Official Methods of Analysis of AOAC International

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A.O.A.C 2005

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Of fi cial Methods of Anal y sis

of AOAC IN TER NA TIONAL

18th Edi tion, 2005

Current through Revision 1, 2006

Dr. Wil liam Horwitz, Ed i tor

Dr. George W. Latimer, Jr., Assistant Editor

Pub lished by

AOAC IN TER NA TIONAL SUITE 500

481 NORTH FRED ER ICK AV E NUE

GAITHERSBURG, MARY LAND 20877-2417, USA

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COPYRIGHT Ó 1920, 1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965

BY THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS

1970, 1975, 1980, 1984, 1990

BY THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS AND

1995, 1996, 1997, 1998, 1999, 2000, 2002, 2003, 2005, 2006

BY AOAC IN TER NA TIONAL

The meth ods of the As so ci a tion were also copy righted in 1916, when they were pub lished in the Jour nal of the As so ci a tion of Of fi cial Ag ri cul tural Chem ists.

All rights re served. No part of this book may be re pro duced in any form or by any means with out the writ ten per mis sion of the As so ci a tion.

Printed in the United States of Amer ica Printed on acid-free pa per

ISBN 0-935584-77-3

A copy of the 18th Edition of this pub li ca tion is on file with the Of fice of the Fed eral Reg is ter.

U.S. Gov ern ment Agencies may ap ply to the Di rec tor of the Of fice of the Fed eral Reg is ter for ap proval to in cor po rate this edi tion by ref er ence in their reg u la tions. The pro ce dures that Fed eral agen cies must fol low in ap ply ing for the Di rec tor’s ap proval are in Ti tle 1, Part 51, of the Code of Fed eral Reg u la tions.

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Im por tant No tices

DIS CLAIMER

METHODS

An a lyt i cal meth ods and pro ce dures in this com pen dium have un der gone sys tem atic interlaboratory stud ies to de ter mine the per for mance char ac ter is tics for the in tended an a lyt i cal ap pli ca tion. AOAC IN TER NA TIONAL mem bers and other vol un teers have re viewed the an a lyt i cal re sults and de ter mined that a par tic u lar method is ap pro pri ate for the analyte and ma trix stated, pro vided the anal y sis is con ducted by a com pe tent an a lyst as writ ten. No war ranty, im plied or ex pressed, is made by AOAC IN TER NA TIONAL on the meth ods de scribed, their safety, or prod ucts men tioned. AOAC IN TER NA TIONAL, its mem bers, and non mem ber vol un teers who have aided in the de vel op ment and val i da tion of meth ods in cluded in this vol ume as sume no re spon si bil ity for any eco nomic, per sonal in jury, or other dam age that may oc cur to in di vid u als or or ga ni za tions be cause of the use of these meth ods.

COM MER CIAL PROD UCTS

Names of man u fac tur ers, sup pli ers, and trade names are fur nished solely as a mat ter of iden ti fi ca tion and con ve nience and re flect the con di tions within which each method was de vel oped in the orig i na tor’s lab o ra tory. In clu sion of this in for ma tion does not im ply AOAC pro mo tion, ap proval, en dorse ment, or cer tif i ca tion. The same or equiv a lent prod ucts, in stru ments, sup plies, ap pa ra tus, or re agents avail able from man u fac tur ers and sup pli ers other than those named, or other brands from other sources, may serve equally well if proper val i da tion in di cates their use is sat is fac tory.

WARNING

Do not per form anal y ses us ing AOAC

^®

Of fi cial Methods

^SM

un less you are knowl edge able about their po ten tial dan gers or haz ards and have re ceived ap pro pri ate train ing. Do not han dle in stru ments, sup plies, ap pa ra tus, re agents, biohazards, or other prod ucts when un fa mil iar with their op er a tion or the po ten tial haz ards as so ci ated with their use.

If a method re quires the use of po ten tially haz ard ous equip ment or prod ucts, see man u fac turer’s safety and cau tion ary in struc tions. Ma te rial Safety Data Sheets (MSDS), or the equiv a lent, must be read and un der stood prior to the use of ma te ri als spec i fied by a method.

Al ways use fume hoods, proper ven ti la tion, and pro tec tive cloth ing and equip ment when re quired.

See Ap pen dix B, “Lab o ra tory Safety,” for fur ther in for ma tion on safety.

REF ER ENCING AOAC

^®

OF FI CIAL METHODS

^SM

Each AOAC

^®

Of fi cial Method

^SM

has its own per ma nent method num ber that is part of the ti tle block. The para graph num ber lo cated in the up per left is only a lo ca tor num ber and not the method num ber. For ex am ple:

49.2.18A

AOAC Of fi cial Method 2005.08 Aflatoxins in Corn, Raw Peanuts,

and Peanut Butter

2005.08 is the per ma nent num ber of the method and 49.2.18A is the section num ber used to fa cil i tate lo cat ing meth ods. Per ma nent num bers in di cate the year the method was ap proved (in this case 2005) fol lowed by the or der in

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which the method was ap proved that year (in this ex am ple, it is the 8th method ap proved in 2005). The first set of numbers in the section number indicates the chapter (in this case, Chapter 49), the second set indicates the subchapter (in this case, subchapter 2), and the last set indicates the order in which the method appears in the subchapter (in this case, it is the 18th method).

When ref er enc ing AOAC

^®

Of fi cial Methods

^SM

, only the per ma nent num ber should be ref er enced as seen in this ex am ple:

(1) Of fi cial Methods of Anal y sis of AOAC INTERNATIONAL (2005) 18th Ed., AOAC IN TER NA TIONAL,

Gaithersburg, MD, USA, Of fi cial Method 2005.08 RE VI SIONS

AOAC pub lishes an nual re vi sions to the Of fi cial Methods of Anal y sis of AOAC IN TER NA TIONAL. Re vi sions con tain new meth ods ap proved since the last pub li ca tion and re vi sions to ex ist ing meth ods. You will au to mat i cally re ceive no tice of the avail abil ity of new re vi sions and be sent an ad vance in voice al low ing you to pur chase the re vi sions with out ob li ga tion. If you main tain the re vi sion ser vice, your 18th Edi tion will be cur rent.

For additional revisions received after the first printing (2005) of the 18th Edition, visit AOAC's Web site at www.aoac.org for the Official Methods of Analysis online. These revisions will be included in a subsequent printing.

(Note: In di vid ual cop ies of re vi sions will be pro vided on an an nual ba sis only. AOAC IN TER NA TIONAL will not re tain back cop ies of re vi sions for sale. If you do not pur chase re vi sions on an an nual basis, you will need to buy an en tire new book to ob tain miss ing re vi sions.)

