Tiêu chuẩn iso 15302 2017

© ISO 2017 Animal and vegetable fats and oils — Determination of benzo[a]pyrene — Reverse phase high performance liquid chromatography method Corps gras d’origines animale et végétale — Détermination[.]

A nimal and veg etable fats and oils

Corps gras d’origines a imale et v g tale — Déte mination du

benzo[a] pyr ne — Méth de p r chromato ra hie liquide à h ute

pe frma c à p larité de p ase inve sée

T ir d edition

2 17-0

Reference n mb r

ISO 1 3 2:2 17(E)

COPYRIGHT PROTECTED DOCUMENT

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F reword i v

1 Sc ope 1

2 Nor mati ve r eferenc es 1

3 Terms an definitions 1

4 Principle 1

5 Re g ents 1

6 A pparatus 3

7 S mpl ng 4

8 Preparatio of test sample 4

9 Proc ed re .4

10 Ex pres io of r esults 6

1 1 Calculation of r elative r esp nse factors 6

1 2 Calculation of benzo[a]pyr ene content 7

11 Precision 7

1 1 Repeatabi ty 7

1 2 Reprod cibi ty 7

12 Test rep r t 7

A nne x A (informative)Results of inter laborator y tests 8

Biblog raphy 9

ISO (he Int ernational Org nization for Stan ardization) is a worldwidefede ation of national s an ards

b dies (ISO membe b dies) The work of pr p ring Int ernational Stan ards is normaly car ied out

through ISO t ech ical committ ees Each membe b dy int er st ed in a subje t for w hich a t ech ical

committ ee has be n es a lshed has the right t o be r pr sent ed on that committ ee Int ernational

org nizations, g ove nmental an non-g ove nmental, in laison with ISO, also take part in the work

ISO cola orat es closely with the Int ernational Ele trot ech ical C mmis ion (IEC) on al matt ers of

ele trot ech ical s an ardization

The proc d r s used t o develo this document an those int en ed for it furthe maint enanc ar

desc ibed in the ISO/IEC Dir ctives, Part 1 In p rticular the dife ent a pro al c it eria ne ded for the

dife ent ty es of ISO document should be not ed This document was draft ed in ac ordanc with the

edit orial rules of the ISO/IEC Dir ctives, Part 2 ( e www iso org dir ctives)

A tt ention is drawn t o the p s ibi ity that some of the element of this document ma be the subje t of

p t ent right ISO shal not be held r sponsible for identifying any or al such p t ent right Detais of

any p t ent right identified d ring the develo ment of the document wi be in the Introd ction an / r

on the ISO ls of p t ent de larations r c ived ( e www iso org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

F or an ex lanation on the v lu tary natur of s an ards, the meaning of ISO spe if ic t erms an

ex r s ions r lat ed t o conformity as es ment, as wel as information a out ISO’ s adhe enc t o the

World Trade Org nization (WTO) principles in the Te hnical Bar ie s t o Trade (TBT) se the folowing

URL: www iso org iso / for word html

This document was pr p r d b Te h ical C mmitt ee ISO/TC 34, Fo d produc ts , Subcommitt ee SC 1 ,

Animal a d vegeta le f ts a d oil s

This third edition canc ls an r plac s the se on edition (ISO 1 3 2:2 0 ), of w hich it cons itut es a

minor r vision The sco e ofthisdocument has be n r vised t o ex clu e it a plcation t o mi k an mi k

prod ct an their de iv tives

A nimal and veg etable fats and oils — Determination of

This document spe if ies a method for the det ermination of benzo[a ]p r ne in c u e or r fined edible

ois an fat b r verse-phase high pe formanc lq id chromat ogra hy (HPL ) using fluorimetric

det ection in the rang e 0,1µg kg t o 5 µg kg

Mik an mik prod ct (or fat coming from mi k an mik prod ct ) ar ex clu ed from the sco eof this

document

2 Normati ve referenc es

The folowing document ar r fe r d t o in the t ext in such a wa that some or al of their cont ent

cons itut es r q ir ment of this document F or dat ed r fe enc s, only the edition cit ed a ples F or

un ated r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amendment ) a ples

ISO 66 1, Animal a d vegeta le fts a d oil s — Prep ration o tes t s ample

3 Terms and definitions

F or the purposes of this document, the fol owing t erms and def initionsa ply

ISO an IEC maintain t erminolo ical data ases for use in s an ardization at he folowing ad r s es:

