Table of Contents Table of Contents
Previous Page  206 / 311 Next Page
Information
Show Menu
Previous Page 206 / 311 Next Page
Page Background

© 2015 AOAC INTERNATIONAL

” PJ GLHWDU\ VWDUFK XVH PJ IRU VDPSOHV FRQWDLQLQJ

GLHWDU\ VWDUFK LQWR VFUHZ FDS JODVV WXEHV 7HVW SRUWLRQ :

E

is for

WKH DQDO\VLV RI HQ]\PDWLFDOO\ UHOHDVHG JOXFRVH DQG :

F

is for the

GHWHUPLQDWLRQ RI IUHH JOXFRVH ,Q DGGLWLRQ WR XQNQRZQV ZHLJK WHVW

SRUWLRQV :

E

:

F

RI ' JOXFRVH DQG SXUL¿HG FRUQ VWDUFK ZKLFK

VHUYH DV TXDOLW\ FRQWURO VDPSOHV

C

(

g

). Also include two tubes with

QR WHVW SRUWLRQ WR VHUYH DV UHDJHQW EODQNV SHU HDFK DQDO\WLFDO UXQ

IRU IUHH JOXFRVH RU HQ]\PDWLFDOO\ UHOHDVHG JOXFRVH IUHH JOXFRVH

(

2

'LVSHQVH P/ RI

0 VRGLXP DFHWDWH EXIIHU

C

(

a

), into

each tube.

(

3

7R WXEHV ZLWK WHVW SRUWLRQV GHVLJQDWHG :

E

and to each of the

UHDJHQW EODQNV WR EH XVHG ZLWK DQDO\VLV RI HQ]\PDWLFDOO\ UHOHDVHG

JOXFRVH IUHH JOXFRVH DGG D YROXPH RI KHDW VWDEOH Į DP\ODVH

C

(

b

WR GHOLYHU FD

WR OLTXHIRQ XQLWV RU

WR %$8

RI HQ]\PH DFWLYLW\ W\SLFDOO\ P/ HQ]\PH DV SXUFKDVHG GR QRW

DGG WKH DP\ODVH WR :

F

DQG WR WKH UHDJHQW EODQNV WR EH XVHG ZLWK IUHH

JOXFRVH GHWHUPLQDWLRQV &DS WXEHV DQG YRUWH[ WR PL[

Note:

Vortex tube so that the solution column extends to the

cap, washing the entire interior of the tube and dispersing the test

portion.

(

4

,QFXEDWH DOO WXEHV IRU K DW

ƒ& LQ D IRUFHG DLU RYHQ

YRUWH[LQJ WXEHV DW

DQG PLQ RI LQFXEDWLRQ

(

5

) Cool tubes at ambient temperature on bench for 0.5 h. At

this point, separate tubes designated for free glucose analysis (tubes

FRQWDLQLQJ :

F

WHVW SRUWLRQV DQG UHDJHQW EODQNV ZLWK QR HQ]\PH

from the rest of the run. Those designated for free glucose should

VNLS VWHSV

6

) and (

7

) and continue with steps (

8

±

13

).

(

6

$GG P/ RI GLOXWHG DP\ORJOXFRVLGDVH VROXWLRQ

C

(

c

), to :

E

WHVW DQG TXDOLW\ FRQWURO VDPSOHV DQG UHDJHQW EODQNV 9RUWH[ WXEHV

(

7

,QFXEDWH WXEHV IRU K LQ D ZDWHU EDWK DW ƒ& YRUWH[LQJ DW

1 h of incubation.

(

8

$GG P/ ZDWHU WR HDFK WXEH &DS DQG LQYHUW DW OHDVW

four times to mix completely. Proceed immediately through

steps (

9

±

13

).