IN QUIRIES

In quiries re gard ing meth ods pub lished in this book should be di rected to AOAC IN TER NA TIONAL, Of fi cial Methods Pro gram, 481 N. Fred er ick Ave, Suite 500, Gaithersburg, MD 20877-2417, USA. Tele phone +1-301-924-7077. Fax +1-301-924-7089. E-mail: [email protected]. Please note that due to the time nec es sary to an swer any tech ni cal ques tions re gard ing an AOAC

^â

Of fi cial Method

^SM

, AOAC staff pro vides tech ni cal as sis tance re gard ing meth ods in OMA to AOAC mem bers only.

In quiries re gard ing pur chase of Of fi cial Methods of Anal y sis or the re vi sion ser vice, the Jour nal of AOAC IN TER NA TIONAL, or other AOAC pub li ca tions should be di rected to AOAC IN TER NA TIONAL, Fulfillment, 481 N. Fred er ick Ave, Suite 500, Gaithersburg, MD 20877-2417, USA. Tele phone +1-301-924-7077. Fax +1-301-924-7089. E-mail: [email protected].

COM MENTS ON METHODS

AOAC IN TER NA TIONAL adopts meth ods that show by their per for mance data what can be ex pected of them. As

an a lysts use AOAC

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Of fi cial Methods

^SM

, they gen er ate ad di tional in for ma tion and data con cern ing ap pli ca bil ity,

spec i fic ity, sen si tiv ity, re li abil ity, and ac cu racy of the meth ods. An a lysts are re quested to ad vise AOAC

IN TER NA TIONAL about their ex pe ri ences with the AOAC

^®

Of fi cial Methods

^SM

pub lished in this book. In par tic u lar,

an a lysts should no tify AOAC IN TER NA TIONAL of prob lems in the per for mance of an Of fi cial Method

^SM

, which

may in di cate the method should be re vised or re stud ied. Di rect com ments to AOAC IN TER NA TIONAL, Of fi cial

Methods Pro gram, 481 N. Fred er ick Ave, Suite 500, Gaithersburg, MD 20877-2417, USA. Tele phone

+1-301-924-7077. Fax +1-301-924-7089. E-mail: [email protected].

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Pref ace

E lec tronic pub lish ing ar rived just in time to save the Of fi cial Methods of Anal y sis of AOAC IN TER NA TIONAL from its own suc cess. Each of the two vol umes of the pre vi ous 17th Edition has grown to an al most un man age able size. Now, for the first time, the re sults of 122 years of re view and ap proval of col labor atively stud ied meth ods are captured on the Internet. The older meth ods are still there, but their use is prob a bly con fined to teach ing “ag ri cul tural chem is try.” The prop er ties of meth ods do not de te ri o rate with age and the “clas si cal” analytes still need to be de ter mined. But the change in em pha sis with the reg u la tory winds is ob vi ous: Mi cro bi ol ogy and nu tri tion have blos somed as reg u la tory em pha sis has shifted from eco nom ics to safety and health. New drug and food ad di tive ap proval, based on preclearance of man u fac tur ing op er a tions and con tin u ous qual ity con trol as well as safety and ef fi cacy, have re duced the need for reg u la tory con trol through mar ket sam pling and anal y sis.

Most no ta ble has been the shift from clas si cal stoichiometric chem is try, based on the bal ance and buret, to cal i bra tion chem is try, based on an in stru men tal com par i son of a re sponse of an analyte with that of a stan dard. This shift was ini ti ated by the re mark able sep a ra tion pow ers of chro ma tog ra phy al low ing an analyte to be sep a rated from in ter fer ing com po nents be fore be ing mea sured by the in stru ment.

Chro ma tog ra phy moved an a lyt i cal chem is try from the realm of gram quan ti ties into mi cro gram quan ti ties but not with out un rec og nized sam pling prob lems. The sen si tiv ity, sta bil ity, and speed of mod ern elec tron ics per mit the per for mance of an a lyt i cal work au to mat i cally, from the mea sure ment of the test por tion, through de tec tion, am pli fi ca tion, and in ter pre ta tion of the sig nal, to the print ing of the an a lyt i cal re port.

The an a lyt i cal prob lem has shifted from mea sure ment to con trol. Much of the an a lyt i cal op er a tion has moved from an op er a tor to a black box in a com puter. Changes in phys i cal prop er ties, such as light in ten si ties or ion con duc tances, are mea sured au to mat i cally and con verted into an a lyt i cal re ports con tin u ously, chang ing the lab o ra tory into an au to mated fac tory. But the fa cil ity for au to mated per for mance al lows the re spon si bil ity for re li abil ity to eas ily shift from the an a lyst to the in stru ment. This is also true of the blind ap pli ca tion of com puter pro grams with no re view of the ap pli ca bil ity of the pro gram to the prob lem. The com puter has the abil ity not only to ex tract hid den in for ma tion from a jun gle of back ground, but also to for mu late spu ri ous peaks that it has been pro grammed to guess ought to be there.

AOAC ini ti ated the pro ce dure of val i da tion of meth ods through interlaboratory stud ies. These stud ies pro duce re sults from a sin gle sam ple of method per for mance in the hands of an as sumed ran dom sam ple of lab o ra to ries. Un for tu nately, time and ex pense rarely per mit per form ing ad di tional stud ies. There fore, the ini tial stud ies usu ally stand as the sole pub lished ev i dence of sat is fac tory interlaboratory per for mance.

AOAC mem bers are in ves ti gat ing sur ro gates for this nec es sary, but lengthy and costly pro ce dure.

Seventy-seven new meth ods have been added to this edi tion, pre dom i nantly mi cro bi o log i cal or chro mato graphic in na ture, all of them sub jected to the rig ors of an interlaboratory study. Many of these meth ods in cor po rate in ter nal con trols to en sure that the re ac tions are pro ceed ing as in tended. Most ap peal ing is the in tro duc tion of sys tem suit abil ity spec i fi ca tions into chro mato graphic sys tems that per mit flex i bil ity with out sac ri fic ing re li abil ity. For over a cen tury, the guid ing prin ci ple in the ap pli ca tion of stan dard meth ods has been to fol low in struc tions to the let ter to ob tain re sults equiv a lent to those orig i nally ob tained. But the com pe ti tion for im prove ments in sys tems ad vanced the sci ence of sep a ra tion and

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de tec tion so rap idly that suit abil ity spec i fi ca tions for in tro duc ing flex i bil ity with out sac ri fic ing per for mance had to be in vented.

In ter nal con trols re quire that the meth ods meet re peat abil ity per for mance spec i fi ca tions. An ap pre cia ble frac tion of the new mi cro bi o log i cal meth ods are screen ing tests in volv ing pre as sem bled immunoassays kits. Rel a tively quickly, these kits sep a rate lab o ra tory sam ples that can be dis carded as neg a tive from those that pre sum ably con tain patho genic or gan isms, re quir ing the ap pli ca tion of con fir ma tory tests. These kits also in vari ably con tain the re quire ment for ac com pa ny ing pos i tive and neg a tive con trols that pro vide con cur rent as sur ance of proper per for mance.

