Tiêu chuẩn iso 18465 2017

© ISO 2017 Microbiology of the food chain — Quantitative determination of emetic toxin (cereulide) using LC MS/MS Microbiologie de la chaîne alimentaire — Détermination quantitative de la toxine éméti[.]

Microbiolog y ofthe food chain —

Micro iolo ie de la ch îne alimentaire — Déte mination q a titative

de la to ine émétique ( ér ulide) p r CL- SM/SM

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© ISO 2017, P blshed in Sw itz rlan

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written permis ion Permis ion c n be req esed from either ISO at the ad r es below or ISO’s member bod y in the c u try of

F reword i v

Introduction v

1 Sc ope 1

2 Nor mati ve r eferenc es 1

3 Terms an definitions 1

4 General pr inciple 1

5 Re g ents 1

6 A pparatus and equipment 3

7 Proc ed re .4

7.1 Sample pr eparation 4

7.2 Stan ar d pr ep ration 4

7.3 L -MS analysis 5

7.3.1 L con itions 5

7.3.2 MScon itions an tu ing p ramete s 5

7.3.3 Transitions (multiple r eaction monitoring , MRM) 6

8 Calculation

6 9 Qual ty c ontrols 7

10 Precision 8

1 1 General 8

1 2 Repeatabi ty 8

1 3 Reprod cibi ty 9

A nne x A (informative)Results of the inter laborator y stud y 10

A nne x B (informative)P os ible transitio s of cer eul de in MS [3] 12

Biblog raphy 14

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

For an ex lanation on the meaning of ISO spe ific terms an ex r s ions r lat ed to conformity as es ment,

as wel as information a out ISO’s adhe enc to the Wor ld Trade Org nization (WTO) principles in the

Te h ical Bar ie s to Trade (TBT) se the folowing URL: www iso org iso/ for word html

This document was pr p r d b the Euro ean C mmitt ee for Stan ardization (CEN) Te h ical

Committ ee CEN /TC 2 5, Fo d An lys i s — Hori zo tal meth ds , in col a oration with ISO Te h ical

Committ ee ISO/TC 34, Fo d produc ts , Subcommitt ee SC 9, Mic ro iolo y, in ac ordanc with the

a r ement on t ech ical co pe ation betwe n ISO and CEN (V ien a Agr ement)

Ce eulde, the emetic t oxin prod c d in fo ds b c rtain s rains of B ac ilus c er us , is a heat an acid

s a le t oxin that ca uses na usea and v miting w hen ing est ed In ve y rar cases, peo le can die aft er

ing es ion of the t oxin Due t o it s a i ty, the t oxin ma s i l be pr sent even w hen B c er us can no

long er be det ect ed The pr senc of c r ulde se ms t o be lnked t o s ar h-rich fo ds l ke ric (dishes)

an p s a (dishes) Howeve , r c nt data sugg es that the oc ur enc of fo d b rne out br aks d e t o

c r ulde is mor common t o fo ds in g ene al

9]

The t oxin has a cyclc s ructur and consis s of in

t otal 1 monome s as a r peat of (D-O-Leucine-D-Alanine-L-O- Valne-L-Valne) S ve al methods

ha e be n develo ed for the det ection an / r q antification of the t oxin Some of these methods ar

nonspe ific bio-as ay s

[3, 4]

an othe methods ar spe ificaly based on the chemical analy sis using

lq id chromat ogra hy with mas spe trometry (L -MS /MS)for the det ection an q antification of the

t oxin

[5, 6, 7, 8]

The chemical methods ar mor spe ific for c r ulde an ha e, the efor , be n chosen as

the s arting p int for s andardization of a method for the q antification of c r ulde R ec ntly, r sear h

has be n done for the chemodive sity of c r ulde A t leas 1 c r ulde v riant we e det ect ed b

UHPL -TOFMS and ion-tra MS seq encing, among w hich the pr viously u k own isoc r uldes

A –G

[10]

Microbiolog y ofthe food chain — Quantitative

This document desc ibes the q antitative analy sis of the emetic t oxin c r ulde using high pe formanc

lq id chromat ogra hy (HPL ) or ultra pe formanc lq id chromat ogra hy (UHPL ) con e t ed t o a

tan em mas spe tromet er (L -MS /MS)

This document is a plca le t o the analy sis of the t oxin in prod ct int en ed for h man consumption

2 Normati ve r eferences

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 at ed r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amen ment ) a ples

ISO 3 9 , Wate fr a aly tic al la orator y use — S ec ific atio a d tes t meth ds

ISO 1 42, L b rator y glas swar — One -mark v lumetric flask s

3 Terms and definitions

F or the purposes of this document, the folowing t ermsand definitions a ply

ISO an IEC maintain t erminolo ical data ases for use in s an ardization at the 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

cereul de

t oxin cyclo[D-O-Leucine-D-Alanine-L-O-Valne-L- Valne ]

3prod c d b c rtain s rains of the spe ies of

is used as an int ernal s andard The comp nent in the solution ar sep rat ed using HPL or UHPL

an subseq ently det ect ed using tan em mas spe trometry (L -MS /MS) F or MS, the ele tro spra

ionization t ech iq e (ESI) is used, using the positive mode The level of emetic t oxin (c r ulde) is