(

9

) (

a

)

Volume by sum of volume additions

.—Transfer ca

P/ WHVW VDPSOH VROXWLRQV WR PLFURFHQWULIXJH WXEHV DQG

centrifuge at 1000 ×

g

for 10 min. If the sample remains cloudy

after centrifugation, centrifuge an additional 10 min at 10 000 ×

g

WR FODULI\ WKH VROXWLRQ EHIRUH SURFHHGLQJ 6ROXWLRQV PD\ LQFUHDVH LQ

WHPSHUDWXUH GXULQJ FHQWULIXJDWLRQ DOORZ FHQWULIXJHG VROXWLRQV WR

come to room temperature before preparing dilution.

(

b

)

9ROXPH XVLQJ YROXPHWULF ÀDVNV

²4XDQWLWDWLYHO\ WUDQVIHU WHVW

VDPSOH VROXWLRQV ZLWK ¿OWUDWLRQ WKURXJK D KDUGHQHG SDSHU ¿OWHU ZLWK

—P UHWHQWLRQ DQG ULQVHV ZLWK ZDWHU WR P/ YROXPHWULF ÀDVNV

(

10

3UHSDUH GLOXWLRQV DV QHHGHG ZLWK GLVWLOOHG RU GHLRQL]HG

ZDWHU 6ROXWLRQV IURP FRQWURO VDPSOHV DQG WHVW VDPSOHV HVWLPDWHG

WR JLYH JUHDWHU WKDQ —J JOXFRVH P/ FRQFHQWUDWLRQV RI IUHH DQG

released glucose should be diluted 1 in 10 if processed as in (

9

)(

a

)

or 1 in 5 if processed as in (

9

)(

b

5HDJHQW EODQNV VKRXOG EH GLOXWHG

WR SURYLGH VROXWLRQV ZLWK WKH VDPH GLOXWLRQV DV XVHG ZLWK WKH WHVW

VROXWLRQV VR WKDW WKH GLOXWHG UHDJHQW EODQN VROXWLRQV FDQ EH XVHG

WR PDNH FRUUHFWLRQV IRU VLPLODUO\ GLOXWHG WHVW VROXWLRQV 'LOXWLRQV

PD\ EH SUHSDUHG XVLQJ YROXPHWULF ÀDVNV RU E\ DFFXUDWH SLSHWWLQJ ,I

GRQH E\ SLSHWWLQJ XVH D PLQLPXP RI

P/ WHVW VDPSOH RU FRQWURO

VROXWLRQ WR PLQLPL]H WKH LPSDFW RI YDULDWLRQ LQ SLSHWWLQJ VPDOO

YROXPHV

(

11

3LSHW

P/ LQ GXSOLFDWH RI JOXFRVH ZRUNLQJ VWDQGDUG

VROXWLRQV

DQG

—J P/ JOXFRVH

C

(

f

), and

UHDJHQW EODQN TXDOLW\ FRQWURO VDPSOH DQG WHVW VDPSOH VROXWLRQV

LQWR WKH ERWWRPV RI î PP JODVV WHVW WXEHV XVLQJ WZR WXEHV

VROXWLRQ $GG P/*232' UHDJHQW

C

(

e

)(

1

), to each tube. Vortex

WXEHV 3ODFH WXEHV LQ D UDFN DQG FRYHU ZLWK SODVWLF ¿OP WR VHDO

Note

$OWHUQDWLYH WR WKH XVH RI WKH *232' PHWKRG SURFHHG ZLWK

alternate glucose determination method,

C

(

e

)(

2

), for measurement

RI JOXFRVH LQ ZRUNLQJ VWDQGDUGV UHDJHQW EODQN FRQWURO VDPSOH DQG

test sample solutions.

(

12

) Incubate in a 50°C water bath for 20 min.

(

13

6HW VSHFWURSKRWRPHWHU WR PHDVXUH DEVRUEDQFH DW

QP

$IWHU WKH LQFXEDWLRQ LV FRPSOHWH ]HUR WKH VSHFWURSKRWRPHWHU ZLWK

WKH *232' UHDFWHG —J P/ ZRUNLQJ VWDQGDUG VROXWLRQ 5HDG

DEVRUEDQFHV RI UHPDLQLQJ *232' UHDFWHG ZRUNLQJ VWDQGDUG

VROXWLRQV DQG UHDJHQW EODQN FRQWURO VDPSOH DQG WHVW VDPSOH

VROXWLRQV $OO UHDFWHG VROXWLRQV PXVW EH UHDG ZLWKLQ PLQ RI WKH

HQG RI WKH *232' LQFXEDWLRQ 7KH GXSOLFDWH DEVRUEDQFH YDOXHV DUH

DYHUDJHG IRU HDFK UHDJHQW EODQN WHVW VDPSOH DQG FRQWURO VDPSOH

solution and used in

Calculations

.