This edi tion joins the uni ver sal move ment to ward the use of the in ter na tional sys tem (SI) of units, many of which are un fa mil iar to U.S. sci en tists. Dur ing a tran si tion pe riod, both the com mon sys tem as well as the SI sys tem will be given. Note that the term “nor mal ity” is be ing re placed by “moles per li ter.” An other ed i to rial change be ing in tro duced is to move away from the ten dency to des ig nate any thing be ing worked on as the

“sam ple.” In stead, the se quence of “lab o ra tory sam ple” ÷ “test sam ple” ÷ “test por tion” is be ing in tro duced. This vo cab u lary is be ing used by the In ter na tional Un ion Of Pure and Ap plied Chem is try (IUPAC) and the In ter na tional Or ga ni za tion for Stan dard iza tion (ISO), which does not per mit the un mod i fied term “sam ple” to be used in con junc tion with sub se quent chem i cal op er a tions.

An im por tant fea ture of the 18th Edi tion is the in ter na tional source of many of the meth ods, with many coun tries and in ter na tional or ga ni za tions con trib ut ing their ex per tise to method stan dard iza tion. It is also grat i fy ing to see the in tro duc tion of qual ity con trol fea tures into the meth ods, which pro vide the an a lyst with guides to proper per for mance. On the other hand, the ease with which re sults are ob tained from com put ers also per mits the in tro duc tion of un an tic i pated er rors, de tected only by the un rea son able ness of the re sults. In the ab sence of a blue print of what is to be ex pected, gross er rors may be made. The in tro duc tion of qual ity as sur ance prin ci ples into the lab o ra tory may as sist in min i miz ing such oc cur rences.

Nu mer ous in di vid u als, vol un teer sci en tists, and pro fes sional staff have con trib uted en thu si as ti cally to this cen tury-old pro gram of method val i da tion. The an a lyt i cal com mu nity is grate ful for their con tin ued valu able ef forts.

