Tiêu chuẩn iso 19563 2017

© ISO 2017 Determination of theanine in tea and instant tea in solid form using high performance liquid chromatography Détermination de la théanine dans le thé et le thé instantané sous forme solide e[.]

Determination oftheanine in tea and

instant tea in solid form using hig

Déte mination de la th a ine da s le th et le th insta ta é sous

frme solide en utilisant la chromato ra hie en p ase liquide à h ute

pe frma c

Fir t edition

2 17-0

Refer ence n mb r

ISO 1 563:2 17(E)

COPYRIGHT PROTECTED DOCUMENT

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F reword i v

Introduction 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 2

6 A pparatus 3

7 S mpl ng 3

8 Preparatio of test samples 3

9 Proc ed re .3

9.1 General 3

9.2 Dete mination of dry mate content 4

9.3 Tes p rtion 4

9.3.1 Sample pr ep rations 4

9.3.2 Sample clean-up (using a poly mide column)— Optional 4

9.4 Dete mination 5

9.4.1 A djustment of the a p ratus 5

9.4.2 HPL analysis

5 9.4.3 Identification 5

10 Calculation 6

11 Precision 6

1 1 Inter la or atory tes 6

1 2 Repeatabi ty 6

1 3 Reprod cibi ty 6

12 Test rep r t 7

A nne x A (informative)T ypical the nine calbration g raph 8

A nne x B (informative)T ypical chromatog rams 9

A nne x C (informative) Results of inter lab ratory tests 12

Biblog raphy 13

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

The committ ee r sp nsible for thisdocument is ISO/TC34, Fo d produc ts , Subcommitt ee SC 8, Tea

Theanine (N-ethy l-γ -L-glutamine) is a non-prot ein de ived (fr e) amino acid In the plant world, it is a

uniq e amino acid foun in Camel a g en s and the mushro m B oletus b dius an Ilex g ay us a The mos

common an the mos prominent oc ur enc isin Camel a s inens i s (L )O K untz spe ies

Theanine cons itut es c irc a 0,1 % t o 2 % w/w of the dry weight in t ea lea es (Camel a s inens i s) an

is a pro imat ely 5 % of the t otal fr e amino acids S p ration of L- an D-theanine b HPL is not

achieved b this method; howeve , L-theanine is the major form (mor than 9 % ) in al t eas

[2][3]

Theanine has be n as ociat ed with the ty ical t ea umami tast e an is also being s u ied t o ev luat e

pot ential beneficial efe t t o health

The umami tast e charact eris ic has be n as ociat ed with theanine w hich is also being ev luat ed for it

pot ential benefit t o health This is also c eating int er s in theanine as an imp rtant no el fo d and

dietary sup lement ingr dient

Determination oftheanine in tea and instant tea in solid

This document spe if ies a high-pe formanc lq id chromat ogra hic (HPL ) method for the

det ermination of theanine cont ent in t ea (Camel a s inens i s) It is a plca le t o b th t ea an ins ant t ea

samples S p ration of L- an D-theanine is not pos ible using this method; howeve , the L-enantiome

is the major form in t ea

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 1 7 , Tea — Prep ratio o gro nd s ample o know n dr y mat e c ontent

ISO 1 7 , Tea — Dete min tion o los s in mas s at 10 3 °C

ISO 1 3 , Tea — S mpln

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

ISO 7 1 , Ins ta t tea in s old frm — Dete min tio o moi s tur c ontent (los s in mas s at 10 3 °C)

ISO 7 1 , Ins ta t tea in s old frm — S mpln

3 Terms and definitions

No t erms an def initions ar lst ed in this document

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

4 Principle

A r ve sed-phase (RP) C1 column isused w hich is ca a leof sep rating mor polar comp u ds using

wat er as an eluent an UV det ection at 2 0 nm Theanine is separat ed from the othe amino acids;

howeve , the p t ential co-elution of theanine with methionine is not r lev nt d e t o the ve y smal

conc ntration of methionine in t ea It is r commen ed that p ly mide column extract clean-up is used

t o r mo e int erfe ing p ly henols for column prot ection w hen sep ration of theanine from othe

cons ituent in the extract sample is difficult Alt ernatively, the column should be washed aft er each t ea

sample using ac t onitri e

5 Reag ents

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

5.1 Water , conforming to g r ade1 of ISO 3 96

5.2 Ac etonitrie, HPL g rade (howeve , supe g rade ispreferable)

5.3 Methanol, r eg ular la or atory distiled g rade

5.4 Pol yamide (Poly mide CC 6

1)