F. Calculations

'HWHUPLQH WKH TXDGUDWLF HTXDWLRQ WKDW ¿WV WKH DEVRUEDQFHV RI WKH

ZRUNLQJ VWDQGDUG VROXWLRQV 7KH DEVRUEDQFH YDOXHV

A

CF

or

A

CE

, are

WKH LQGHSHQGHQW YDULDEOHV

X

), and actual glucose concentrations

DUH WKH GHSHQGHQW YDULDEOHV

Y

,QGLYLGXDO DEVRUEDQFH YDOXHV RI WKH

ZRUNLQJ VWDQGDUG VROXWLRQV QRW DYHUDJHV DUH XVHG 7KH HTXDWLRQ

has the form:

—J *OXFRVH P/

A

CF or CE

2

×

Q A

CF or CE

×

S I

)

&DOFXODWH GLHWDU\ VWDUFK FRQWHQW LQ WHVW VDPSOH DV UHFHLYHG DV

follows:

)UHH JOXFRVH

A

CF

2

×

Q A

CF

×

S I

) ×

V

F

×

DF

F

î

î

W

F

î

î

'LHWDU\ VWDUFK

>

A

CE

2

×

Q A

CE

×

S I

) ×

V

E

×

DF

E

î

î

W

E

î

î @ ± IUHH JOXFRVH

where subscript

F

UHSUHVHQWV YDOXHV IRU VDPSOHV DQDO\]HG IRU IUHH

glucose and subscript

E

UHSUHVHQWV YDOXHV IRU VDPSOHV WUHDWHG ZLWK

DP\ODVH DQG DP\ORJOXFRVLGDVH

A

CF

, A

CE

DEVRUEDQFH RI UHDFWLRQ

solutions minus the absorbance of the appropriately diluted reagent

EODQN YDOXHV DUH DYHUDJHV RI WKH WZR UHSOLFDWHV IRU HDFK WHVW

VROXWLRQ

Q

TXDGUDWLF VORSH WHUP

S

OLQHDU VORSH WHUP DQG

I

LQWHUFHSW RI WKH VWDQGDUG FXUYH WR FRQYHUW DEVRUEDQFH YDOXHV WR —J

JOXFRVH P/

V

F

,

V

E

¿QDO VDPSOH VROXWLRQ YROXPH FD

P/

for

V

F

DQG

P/ IRU

V

E

LI GRQH E\ VXPPDWLRQ RI YROXPHWULF

DGGLWLRQV RWKHUZLVH E\ VL]H RI YROXPHWULF ÀDVN XVHG

DF

GLOXWLRQ IDFWRU H J P/ VDPSOH VROXWLRQ GLOXWHG LQWR P/

J

—J FRQYHUVLRQ IURP —J WR J

W

E

, W

F

WHVW SRUWLRQ ZHLJKW DV UHFHLYHG

IDFWRU WR FRQYHUW IURP

measured glucose as determined, to anhydroglucose, as occurs in

starch.

If test samples are run in duplicate portions, the free glucose %

LQ WKH GLHWDU\ VWDUFK HTXDWLRQ LV WKH DYHUDJH IUHH JOXFRVH YDOXH

determined for the test sample.

5HIHUHQFHV

)RRG &KHPLFDOV &RGH[

WK (G 7KH

8QLWHG 6WDWHV 3KDUPDFRSHLDO &RQYHQWLRQ

5RFNYLOOH 0' 86$ $SSHQGL[ 9 (Q]\PH

$VVD\V Į $P\ODVH $FWLYLW\ %DFWHULDO

SS ±