—Wil liam Horwitz, Ed i tor

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Contents

List of Changes for Revision 1, 2006. . . iii

Im por tant No tices. . . vi

Pref ace . . . viii

About the As so ci a tion . . . xiv

Guide to Method For mat . . . xvi

Def i ni tion of Terms and Ex plan a tory Notes . . . xvii

AOAC^®Of fi cial Methods^SM Val i da tion Pro gram . . . xxiv

1. Ag ri cul tural Liming Ma te rials

1.1 Liming Ma te rials—Gen eral. . . 1

1.2 Cal cium Sil i cate Slags . . . 3

1.3 Gravimetric El e men tal Anal y ses . . . 4

1.4 Chelometric El e men tal Anal y ses . . . 6

1.5 Colorimetric El e men tal Anal y ses . . . 6

2. Fer til izers

2.1 Fer til izers—Gen eral . . . 1

2.2 Wa ter . . . 4

2.3 Phos pho rus . . . 5

2.4 Ni tro gen . . . 12

2.5 Po tas sium . . . 21

2.6 Other El e ments . . . 25

2.7 Peat . . . 36

2.8 Soils . . . 40

3. Plants

3.1 Gen eral Methods . . . 1

3.2 Metals . . . 2

3.3 In di vid ual Metals . . . 5

3.4 Non metals . . . 13

3.5 Other Con stit u ents . . . 23

3.6 Pig ments . . . 28

3.7 To bacco . . . 30

4. An i mal Feed

4.1 An i mal Feed—Gen eral . . . 1

4.2 Pro tein . . . 24

4.3 Urea . . . 36

4.4 Ni tro gen . . . 37

4.5 Fat . . . 39

4.6 Fi ber . . . 44

4.7 Sugars . . . 55

4.8 Min erals . . . 56

4.9 Mi cros copy . . . 66

4.10 Ad di tives . . . 68

5. Drugs in Feeds

5.1 Feeds—Gen eral Methods . . . 1

5.2 Chem i cal Methods for An ti bi otics. . . 34

5.3 Mi cro bi o log i cal Methods for An ti bi otics . . . 41

Com mon and Chem i cal Names of Drugs . . . 60

6. Dis in fec tants

6.1 Phe nol Co ef fi cient Methods . . . 1

6.2 Hard Sur face Car rier Test Methods . . . 3

6.3 Other Tests. . . 17

7. Pes ti cide For mu la tions

7.2 In or ganic and Organometallic Pes ti cides and Adjuvants . . . 8

7.3 Fun gi cides . . . 22

7.4 Her bi cides . . . 42

7.5 Pes ti cides Re lated to Nat u ral Prod ucts and Their Syn er gists . . . 52

7.6 Organohalogen Pes ti cides . . . 63

7.7 Thiophosphorus and Other Organophosphorus Pes ti cides . . . 92

7.8 Mis cel la neous Pes ti cides . . . 115

Com mon and Chem i cal Names of Pes ti cides . . . 121

8. Haz ard ous Sub stances. . . 1

9. Metals and Other El e ments at Trace Levels in Foods

9.1 Multielement Methods . . . 1

9.2 Sin gle El e ment Methods. . . 22

10. Pes ti cide and In dus trial Chem i cal Res i dues

10.1 Gen eral Multiresidue Methods . . . 1

10.2 Organo chlorine Res i dues . . . 17

10.3 Organophosphorus Res i dues. . . 26

10.4 Fu mi gant Res i dues . . . 40

10.5 Carbamate Res i dues . . . 41

10.6 In di vid ual Res i dues . . . 48

10.7 Pes ti cides in Wa ter. . . 78

Com mon and Chem i cal Names of Pes ti cides . . . 97

11. Wa ters; and Salt

11.1 Wa ter . . . 1

11.2 Salt. . . 31

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12. Microchemical Methods

. . . 1

13. Ra dio ac tiv ity

. . . 1

14. Vet er i nary An a lyt i cal Tox i col ogy

. . . 1

15. Cos metics

15.2 De odor ants and An ti per spi rants. . . 6

15.3 De pil a tories . . . 11

15.4 Face Pow der . . . 11

15.5 Hair Prep a ra tions . . . 12

15.6 Sun tan Prep a ra tions . . . 13

16. Ex tra ne ous Ma te rials: Iso la tion

16.1 Gen eral . . . 1

16.2 Bev er ages and Bev er age Ma te rials . . . 6

16.3 Dairy Prod ucts . . . 8

16.4 Nuts and Nut Prod ucts . . . 13

16.5 Grains and Their Prod ucts . . . 15

16.6 Baked Goods . . . 21

16.7 Break fast Ce reals . . . 23

16.8 Eggs and Egg Prod ucts . . . 24

16.9 Poul try, Meat, and Fish and Other Ma rine Prod ucts . . . 25

16.10 Fruits and Fruit Prod ucts . . . 28

16.11 Snack Food Prod ucts . . . 31

16.12 Sugars and Sugar Prod ucts . . . 31

16.13 Veg e ta bles and Veg e ta ble Prod ucts . . . 32

16.14 Spices and Other Con di ments . . . 39

16.15 Mis cel la neous . . . 48

16.16 An i mal Ex cre tions . . . 56

16.17 Mold . . . 66

16.18 Fruits and Fruit Prod ucts . . . 69

16.19 Veg e ta bles and Veg e ta ble Prod ucts . . . 70

17. Mi cro bi o log i cal Methods

17.1 Eggs and Egg Prod ucts . . . 1

17.2 Chilled, Frozen, Pre cooked, or Pre pared Foods, and Nutmeats . . . 4

17.3 Coliforms . . . 27

17.4 Esch e richia coli. . . 50

17.5 Staph y lo coc cus . . . 81

17.6 Ste ril ity (Com mer cial) of Foods (Canned, Low Acid) . . . 99

17.7 Clostridium. . . 104

17.8 Ba cil lus. . . 113

17.9 Sal mo nella. . . 117

17.10 Lis te ria. . . 200

17.11 Vibrio. . . 237

17.12 Vi ruses . . . 242

17.13 So matic Cells. . . 244

17.14 Bacillus anthracis . . . 246

18. Drugs: Part I

18.2 Sol vents . . . 2

18.3 Halogenated Drugs . . . 3

18.4 In or ganic Drugs . . . 5

18.5 An ti his ta mines. . . 18

18.6 Alkanolamines . . . 21

18.7 Phenethylamines. . . 27

18.8 Aminobenzoates . . . 28

18.9 Syn thetics . . . 32

18.10 Microchemical Tests. . . 44

18.11 Mi cros copy . . . 54

18.13 Antifungal . . . 55

18.14 Antiparkinsonian . . . 56

18.15 Antihypertensive. . . 57

19. Drugs: Part II

19.1 Acids . . . 1

19.2 Phe no lic Drugs . . . 2

19.3 An al ge sics and Antipyretics . . . 7

19.4 Hyp notics and Sed a tives. . . 13

19.5 An ti co ag u lants . . . 20

19.6 Sul fona mides . . . 24

19.7 Thiazides . . . 27

19.8 Other Sul fur-Containing Drugs . . . 30

20. Drugs: Part III

20.1 Opium Al ka loids . . . 1

20.2 Tropane Al ka loids . . . 6

20.3 Xanthine Al ka loids . . . 7

20.4 Ip e cac Al ka loids . . . 8

20.5 Ephedra Al ka loids . . . 9

20.6 Er got Al ka loids . . . 11

20.7 Physostigmine Al ka loids . . . 14

20.8 Chinchona Al ka loids . . . 16

20.9 Rau wol fia Al ka loids . . . 18

20.10 Other Al ka loids . . . 25

20.11 Dig i talis . . . 27

20.12 Other Nat u ral Prod ucts . . . 30

21. Drugs: Part IV

21.1 Nat u ral Estrogens . . . 1

21.2 Syn thetic Estrogens . . . 3

21.3 Progestational Ste roids . . . 6

21.4 Adrenocortico Ste roids . . . 8

21.5 Thy roid . . . 14

22. Drugs: Part V. . . 1

23. Drugs and Feed Ad di tives in An i mal Tis sues

. . . 1

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24. Fo ren sic Sci ences

. . . 1

25. Baking Pow ders and Baking Chem i cals

. . . 1

26. Dis tilled Li quors

26.1 Spirits . . . 1

26.2 Cor dials and Li queurs . . . 19

27. Malt Bev er ages and Brewing Ma te rials

27.1 Beer . . . 1

27.2 Bar ley . . . 23

27.3 Malt . . . 24

27.4 Ce real Ad juncts . . . 31

27.5 Hops . . . 34

27.6 Brewing Sugars and Syrups . . . 36

27.7 Wort . . . 38

27.8 Yeast . . . 39

27.9 Brewers’ Grains . . . 41

28. Wines

28.1 Gen eral . . . 1

28.2 Pre ser va tives. . . 17

28.3 Fla vors . . . 18

29. Nonalcoholic Bev er ages and Con cen trates. . . 1

30. Cof fee and Tea

. . . 1

31. Ca cao Bean and Its Prod ucts

31.1 Gen eral . . . 1

31.2 Shell. . . 3

31.3 Choc o late Li quor . . . 9

31.4 Fat. . . 9

31.5 Other Con stit u ents . . . 12

32. Ce real Foods

32.1 Wheat Flour . . . 1

32.2 Wheat, Rye, Oats, Corn, Buck wheat, Rice, Bar ley, and Soy beans and Their Prod ucts Ex cept Ce real Ad juncts . . . 27

32.3 Bread . . . 49

32.4 Baked Prod ucts . . . 54

32.5 Mac a roni, Egg Noo dles, and Sim i lar Prod ucts . . . 56

33. Dairy Prod ucts

33.1 Sam pling . . . 1

33.2 Milk . . . 4

33.3 Cream . . . 52

33.4 Evap o rated and Con densed Milk . . . 58

33.5 Dried Milk, Non fat Dry Milk, and Malted Milk . . . 59

33.6 But ter . . . 63

33.7 Cheese . . . 68

33.8 Ice Cream and Frozen Des serts . . . 82

34. Eggs and Egg Prod ucts

. . . 1

35. Fish and Other Ma rine Prod ucts

. . . 1

36. Fla vors

36.2 Va nilla Ex tract and Its Sub sti tutes . . . 1

36.3 Lemon, Or ange, and Lime Ex tracts, Fla vors, and Oils. . . 12

36.4 Al mond Ex tract . . . 18

36.5 Cassia, Cin na mon, and Clove Ex tracts . . . 20

36.6 Fla vor Ex tracts and Toi let Prep a ra tions . . . 21

37. Fruits and Fruit Prod ucts

. . . 1

38. Gel a tin, Des sert Prep a ra tions, and Mixes

. . . 1

39. Meat and Meat Prod ucts. . . 1

40. Nuts and Nut Prod ucts

. . . 1

41. Oils and Fats. . . 1

42. Veg e ta ble Prod ucts, Pro cessed

42.1 Canned Veg e ta bles . . . 1

42.2 Dried Veg e ta bles . . . 9

42.3 Frozen Veg e ta bles . . . 10

43. Spices and Other Con di ments

. . . 1

44. Sugars and Sugar Prod ucts

44.1 Sugars and Syrups . . . 1

44.2 Mo las ses and Mo las ses Prod ucts . . . 18

44.3 Con fec tion ary . . . 24

44.4 Honey . . . 25

44.5 Ma ple, Sap, Ma ple Syrup, Ma ple Syrup Prod ucts. . . 37

44.6 Sugar Beets . . . 47

44.7 Corn Syrups and Other Starch Derived Sweeteners. . . 48

45. Vi ta mins and Other Nu tri ents

45.1 Chem i cal Methods . . . 1

45.2 Mi cro bi o log i cal Methods . . . 53

45.3 Bioassay Methods . . . 70

45.4 Nu tri tionally Re lated Com po nents . . . 81

46. Color Ad di tives

46.1 Sep a ra tion and Iden ti fi ca tion of Color Ad di tives in Foods, Drugs, and Cos metics . . . 1

46.2 In ter me di ates . . . 14

46.3 Sub sid iary and Lower Sulfonated Dyes . . . 21

46.4 Metals and Other El e ments . . . 22

46.5 Halo gens . . . 25

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47. Food Ad di tives: Di rect

