21' Arsenic in ,ood' A.ACI .&&icial ethod o& Analysis' 1;th edition)

>) A.AC >;)1' Arsenic' Cadmium' Lead' Selenium' and Qinc in Human and Pet ,oods' A.ACI .&&icial ethod o& Analysis' 1;th

edition)


10/11

CRD 23 1

Anne> F."$ ": In A !n )!#e HPLCBICPBMS , 0eth"(

LC conditionsN

ColumnNCAPC5LL PA C1;'particle sie Em' B) mm i)d) 8 2 mm !Shiseido Ltd)' *o7yo' $apan#' or e9ui"alent)

obile PhaseNSolution composed o& )

In(ection "olumeN2 EL)

Column temperatureNRoom temperature' 2F3FC)

ICP-S conditionsN

onitoring ionNm/ ?' single ion monitoring).ptimie the condition &or detection o& arsenic'

&'()* 0.5 ) +, -+/''/ (' / - ( ( 10 L ) ' '.

P'-' ('-+/' ' * 500 : ( /('' () (() *(' / ('

'*.

A// 2 L +, 0.15 +;L (( (/.

H' 100


11/11

CRD 23 11

e)g)' Agilent ?ce ICP-S systemR, po%erN1) 7)Plasma gas !Argon gas#N1 L/min)Carrier gas !Argon gas#N)? L/min)Au8iliary gas !Argon gas#N)> L/min)a7e up gas !Argon gas#N)B3 L/min)

Identi&y the pea7 o& each chemical &orm o& arsenic in the order o& elution and the retention time !T1T2T3

statistical sample

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