5.6 Synthetic Cereulde

1)

5.7 A mmo ium for mate, pr o analyse g rade

5.8 Mo ie phase A , consis ing of 1 mmol/l ammonium formate (5.7) with 0,1 % (v/v) formic acid

-Cereul de stock solution IS-A , ρ = 1 0 0 0 ng /ml (in methanol)

Weigh 1 mg t o the near s 0,0 mg

1

C

6-Ce eul de (5.5) int o a glas v lumetric flask (6.1 ) of 1 0 ml

an dis olve, make up t o the mark with methanol (5.3) This solution is not cor e t ed for the purity of

the comp un St or the solution in the fr e e (6.1 )

NOTE Cereulide (la el ed an non-la eled) st ock an stan ard solutions are e tremely sta le, me ning

over thre year when stored in a fre zer (6.1 )

of 1 0 ml, make up t o the mark with methanol (5.3) an mix the solution St or the solution in the

make up t o the mark with ac t onitrie (5.2) and mix St or the solution in the fr e e (6.1 )

of 1 0 ml, make up t o the mark with ac t onitri e (5.2)an mix St or the solution in the fr e e (6.1 )

5.14 Cereulde stock solutio Cer -A , ρ = 1 0 00 ng /ml (in methanol)

Weigh 5 mg t o the near s 0,0 mg synthetic c r ulde (5.6) int o a glas v lumetric flask (6.1 ) of 5 ml

an dis olve, make up t o the mark with methanol (5.3) This solution is not cor e t ed for the purity of

the comp un St or the solution in the fr e e (6.1 )

5.15 Cereulde standar d solutio Cer-B, ρ = 1 0 ng /ml (in ac tonitrie)

Pipett e (6.1 ) 5 0μl c r ulde st ock solution A (5.14) in a glas v lumetric flask (6.1 ) of 5 0 ml, make

up t o the mark with ac t onitri e (5.2)an mix the solution St or the solution in the fr e e (6.1 )

1) C iralix 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 pr od ct

5.16 Cereulde stock solutio Cer -C, ρ = 10 ng /ml (in ac tonitrie).

Pipett e (6.1 ) 1 ml c r ulde s an ard solution B (5.1 ) in a glas v lumetric flask (6.1 ) of 1 0 ml,

make upt o the mark with ac t onitrie (5.2)an mix the solution St or the solution in the fr e e (6.1 )

5.17 P ositi ve c ontr ol sample or spiked sample (level a pr ox imately 1 ng /g )

6.1 Tandem mas spectr ometer, eq ipped with ESI inte fac (in p sitive mode) an multiple

r eaction monitoring (MR M)mode

LC sy stem, pump syst em (HPL or UHPL ), deg s e , aut osample , column o en

L -MS sof ware, suita le of data cole tion, int egration

6.3 Centrifug e, ca a le of a c ntrifugal force of 10 0g to 1 50 g for 5 ml tubes

6.4 Centrifug e, ca a le of a c ntrifugal force of 10 0 0 to 1 0 0 for 2ml tubes

6.5 Centrifug e tubes, (plas ic)with closing ca , 2 ml disp sa le

6.6 Centrifug e tubes, (glas ) with leakag e fr ee screw ca 5 ml

6.7 Hor izo tal mechanical shaker, ca a le of holding 5 ml c ntrifug e tubes

6.8 A nal ytical balanc e, ac uracy to the near es 0,0 mg

6.9 Gr inder, e.g mixe , blen e , c y g enic mixe

6.10 Calbrated plung er pipetes, rang es from 1 μ l to 1 0 μl, 1 0 μl to 1 0 0 μl, an 1 0 0 μ l

to 5 0 0 μl, 20 0 μ l to 1 0 0μl

6.11 Glas v lumetr ic flasks, v lume of 5 ml, 1 0 ml an 5 0 ml ac or ding to ISO 1 42

6.12 Glas autosample vials, with sna /scr ew ca 2 ml

6.13 PTFE membrane fiter , diamete of 25 mm and 0,45 μ m p r e siz

6.14 Vor te x mixer

6.15 F reezer, ca a le of temper atur es below −1 °C, pr eferably below − 8 °C

2) Su elco Dis overy® is an example of a suita le 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 pr od ct

3) Water B H C-1 is an example of a suitable prod ct a vaila le commercialy 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

7 Proc edure

7.1 S mple preparation

St or the samples (befor an d ring the ex e iment) in the fr e e (6.1 ) t o pr vent growth of mic

o-org nisms

Take (1 0 ± 2 ) g of a r pr sentative p rt of the sample and trans e it t o a sample jar In case the

amou t of sample is l mit ed (for ex mple in case of samples inv lved in fo d p isoning), take as much

sample as p s ible and tr at identical as the othe samples In this case, a not e should be mentioned

w hen r p rting the r sult Homog eniz the sample b grin ing (6 9)