47.2 An ti ox i dants . . . 1

47.3 Chem i cal Pre ser va tives . . . 7

47.4 Emul sifying Agents . . . 37

47.5 En zymes . . . 39

48. Food Ad di tives: In di rect

. . . 1

49. Nat u ral Toxins

49.1 Myco toxins . . . 1

49.2 Aflatoxins . . . 2

49.3 Af la toxin M₁. . . 41

49.4 Deoxynivalenol . . . 48

49.5 Fumonisins . . . 50

49.6 Ochratoxins . . . 57

49.7 Patulin . . . 69

49.8 Sterigmatocystin . . . 74

49.9 Zearalenone . . . 76

49.10 Sea food Toxins . . . 80

49.11 Plant Toxins . . . 96

50. In fant For mulas, Baby Foods, and Enteral Prod ucts

. . . 1

51. Dietary Supplements

50.1 Ephedra Alkaloids . . . 1

50.2 Glucosamine . . . 7

50.3 b-Carotene . . . 10

Ap pen dix A

Stan dard So lu tions and Ref er ence Ma te rials . . . 1

Ap pen dix B

Lab o ra tory Safety . . . 1

Ap pen dix C

Ref er ence Ta bles . . . 1

Ap pen dix D

Guide lines for Col lab o ra tive Study Pro ce dures to Val i date Char ac ter is tics of a Method of Anal y sis . . . 1

Ap pen dix E

Lab o ra tory Qual ity As sur ance . . . 1

Sub ject In dex. . . 1

In dex of Method Numbers

. . . 1

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About the As so ci a tion

D

^uring the past 122 years, AOAC IN TER NA TIONAL (for merly the As so ci a tion of Of fi cial An a lyt i cal Chem ists and before that the Association of Official Agricultural Chemists) evolved from a group of chem ists in the U.S. De part ment of Ag ri cul ture and the individual states into an in de pend ent sci en tific As so ci a tion of an a lyt i cal sci en tists with mem bers through out the world. To day, AOAC is the leader in pro vid ing val i dated meth ods, pro fi ciency test sam ples, ac cred i ta tion cri te ria, and sci en tific in for ma tion to in dus try, gov ern ment agen cies, and ac a demic in sti tu tions.

MIS SION

AOAC IN TER NA TIONAL ad vances the global chem is try and mi cro bi ol ogy an a lyt i cal com mu nity by pro mot ing meth ods val i da tion and qual ity mea sure ments.

AOAC METHODS VAL I DA TION PRO GRAMS To fur ther its mis sion, the As so ci a tion’s pri mary pro grams fo cus on the val i da tion of chem i cal and mi cro bi o log i cal an a lyt i cal meth ods. These val i da tion pro grams are: the AOAC® Of fi cial Methods^SM Pro gram, the pro gram of choice when the high est level of con fi dence is de sired; the AOAC® Peer-Verified Methods^SM Pro gram, used when speed of val i da tion is es sen tial and a lesser de gree of con fi dence is ac cept able; and the AOAC® Per for mance Tested Methods^SM Pro gram used to test the per for mance of test kits.

Over 800 vol un teer sci en tists, work ing in in dus try, gov ern ment, and ac a demic lab o ra to ries world wide largely ac com plish the ac tual work of val i da tion.

The meth ods found in this 18th Edi tion of the Of fi cial Meth ods of Anal y sis of AOAC IN TER NA TIONAL have been val i dated within the AOAC® Of fi cial Meth ods^SM Pro gram. Can di dates for AOAC®

Of fi cial Method^SM sta tus are sub jected to col lab o ra tive study by eight or more lab o ra to ries, ac cord ing to in ter na tion ally rec og nized stan dards and re ceive rig or ous sci en tific re view of per for mance re sults (see page xxiv for ad di tional de tails of the AOAC® Of fi cial Meth ods^SM Val i da tion Pro gram).

AOAC val i dated meth ods are used by gov ern ment, in dus try, and ac a de mia through out the world for anal y sis of a va ri ety of com mod i ties—par tic u larly those re lated to food, ag ri cul ture, pub lic health and safety, and the en vi ron ment. In fact, many of the val i dated meth ods in this edi tion have been adopted by in dus try and gov ern ment agen cies as de facto stan dards in the op er a tion of their lab o ra to ries.

OTHER PRO GRAMS

The As so ci a tion also has sev eral pro grams that as sist lab o ra tory man ag ers in mea sur ing the ac cu racy of an a lyt i cal re sults, im prove

pro fes sional de vel op ment, and pro vide the op por tu nity for sci en tists to in ter act.

THE AOAC® LAB O RA TORY PRO FI CIENCY TESTING PRO GRAM

The AOAC® Lab o ra tory Pro fi ciency Testing Pro gram pro vides an in de pend ent as sess ment of the ac cu racy and re li abil ity of an a lyt i cal re sults in the anal y sis of a wide range of analytes and ma trix. Pro gram mod ules in clude Stan dard Mi cro bi ol ogy, HACCP, Nu tri tional La beling, and Pes ti cide Res i dues in Fruits and Veg e ta bles.

TECH NI CAL DI VI SIONS

The goals of the AOAC tech ni cal di vi sions are to im prove the over all qual ity of lab o ra tory op er a tions and fos ter har mo ni za tion of lab o ra tory pro ce dures and sys tems. The Tech ni cal Di vi sion for Lab o ra tory Man age ment helps lab o ra tory man ag ers im prove the op er a tions of their lab o ra to ries through the ex change of ideas and through pro fes sional de vel op ment ac tiv i ties. The Tech ni cal Di vi sion on Ref er ence Ma te rials im proves the qual ity of an a lyt i cal mea sure ments through the use of ref er ence ma te ri als.