— IECEle tro edia: a aia le at ht p:/ www ele tro edia org

— ISO Onlne brow sing plat orm:a ai a le at ht p:/ www iso org o p

3.1

benzo[a ]pyrene co tent

mas fraction of benzo[a ]p r ne in the t es p rtion, as det ermined using the method spe if ied in this

document

Note 1to entry: T e content is e pres ed in mic o rams per kilo ram

4 Principle

A t es p rtion is dis olved in l ght petroleum an benzo[b]chry sene is ad ed as int ernal s an ard A

suita le amou t of sample is adsorbed on an alumina column an elut ed with lght petroleum t o r mo e

any benzo[a ]p r ne pr sent

Use only r ag ent of r co niz d analytical grade, unles othe wise spe if ied Whe e analytical grade

solvent othe than the r commen ed ones ar used, a ful blan analy sis shal be car ied out an the

r sult of this blan analy sis r port ed

SAF TY PRECAUTIONS — A ttentio is drawn to re ulations w hich specify handl ng proced res

for dang erous substances User sho ld be aware of and comply with technical, org nizatio al,

and per o al safety me sures

5.1 Water , double dis i ed, fiter ed thr ough a membrane fite of p r e siz 0,45 µm; deioniz d wate

o tained b purifying demineralz d wate sys ems ma y also be used

5.2 Light petroleum, (b i ng p int rang e 40 °C to 60 °C), or hexane, r edis i ed o e potas ium

hydr oxide pelet (4 g /l)

5.3 Ac etonitrie, suitable for HPL

5.4 Tetrah ydr ofuran, suitable for HPL

5.5 Ac etonitrie-tetrahydrofuran mix tur e, pr ep r ed b mixing 9 ml ac tonitrie (5.3) and 10 ml

tetrahydr ofuran (5.4)

5.6 Toluene, suita le for HPL

5.7 Sodium sulfate, g r an lar, an ydr ous

5.8 A lumina acti vity g rade 4, pr ep r ed fr om neutr al aluminium ox ide, activity g r ade supe 1

1)

,

deactiv ted b the ad ition of 1 ml wate (5.1)to 9 g of alumina

Due t o the dife enc s in activity of alumina of v rious bran s, a che k is r commen ed t o conf irm that

the deactiv tion proc d r is a pro riat e for t otal benzo[a ]p r ne r co e y from a r fe enc sample

CAUTION — THE DEACTIV TION REACTION IS EXOTHERMIC AND PRES URE CAN BUILD UP

Shake thecontaine for a out 1 min an alow the cont ent t o eq i ibrat e for 24 h St or the alumina in

a closed ves el at ambient t empe atur

5.9 Benzo[a]pyrene

2)

, of purity 9 ,0 % b mas

CAUTION — BENZO[a ]PYRENE IS A KNOWN CARCINOGEN CARRY OUT AL WORK WITH IT IN A

FUME HOOD, WEARING GLOVES TO MINIMIZE EXPOSURE

5.9.1 Benzo[a]pyrene stock solutio in toluene, 0,5 mg /ml

Weigh, t o the near s 0,1 mg, a out 1 ,5 mg of benzo[a ]p r ne in a 2 ml grad at ed flask Dis olve it in

t oluene (5.6) an make up t o the mark with that solvent

St or the solution in the dark at 4 °C w he e it is s a le for at leas 6 months

5.9.2 Benzo[a]pyrene standard solutio s

Pr p r two s an ard solutions containing a pro imat ely 0,2 µg ml an 0,0 µg ml of benzo[a ]p r ne,

r spe tively, b diuting alq ot of the st ock solution (5 9.1) with ac t onitrie

1) “A luminium ox ide 9 active neutr al” is an example of a suitable pr od ct a vaila le commer cialy T is information

is g iven for the con enience of user of this document an do s not constitute an en or ement by ISO of this prod ct

E uivalent pr od cts ma y b used if they can b show n to le d to the same r esults

2) A suitable r eference material is a vaila le fr om the Joint Rese r ch Centr e of the E r ope n Commis ion, Institute

for Refer ence Materials an Me sur ements (IRMM) T is information is g iven for the con venience of user of this

document an do s not constitute an en or ement by ISO of this pr od ct

5.10 Benzo[b]chrysene

3)

inter nal standard solution in ac tonitrie

Pr p r a st ock solution containing, t o the near s nano ram, a pro imat ely 1 ng µl Di ut e

this solution b a fact or of 1 in a v lumetric flask t o o tain an int ernal s an ard solution with a

conc ntration of a pro imat ely 1ng µl

NOTE T is solution can also b prepared by dis olving b nzo[b]chrysene

3)