Weigh 2,5 g, t o the near s mg (6.8), of the homog eniz d sample int o a c ntrifug e tube (6.6) an

pipett e (6.1 ) 5 0 μl int ernal s an ard solution IS-C (5.1 ); close the tube with the sc ew t op Mix

(6.14) a out 1 s an let the tubes r s for 3 min Glas tubes should be used espe ialy w hen solutions

ar st or d for a long er time F or short contact times, plas ic tubes can be used as wel Ad 2 ,5 ml

ac t onitrie (5.2) an close the tube a ain with the sc ew t op

Plac the tube()horizontaly on the shake (6.7) an shake f irmly for a pro imat ely 1 h Aft er shaking,

c ntrifug e the tubes for 1 min at 1 0 0 t o 1 5 0 (6.3) If the solution is clear without flo ting

p rticles, no filtration st ep is ne es ary If not, filt er the lq id phase using P FE membrane f ilt ers

(6.1 ), or trans e 2 ml in a c ntrifug e tube(6.5) an c ntrifug e the solution at 1 0 0 t o 1 0 0 (6.4)

for 1 min

Fi l an a ut o sample vial (6.1 ) an close the vial with a ca The samples ar now r ady for analysis

7.2 Standard preparation

The s an ards should be pr par d dir ctly in the vials Pipett e (6.1 ) the v lumes as spe if ied in

Ta le 1 with c r ulde st ock solution Cer-C (5.1 ), ac t onitrie (5.2) ( e NOTE) an

1

C

6-c r ulde

st ock solution IS-D (5.1 )int o a vial (6.1 ) an close the vial with a ca Mix (6.14)the solution for 2 s

NOTE If ad itional cle n u is ne ded, heptane e traction may b used t o red ce the int erfering (fat y)

components Af er ad ing 2 ,5 ml acet onitrile, ad 1 ,0 ml heptane an close the tub s with a s rew t op Place

the tub (s) horizontal y on the shaker (6.7) an shake f irmly for ap ro imat ely 1 h Centrifug the tub s for 1

min at 1 0 0 t o 1 5 0 (6.3) Proce d with the sample preparation with the lower acet onitrile layer

Table 1 — Preparatio of cal bratio standard solutions

Stan ard Ce -C (5.1 ) A cet onitri e (5.2) IS-D (5.1 )

(5.1 ) to the s an ards (1 0 0 μl)

R eanaly se the sample from s art w hen a r sult is out ide the calbration rang e be ause of it high

conc ntration, weighing les ofthe sample < ,5g

If a l mit ed amount of sample is a ai a le, use an eq iv lent ratio of int ernal s andard an solutions

The f inal conc ntration of

1

C

6-Ce eulde (IS) in the sample extract an s an ards isρ = 1,7 ng ml

The conc ntrationsin Ta le 1 depen on the ex ct weighed amou t of c r ulde Cer-A (5.14)an

1

C

6-

Ce eulde IS-A (5.1 )

7.3 L C-MS anal ysis

7.3.1 L c onditio s

Any suita le L sy st em can be used Paramet ers, flow, r t ention time an gradient ar ins rument/ty e

column/man factur r depen ent an shal be det ermined b o timizing the L sy st em Both isoc atic

an gradient elution methods can be used The p ramet ers displa ed below can be used as initial

con itions for o timization

Inje tion v lume: 1 μl t o 2 μl

Autosample tra

tem-pe atur :

5 °C t o 1 °C

Mo ie phase A: 1 mmol ammonium format e (5.7) with 0,1 % (v v) formic acid in w at er (5.1)

Mo ie phase B: A cet onitrie (5.2)with 0,1 % (v v)formic acid (5.4)

7.3.2 MS c onditio s and tuning parameter s

The MS p ramet ers can v ry; they ar ins rument/man factur r depen ent an shal be o tained b

tuning the ins rument befor analy sis The paramet ers displa ed below can be used as initial con itions

for o timization; ex mples displa ed ar from a Wat ers

4) Water ® Micromas ® is an example of a suitable pr od ct 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

Mas rang e calbration: 5 t o 1 3 0 m/ z

7.3.3 Transitio s (multiple reactio mo itor ing, MRM)

The transitions mentioned can deviat e depen ing on the mas spe tromet er They ar r commen ed

but othe transitions can also be used A l s of p s ible transitions isa ai a le in An ex B

Table 2 — Cereulde quantificatio MRM (M+NH

4)+

Firs prod ct ion trac 1 1 70,7 > 3 4,4

Quantif ication ion (most a u dant transition)

S con prod ct ion trac 1 1 70,7 > 4 9,4

Qualif ication ion (secon most a u dant ransition)

4)+

Firs prod ct ion trac 1 1 7 6,7 > 1 72,2

Internal stan ard q antification ion (most ab n ant

transition)

S con prod ct ion trac 1 1 7 6,7 > 3 5,4

Qualif ication ion (secon most a u dant ransition)

A calbration l ne is made from the r sult from the c r ulde s an ards as spe ified in Ta le 1 (calbration

rang e) The calbration lne shal be for ed through the origin The calculation method shal be set as an

int ernal s an ard method, an alow s q antification of the samples w hich ar within the rang e