TRAINING COURSES

AOAC IN TER NA TIONAL of fers a se ries of courses that pro vide hands-on train ing in spe cific top i cal ar eas, and as sist an a lyt i cal sci en tists to ac quire the skills they need to ad dress daily chal lenges faced in their lab o ra to ries. Cur rent of fer ings in clude courses on qual ity as sur ance for an a lyt i cal and mi cro bi o log i cal lab o ra to ries, ISO 17025, ISO au dit sys tems, and single laboratory validation.

CO OP ER A TIVE AC TIV ITIES

AOAC IN TER NA TIONAL has es tab lished joint com mit tees, li ai sons, and rep re sen ta tion with nu mer ous sci en tific or ga ni za tions world wide. The As so ci a tion serves as the Sec re tar iat of the In ter Agency Meet ing, an af fil i a tion of 16 in ter na tional or ga ni za tions ac tive in the field of anal y sis and sam pling of food prod ucts and as so ci ated qual ity as sur ance mea sures in conjunction with the Joint FAO/WHO Codex Alimentarius. The As so ci a tion also par tic i pates in the meet ings of the Co dex Alimentarius Com mis sion, the In ter na tional Or ga ni za tion for Stan dard iza tion (ISO), the Eu ro pean Com mu nity for Stan dard iza tion, the World Health Or ga ni za tion, the Pan Amer i can Health Or ga ni za tion, and other in ter na tional groups, both pri vate and gov ern ment spon sored.

Such ar range ments en able AOAC IN TER NA TIONAL to ex press its ba sic pol i cies on the de vel op ment of in ter na tion ally ac cept able

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meth ods of anal y sis and pro vide sec re tar i ats with ba sic in for ma tion re gard ing AOAC’s phi los o phy and pro ce dures.

AOAC-appointed Li ai son Of fi cers co or di nate AOAC ac tiv i ties with na tional, state, pro vin cial, mu nic i pal, lo cal agen cies and in dus tries and their af fil i ated or ga ni za tions, and other method or ga ni za tions that have oral or writ ten co op er a tive agree ments with AOAC IN TER NA TIONAL.

MEET INGS AND EX PO SI TIONS

The AOAC IN TER NA TIONAL An nual Meet ing and Ex po si tion is rec og nized world wide as the most sig nif i cant meet ing for qual ity as sur ance and lab o ra tory man age ment pro fes sion als dealing with regulated commodities. The an nual sci en tific pro gram in cludes cut ting-edge in for ma tion from the world’s most re spected sci en tists and lab o ra to ries. Through the sci en tific sym po sia, poster ses sions, work shops, fo rums, and short courses, meet ing at ten dees en hance their an a lyt i cal ex per tise, share their re search, and strengthen con tacts with their col leagues from around the world. The An nual Lab o ra tory Ex po si tion held at the An nual Meet ing fea tures over 100 dis plays of the state of the art in lab o ra tory prod ucts and ser vices.

SEC TIONS

AOAC IN TER NA TIONAL has 16 Sec tions lo cated in North Amer ica, Eu rope, Ja pan, China, Taiwan, Latin Amer ica, and the Ca rib bean. Sec tions pro vide op por tu ni ties for AOAC mem bers to gather and share in for ma tion on a more lo cal level, to build and ex pand their net work of pro fes sional con tacts, en hance their lead er ship skills, and gain prac ti cal man age ment ex pe ri ence. All Sec tions are man aged by an elected group of lo cal vol un teers.

PUB LI CA TIONS

In ad di tion to the Of fi cial Methods of Anal y sis of AOAC IN TER NA TIONAL, the As so ci a tion pub lishes the Jour nal of AOAC IN TER NA TIONAL and a va ri ety of other pub li ca tions. AOAC’s Jour nal con tains orig i nal fully ref er eed re search ar ti cles and re ports on cur rent col lab o ra tive study data, in clud ing in for ma tion on inter- and intralaboratory per for mance pre ci sion, which en ables the us ers of the AOAC® Of fi cial Methods^SM to make in formed choices about the ap pro pri ate use of a par tic u lar method. In side Lab o ra tory Man age ment, a full-color bimonthly mag a zine con tains tech ni cal and gen eral ar ti cles on lab o ra tory man age ment, reg u la tions,

emerg ing tech nol o gies, in stru men ta tion, and other ar eas per ti nent to lab o ra tory pro ce dures, man age ment, and qual ity con trol.

AOAC’s other pub li ca tions in clude man u als, meth ods com pi la tions, and mono graphs cov er ing sub jects that in clude qual ity as sur ance, sta tis tics, food anal y sis, ag ri cul tural anal y sis, lab o ra tory man age ment, and pes ti cide anal y sis.

AWARDS

Each year, the As so ci a tion pres ents a num ber of awards in rec og ni tion of out stand ing con tri bu tions to an a lyt i cal meth od ol ogy in ar eas of in ter est to AOAC IN TER NA TIONAL, mer i to ri ous ser vice to the As so ci a tion, and out stand ing work in the AOAC®

Of fi cial Methods^SM Pro gram. AOAC IN TER NA TIONAL also awards an an nual schol ar ship to en cour age study in fields that sup port the mis sion of the As so ci a tion.

MEM BER SHIP AND GOV ER NANCE

AOAC IN TER NA TIONAL is an as so ci a tion com prised of nearly 4000 in di vid ual and 300 or ga ni za tional mem bers from more than 90 coun tries. In di vid ual mem bers are lab o ra tory man ag ers, an a lyt i cal chem ists, mi cro bi ol o gists, tox i col o gists, fo ren sic sci en tists, and man age ment ex ec u tives work ing in in dus try, gov ern ment, and ac a de mia. Or ga ni za tional mem bers are cor po ra tions, com mer cial lab o ra to ries, gov ern ment agen cies, and uni ver si ties.

An elected Board of Di rec tors gov erns the As so ci a tion. The Of fi cial Methods Board, the Ed i to rial Board, spe cial and stand ing com mit tees, Ref eree po si tions con cerned with the val i da tion of meth ods, li ai son po si tions with other or ga ni za tions, and a head quar ters staff ad vance and sup port the mis sion of the As so ci a tion.

For fur ther in for ma tion about AOAC IN TER NA TIONAL and its pro grams and ac tiv i ties, con tact the As so ci a tion at:

AOAC IN TER NA TIONAL 481 N. Fred er ick Av e , Suite 500 Gaithersburg, MD 20877-2417, USA Tel: +1-800-379-2622 (Toll free from North Amer ica)

+1-301-924-7077 (World wide) Fax: +1-301-924-7089 E-mail: [email protected] Web site: http://www.aoac.org

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Guide to Method For mat

(Method shown is in com plete to al low space for de scrip tion.)

4.10.03

AOAC Of fi cial Method 996.13 Ethoxyquin in Feeds Liq uid Chro mato graphic Method

First Ac tion 1996 Fi nal Ac tion 1997

(Ap pli ca ble for de ter mi na tion of 0.5–300 mg/g ethoxyquin in dry ex truded pet food or meat meal.)