, Particle siz : 0,0 mm to 0,1 mm for column chr omatog raphy)

5.5 L-the nine referenc e standard, >9 %, anhydr ous

5.6 HPL mobie phases

WARNING — We r glo es and eye protectio and dispense re g ents in a fume cupboard

5.6.1 Mo ie phase A, 1 0 % pur ified wate (5.1)

5.6.2 Mo ie phase B, 1 0 % ac tonitrie (5.2)

5.7 Standar d solutio

5.7.1 Standar d stock solution, corr esp n ing to 1 mg /ml

Weigh (0,0 0 0 ± 0,0 0 1) g of pur L-theanine (5.5) in a 5 ml v lumetric flask dis olve with purif ied

wat er using sonication t o aid dis olution an make up t o the volumewith purif ied wat er

5.7.2 Standar d wor king solution

Thest ock solution isdiut ed with purified wat er t o pr p r the s andard solutions in the conc ntration

rang e of5 µg ml t o 2 0 µg ml as detaied in Ta le 1

Table 1 — Stock solutio diutio s

Standard

solution

Working solution

(µg ml)

Volume taken from the

st ock solution

(ml)

Final volume

(ml)

1) Polyamide C 6 is the r eq ir ed/minimum specification r eq ir ed for the separation which is a vaila le

commer ciall y 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

6 A pparatus

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

6.1 A nal ytical balanc es, ca a le of weighing to an ac uracy of ±0,0 0 1 g

6.2 Mag netic stir r er, ca a le of 5 0 r/min

6.3 Fiter , membr ane fite u its of 0,45 µm pore siz for fitration of mo ie phase an diuted

sample extract A ll membranes used should be che ked using a simple calbration solution to ensur e

that theanine r etention does not oc ur

6.4 Centrifug e, ca a le of 12 3 0 relative c ntrifugal for ce (R C.F.) ( ypicaly 1 5 0 r/min)

6.5 Glas c olumn, 1 cm × 2,2 cm in diamete , (or p ly mide clean-up only)

6.6 One mar k v lumetr ic flasks, of ca acities5 ml an 1 0 ml

6.7 High per formanc e lquid chromatog raph, eq ip ed to pe form binary g radient elution, w ith a

the mos aticaly contr oled column comp rtment an an ultr a violet dete tor set at 2 0 nm

6.8 C romatog raphic c olumn for HPL , P enomenex Aqua

TM2)

2 0 × 4,6 in diamete or 2 mm in

diamete or eq iv lent

NOTE T is method has b en developed with the Aq a ™ which has the minimum specif ication req ired for

this analy sis

The sampl ng methods that hal be used ar detaied in ISO 1 3 for t ea or ISO 7 1 for ins ant t ea

A r pr sentative sample() shal be sent t o the la orat ory It shal not be damag ed or chang ed d ring

transp rt or st orag e

8 Preparation oftest samples

To ensur homog eneity, grin the sample of leaf t ea in ac ordanc with ISO 1 7 an st or samples in

wel-sealed containe s prot ect ed from lght

Grin ing of ins ant t ea samples is only r q ir d with samples ha ing a co rse gran lar s ructur (e.g

fr e e-dried ins ant t ea)

If it is r q ir d t o che k w hethe the r sult ar within the ac epta le r peata i ty lmit ( e 1 2),

car y out two single det erminations in ac ordanc with 9.2 t o 9.4 u de r peata i ity con itions

2) P enomene Aqua

M

is the req ir ed/minimum specification r eq ired for the separation which is 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

9.2 Deter mination of dry mat er c ontent

Calculat e the dry matt er cont ent from the mois ur cont ent (los in mas at 1 3 °C) det ermined on a

p rtion of the t es sample (Clause 8)in ac ordanc with ISO 1 73 for t ea or ISO 7 1 for ins ant t ea

9.3 Test por tion

9.3.1 S mple preparatio s

9.3.1.1 Le f te

9.3.1.1.1 Weigh (1,0 ± 0,0 ) g of a finely g r ou d sample into a 2 0 ml beake an add 1 0 ml of

b i ng wate

9.3.1.1.2 A llow br ew ing for 5 min using a mag netic s irr er an then fite thr ough a fite p pe (Grade

4 or eq iv lent) into a 1 0ml v lumetric flask

9.3.1.1.3 A llow the tea br ew to co l down to ambient temperature, make up to v lume of 1 0 ml w ith

purified wate and fite using a 0,45 µm membrane befor e inje tion A lte natively, a prox imately 1 ml of

the sample solution can be c ntrifug ed at 1 0 0 r/min for 1 min prior to HPL analysis