, 9 ,0 % by mas , in acet onitrile

6 A pparatus

Usual la orat ory a paratusan , in p rticular, the folowing

6.1 Glas c olumn for chromatog raphy , of leng th 3 0 mm an inte nal diamete 1 mm, fited with

sinter ed glas discs, an p lytetrafluor oethylene (P FE) tap

6.2 Wate baths, onemaintained at 3 ±2)°Cand anothe at 6 ± 2) °C

6.3 Flash evaporator, a r otary ev por ator with v cu m an a wate b th set at 40 °C ma y be used C r e

should be taken to pr event cr os contamination Clean the sys em thor oug hly betwe n dete minations

6.4 High per for manc e lquid chromatog raph, consisting of an HPL pump, inje tion v lve with

5 µl sample lo p, r eve se-phase column, ele tr onic integ r ator and chart r ecor de

If an aut osample is used, the sample lo p shal be flushed with ac t onitrie betwe n conse utive

inje tions

6.5 C lumns for HPL anal ysis

6.5.1 Rever se-phase g uard c olumn, ca a le of r esolving benzo[a]pyr ene from co-ex tr actives,

tog ethe with a propriate pr ecolumn [e.g s ainles -s e l pr ecolumn of leng th 7 mm an inte nal

diamete 4,6 mm, p cked with Lichrosor b RP -1 (of particle siz 5 µm)]

4)

6.5.2 HPL C r ever e-phase c olumn, of leng th 2 0 mm an inte nal diamete 4,6 mm ( tainles s e l),

for p lycyclc ar omatic hydr ocar bons (PA Hs)(e.g Chromsphe 5 PA H or Vydac 2 1 TP5)

4)

6.6 Fluor esc enc e detector, with emis ion wa veleng th at 40 nm ( lt 1 nm) an excitation

wa veleng th at 3 4 nm ( lt 1 nm) The dete tor shal be ca a le of the r eq ir ed pe formanc to car y

out the analysis

6.7 Cr imp-to mini vials, of a out 1ml v lume, with P FE-la yered septa an aluminium ca s

6.8 Hand c imper, for c imping the ca s onto the vials

6.9 Disposable pipet es

3) A suitable r efer ence material is a vaila le from the Joint Rese rch Centr e of the E r ope n Commis ion, Institute

for Refer ence Materials an Me sur ements (IRMM) or Dr E r enstorfer GmbH T is information is g iven for the

con enience of user of this document an do s not constitute an en or ement by ISO of this pr od ct

4) Examples of suitable pr od cts a vaila le commer cialy T is information is g iven for the con venience of user of

this document an do s not constitute an en or ement by ISO of this pr od ct E uivalent pr od cts ma y b used if

they can b show n to le d to the same r esults

7 Sampl ng

A r pr sentative sample should ha e be n sent t o the la orat ory It should not ha e be n damag ed or

chang ed d ring transport or st orag e

Samplng is not part of the method spe ified in this document A r commen ed samplng method is

given in ISO 5 5

[1]

8 Preparation oftest sample

Pr p r the t es sample in ac ordanc with ISO 66 1

9.1 Cle n up of sample

9.1.1 Weigh, to the neares 0,0 1g , a out 0,400 g of thefat or oi into a g las beake an dis olve in 2 ml

of lght petr oleum (5.2) Add 2 µl ofthe inte nal standar d solution (5.1 ) b means of a micr osyring e

This is equiv lent to 5 µg /kg when calculated on the sample mas If a high level of benzo[a]pyr ene is

ex e ted, then ad 5 µl of the inte nal s an ar d solution (5.10) This is eq iv lent to 1 5 µg /kg when

calculated on thesample mas

9.1.2 Fi thechr omatog r aphy column (6.1)to half its height with lght petroleum (5.2) Ra idly weigh

2 g of alumina (5.8) into a smal beake an trans e the alumina immediately to the column, then g ently

ta the column to efe t setlng of the alumina

9.1.3 Add an ydr ous sodium sulfate (5.7) to the to of the column to form a la ye a out 3 mm de p

9.1.4 Open the ta an alow the lght petroleum to fal to the level of the to of the sodium sulfate la ye

9.1.5 Plac a 2 ml g rad ated flask u de the column

9.1.6 Intr od c the oi solution (9.1.1) on to the column Rinse the column with minimal amou t of

lg ht petr oleum (5.2), alow ing the solvent la ye to ru into the sodium sulfate la ye betwe n rinsing s