See Ta ble 996.13 for the re sults of the interlaboratory study sup port ing ac cep tance of the method.

A. Prin ci ple

Ethoxyquin is ex tracted with acetonitrile. Ex tract is an a lyzed by isocratic liq uid chro ma tog ra phy with flu o res cence de tec tion.

B. Ap pa ra tus

(a) Liq uid chromatograph (LC).—Gen er ating 1500 ± 200 psi; with peak area in te gra tor (man ual or com puter), isocratic LC pump, and col umn heater. Op er ating con di tions: in jec tion vol ume, 20 mL; flow rate, 1.3 mL/min; tem per a ture, 35°C; flu o res cence de tec tor out put, an a log to dig i tal con ver sion; de tec tor set tings: ex ci ta tion, 360 nm; emis sion, 432 nm.

(b) LC col umn.—250 ´ 4.6 mm id, C₁₈ octadecylsilane, 5 mm spher i cal, 100 Å pore size.

C. Re agents

(a) Wa ter.—LC grade.

(b) Acetonitrile.—LC grade.

D. Prep a ra tion of Stan dard So lu tions

(a) Ethoxyquin stan dard stock so lu tion.—400 mg/mL. Weigh the equiv a lent of 0.1000 g liq uid ethoxyquin into 250 mL am ber vol u met ric flask and di lute to vol ume with acetonitrile. (Note: Amount of ethoxyquin needed for prep a ra tion of stock so lu tion is based on pu rity of liq uid, e.g., for pu rity of 93.5%, amount of liq uid ethoxyquin = 0.100/0.935 = 0.1070 g.)

H. Cal cu la tions

Cal cu late con cen tra tion of ethoxyquin, mg/g or ppm, in test sam ple from cal i bra tion curve (us ing lin ear re gres sion with line forced through zero in ter cept) as fol lows:

Ethoxyquin, mg/g or ppm = C F W

´15. ´

where C = ethoxyquin con cen tra tion from LC cal i bra tion curve, mg/mL;

1.5 = vol ume of acetonitrile added to test so lu tion, mL; F = di lu tion fac tor;

W = weight of test por tion, g.

Ref er ence: J. AOAC Int. 80, 725(1997).

CAS-91-53-2 (ethoxyquin) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline Re vised: March 1998

Chem i cal Ab stracts Ser vice Reg is try Num ber.

A unique iden ti fier that may be used to search a num ber

of data-retrieval sys tems.

Method may be di vided into sev eral de scrip tive sec tions.

Chem i cal names of pes ti cides and drugs are given at end of per ti nent chap ter.

Cal cu la tion symbols are iden ti fied and show cor rect units.

Lo ca tor num ber iden ti fies method by chap ter, subchapter, and se quence within the subchapter for easy cross ref er enc ing and access.

4 = chap ter 4;

.10 = subchapter 10;

.03 = the third method found in Chap ter 4, subchapter 10. The lo ca tor num ber is not the per ma nent num ber and is in cluded only for convenient accessibility.

Per ma nent num ber iden ti fies method by year of adop tion or first ap pear ance in Of fi cial Methods of Anal y sis of AOAC IN TER NA TIONAL.

996 = First Ac tion 1996;

.13 = se quence of adop tion in 1996.

Ti tle may in clude analyte and ma trix, type of method, and of fi cial sta tus.

Ap pli ca bil ity state ment ad dresses utility and lim i ta tions on use of method or other in for ma tion.

Ref er ences di rect the user to the pub lished col lab o ra tive study and any sub se quent re vi sions in the method. Other in for ma tive ref er ences may be in cluded.

Spec i fi ca tions for nec es sary lab o ra tory ap pa ra tus and re agent prep a ra tions. See also Def i ni tion of Terms and Ex plan a tory Notes.

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Def i ni tion of Terms and Ex plan a tory Notes

Of fi cial Methods

(1) Of fi cial Methods are des ig nated First Ac tion or Fi nal Ac tion, and, in a few cases, Pro ce dures. A First Ac tion method has un der gone col lab o ra tive study, has been rec om mended by the ap pro pri ate Gen eral Ref eree and has been adopted Of fi cial First Ac tion by the Methods Committee. A method may be adopted Fi nal Ac tion a min i mum of 2 years af ter it has been adopted First Ac tion, and af ter it has been rec om mended by the ap pro pri ate Gen eral Ref eree and Methods Com mit tee, and voted on by the Official Methods Board.

Sampling, test sam ple prep a ra tion pro to col, or other type of in struc tions for which an interlaboratory col lab o ra tive study is im prac ti cal may be adopted, as above, as a Pro ce dure.

All meth ods in this book—First Ac tion, Fi nal Ac tion, or P r o c e d u r e — a r e O f f i c i a l M e t h o d s^{S M} o f A O A C IN TER NA TIONAL.

Re agents

(2) Term “H₂O” means dis tilled or deionized wa ter, ex cept where oth er wise spec i fied, and ex cept where the wa ter does not mix with the de ter mi na tion, as in “H₂O bath.”

(3) Term “al co hol” means 95% eth a nol by vol ume. Al co hol of strength x% may be pre pared by di lut ing x mL 95% al co hol to 95 mL with H₂O. Ab so lute al co hol is 99.5% by vol ume. For mulas of spe cially de na tured al co hols (SDA) used as re agents in the United States un der 27CFR21 are as fol lows:

SDA No. 100 Parts al co hol plus

3-A 5 Parts meth a nol

3-C 5 Isopropyl al co hol

30 10 Parts methanol

“Re agent” al co hol is 95 parts SDA 3-A plus 5 parts isopropanol.

(4) Term “ether” means ethyl ether, per ox ide free by the fol low ing test: To 420 mL ether in sep a ra tor, add 9.0 mL 1%

NH₄VO₃ in H₂SO₄ (1 + 16). Shake 3 min and let sep a rate. Drain lower layer into 25 mL glass-stoppered grad u ate, di lute to 10 mL with H₂SO₄ (1 + 16), and mix. Any or ange color should not ex ceed that pro duced by 0.30 mg H₂O₂ (1 mL of so lu tion pre pared by di lut ing 1 mL 30% H₂O₂ to 100 mL with H₂O) and 9.0 mL 1%

NH₄VO₃ in H₂SO₄ (1 + 16). Per ox ides may be elim i nated by pass ing

£700 mL ether through 10 cm col umn of Woelm ba sic alu mina in 22 mm id tube.