9.3.1.2 Te e x tract or instant te

9.3.1.2.1 Weigh (0,10 ± 0,0 1) g sample into a 5 ml v lumetric flask, dis olve an make up w ith

heated purified wate (a prox imately 5 °C)

9.3.1.2.2 Sonicate for 2 min to 5 min for complete dis olution an fite using a 0,45 µm membrane

befor e inje tion A lte natively, a pr oximately 1 ml of the sample solution can be c ntrifug ed at

1 0 0r/min for 1 min prior to HPL analysis

9.3.2 S mple cle n-up (using a pol yamide c olumn) — Optional

Ce tain t ea samples wi l not elut e with clean chromat ograms When this oc urs in the p sition w he e

theanine elut es in the chromat ogram, the int erfe enc wi l pr vent an ac urat e det ermination of the

peak ar a On these oc asions, it is ne es ary t o car y out the clean-up proc d r s as desc ibed below

in orde t o impro e sep ration an t o prot ect the column

9.3.2.1 Suspen 1,2kg p ly mide in a mix tur e of8 l of wate and 1,5 l of methanol, mix wel an alow

settlng o e nig ht

9.3.2.2 Remo e supe natant an wash with 1,5 l methanol twic Stor e in methanol and mix wel

befor e use

9.3.2.3 Column chr omatog raphy:

9.3.2.3.1 Fi a glas column (1 cm × 2,2 cm in diamete ) eq ip ed with a thin la ye of glas wo l an

sea san w ith swolen p ly mide to a heig ht of 8 cm C n ition with 2 0 ml of wate

9.3.2.3.2 A pply 50 ml of the tea solution to the column an immediately cole t the eluate into a 1 0 ml

v lumetric flask

9.3.2.3.3 Wash the column w ith purified wate u ti the mar k of 1 0 ml is r eached Mix wel an fite

using a 0,45 µm membrane befor e inje tion A lte natively, a pr ox imately 1ml of the sample solution can

be c ntrifug ed at 1 0 0 r/min for 1 min prior to HPL analysis

The poly henols wi l be r tained on the p ly mide w hie the theanine elut es This st ep cor esp nds t o

a twofold diution

9.4 Determination

9.4.1 Adjustment of the apparatus

S t up the chromat ogra h (6.7) in ac ordanc with the man factur r’ s ins ructions an adjus it as

folow s

a) Flow rat e of the mo ie phase (5.6.1 an 5.6.2):1,0ml/min for 4,6 mm columns an 0,2 ml/min for

2mm columns

b) Binary gradient conditions: ac ording t o the elution pro ramme as detaied in Ta le 2

c) Tempe atur of the column (6.8): ambient, o tional 2 °C

d) UV det ect or set at 2 0 nm

e) Inje tion v lume:2 µl (o tional 1 µl t o 5 µl)

Table 2 — Elutio programme

Time

(min)

9.4.2 HPL anal ysis

Onc the flow rat e of the mo ie phase (5.6) an t empe atur [se 9.4.1 c)] ar s a le, condition the

column with a blan gradient run (9.4.1) Then inje t ont o the column 2 µl of each of the s an ard

working solutions (5.7.2), folowed b an eq al v lume of the sample solution (9.3.1.1.3 or 9.3.1.2.2)

R epeat the inje tion of themix ed working s an ard solutions (5.7.2) at r gular int erv ls ( y icaly aft er

six t es solutions) Cole t data using the data cole tion/int egration sy st em of the HPL (6.5) for the

peak of theanine in the s an ard an t es sample solutions

Aft er each b t ch of analy sis, thoroughly clean the column b flushing the chromat ogra hic sy st em an

column with 5 % ac t onitri e, 5 % wat er mo ie phase R eplac column seal ng plugs if discon e t ed

for st orag e

9.4.3 Identificatio

Identify the theanine b comp ring r t ention time from sample chromat ograms with that o tained

from the s andard solutions u de the same chromat ogra hic conditions ( e 9.4.1)

NOTE T pical chromat ograms can b fou d in An e B