9.1.7 Elute the column with lght petr oleum with a flow of a out 1 ml/min, discar ding the firs 2 ml of

eluate an cole ting the nex t 6 ml of eluatein a 1 0 ml r oun -b tomed flask

9.1.8 Ev p r ate solvent fr om the eluate in the wate b th set at 6 °C, to a v lume of a out 0,5 ml

to 1,0 ml, an tr ans e the conc ntr ated solution into a c imp-to miniv ial (6.7) pr e-weig hed to the

near es 0,1 mg

9.1.9 C ntin e the ev p ration from the minivial, in the wate b th (5.1) set at 3 °C u de a g en e

s r eam of nitr og en (a out 2 ml/min) u til near ly dry Rinse the r ou d-b tomed flask with a out 1 ml

of lght petr oleum an trans e the rinsing q antitatively to the minivial, contin ing the ev p ration

u de nitr og en Repeat therinsing an trans e to the minivial onc mor e

9.1.10 C ntin e the ev p ration at 3 °C u de nitrog en unti dry

9.1.11 Weigh the minivial to the near es 0,1 mg , an calculate the mas of the r esidue Sto pe the

minivial with the P FE-la yer ed septum an aluminium ca an s or e at 4 °C

9.2 High per for manc e lquid chromatog raphy

9.2.1 Use a mixtur e of 8 0 ml ac tonitrie (5.3) an 1 0 ml wate (5.1) as elution solvent Degas the

elution solvent to r emo e o yg en in or de to a void fluor esc nc q enching Use helum pur g ing or an

onlnev cuum degas e

9.2.2 Elute at a flow rate of a out 1 ml/min

9.2.3 Calbration cur ve and deter minatio of the r elati ve respo se factor : Prep re five diutions

of the standar d benzo[a]pyrene solutions (5.9.2) such that inje tion of 50 µl of each wil g ive r eading s

corresp n ing to 0,0 ng , 0,04 ng , 0,2 ng , 1,0 ng an 2,0 ng of benzo[a]pyr ene Add to the standar d

solutions 0,5 ng of inte nal s an ar d Fr om these, cons ruct a five-p int calbration curve using the peak

ar eas from the integ rator an chart recorde These calbr ations ar e also used to calculate the r elative

r esp nse factor (1 1) betwe n benzo[a]pyr ene and the inte nal s an ar d

9.3 S mple anal ysis

9.3.1 Inje t 2 0 µl of the ac tonitrie-tetrahydr ofuran mix tur e (5.5) into the minivial containing the

cleansed r esid e (9.1.1 ) Dis olve the r esid e b careful swir ling , a voiding contact of the solvent with

the septum

W ith the calbration curve (9.2.3), benzo[a ]p r ne levels of 0,1 µg kg t o 5 µg kg can be det ermined F or

conc ntrations a o e 1 µg kg, the r sid e solution (9.3.2) should be diut ed furthe with ac t onitri

e-t ee-trahydrofuran (5.5), or a smal e v lume than 5 µl (9.3.2) should be inje t ed

9.3.2 Inje t an ac urately known v lume of a out 50 µl of the dis olved r esid e into the HPL column

and start the chr omatog ram ru ning C re should be taken to ensur e that not mor e than 1,5 mg of resid e

is intr od c d into the column A n example of a chromatog ram is g iven in Fig ur e 1 If a lar g er amou t of

r esid e is present , the amou t of tetrahydrofuran (5.4) shal be adjus ed or the clean-up s ep shal be

r epeated

3 b nzo[k]fluoranthene

Figure 1 — Ty ical chromatogram

10 Ex pres ion of results

10.1 Calculation of r elati ve r esponse factor

From the data o tained (9.2.3), calculat e the r lative r sp nse fact or, f

r , as the arithmetic a e ag e of

five s an ards, using F ormula (1):

f

A

A

r r

is BaP

BaP is

=

ρ

ρ

(1)

w he e

A

BaP

is the peak ar a of benzo[a ]p r ne;

A

is

is the peak ar a of the int ernal s an ard solution;

ρ

BaP

is the conc ntration, in mic o rams pe ltr , of benzo[a ]pyr ne;

ρ

is

is the conc ntration, in mic o rams pe ltr , of the int ernal s an ard solution