(5) The fol low ing listed re agents, un less oth er wise spec i fied, have ap prox i mate strength stated and con form in pu rity with

Rec om mended Spec i fi ca tions for An a lyt i cal Re agent Chem i cals of the Amer i can Chem i cal So ci ety:

As say Sul fu ric acid 95.0–98.0% H₂SO₄ Hy dro chlo ric acid 36.5–38.0% HCl

Ni tric acid 68.0–70.0% HNO₃

Fuming ni tric acid ³90% HNO₃

Ace tic acid ³99.7% CH₃COOH

Hydrobromic acid 47.0–49.0% HBr

Am mo nium hy drox ide 28–30% NH₃

Phos pho ric acid ³85% H₃PO₄

Where no in di ca tion of di lu tion is given, re agent con cen tra tion is the con cen tra tion given above.

(6) All other re agents and test so lu tions, un less oth er wise de scribed in the text, are au to mat i cally re agent grade and con form to re quire ments of the Amer i can Chem i cal So ci ety. Where such spec i fi ca tions have not been pre pared, use high est grade re agent.

When an hy drous salt is in tended, it is so stated; oth er wise the hy drated prod uct is meant.

(7) Un less oth er wise spec i fied, phenolphthalein used as in di ca tor is 1% al co hol so lu tion; methyl or ange is 0.1% aque ous so lu tion; methyl red is 0.1% al co hol so lu tion.

(8) Di rec tions for stan dard iz ing re agents are given in Ap pen dix A, Stan dard So lu tions and Cer tified Ref er ence Ma te rials.

(9) Un usual re agents not men tioned in re agent sec tions or cross ref er enced, other than com mon re agents nor mally found in laboratories, are ital i cized the first time they oc cur in a method.

(10) Com mer cially pre pared re agent so lu tions must be checked for ap pli ca bil ity to a spe cific method. They may con tain un de clared buff ers, pre ser va tives, che lat ing agents, etc.

(11) In ex pres sions (1 + 2), (5 + 4), etc., used in con nec tion with name of re agent, the first nu meral in di cates the vol ume of re agent used and the sec ond nu meral in di cates vol ume of H₂O.

For ex am ple, HCl (1 + 2) means re agent pre pared by mix ing 1 vol ume HCl with 2 vol umes H₂O. When one of the re agents is a solid, ex pres sion means part by weight. The first nu meral rep re sents the solid re agent; the sec ond nu meral H₂O. So lu tions for which the sol vent is not spec i fied are aque ous so lu tions.

(12) In mak ing up so lu tions of def i nite per cent age, it is un der stood that x g sub stance is dis solved in H₂O and di luted to 100 mL. Al though not the o ret i cally cor rect, this con ven tion will not re sult in any ap pre cia ble er ror in any meth ods given in this book.

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© 2006 AOAC IN TER NA TIONAL xviii (13) Chro mic acid clean ing so lu tion is pre pared by (1) add ing 1 L H₂SO₄ to ap prox i mately 35 mL sat u rated aque ous Na₂Cr₂O₇ so lu tion; or (2) add ing 2220 mL H₂SO₄ to ap prox i mately 25 mL sat u rated aque ous CrO₃ so lu tion (170 g/100 mL). Re agents may be tech ni cal high grade. Use only af ter first clean ing by other means (e.g., de ter gent) and drain ing. Mix ture is ex pen sive and haz ard ous.

Use re peat edly un til it is di luted or has a green ish tinge. Dis card care fully with co pi ous amounts of H₂O. Re fer to Ap pen dix B, Lab o ra tory Safety chap ter.

(14) All cal cu la tions are based on in ter na tional atomic weights.

Ap pa ra tus

(15) Burets, vol u met ric flasks, and pipets con form to the fol low ing U.S. Fed eral spec i fi ca tions (avail able from Gen eral Ser vices Ad min is tra tion, Spec i fi ca tion Sec tion, L’Enfant Plaza, Ste 8100, Wash ing ton Navy Yard, Bldg 197, Wash ing ton, DC 20407, USA):

Buret A-A-51248 May 19, 1965

Flask, vol u met ric A-A-51360 Feb ru ary 7, 1977 Pipet, vol u met ric A-A-53890 Feb ru ary 24, 1978

See also Ap pen dix V, “Testing of Glass Vol u met ric Ap pa ra tus,” in the Na tional In sti tute of Stan dards and Tech nol ogy (NIST) Spec i fi ca tion Pub li ca tion 260–54, “Cer tif i ca tion and Use of Acidic Po tas sium Dichromate So lu tions as an Ul tra vi o let Absorbance Stan dard SRM935” (avail able from NIST, Of fice of Stan dard Ref er ence Ma te rials, B316 Chem i cals, Gaithersburg, MD 20899, USA).

(16) Stan dard taper glass joints may be used in stead of stop pers where the lat ter are spec i fied or im plied for con nect ing glass ap pa ra tus.

(17) Sieve des ig na tions, un less oth er wise spec i fied, are those de scribed in U.S. Fed eral Spec i fi ca tion RR-S-366e, No vem ber 9, 1973 (avail able from Gen eral Ser vices Ad min is tra tion).

Des ig na tion “100 mesh” (or other num ber) pow der (ma te rial, etc.) means ma te rial ground to pass through stan dard sieve No. 100 (or other num ber). Cor re sponding in ter na tional stan dard and U.S.

stan dard sieves are given in Ta ble 1.

(18) Term “pa per” means fil ter pa per, un less oth er wise spec i fied.

(19) Term “high-speed blender” des ig nates mixer with 4 canted, sharp-edge, stain less steel blades ro tat ing at the bot tom of 4-lobe jar at 10 000–12 000 rpm, or with equiv a lent shear ing ac tion. Sus pended sol ids are re duced to fine pulp by ac tion of blades and by lob u lar con tainer, which swirls sus pended sol ids into blades. War ing Blender, or equiv a lent, meets these re quire ments.

(20) “Flat-end rod” is glass rod with one end flat tened by heat ing to soft en ing in flame and press ing ver ti cally on flat sur face to form cir cu lar disk with flat bot tom at end.

(21) Des ig na tion and pore di am e ter range of fritted glass ware are: ex tra coarse, 170–220 mm; coarse, 40–60; me dium, 10–15; fine, 4–5.5; Jena des ig na tions and pore di am e ter are: (1) 110 mm; (2) 45;

(3) 25; (4) 8.

(22) Un less oth er wise in di cated, tem per a tures are ex pressed in de grees Cel sius (Cen ti grade).

Sam ple

(23) Ter mi nol ogy and us age for items and op er a tions col lo qui ally des ig nated with the term “sam ple”: Newly adopted

meth ods will avoid the con fus ing us age of the term “sam ple” for any thing the an a lyst is work ing with. The no men cla ture rec om mended by the In ter na tional Un ion of Pure and Ap plied Chem is try (IUPAC), Pure & Appl. Chem. 62, 1193(1990), for an a lyt i cal chem is try, based upon the In ter na tional Or ga ni za tion for Stan dard iza tion (ISO) rec om men da tions, will be uti lized. The crit i cal def i ni tions are:

A lab o ra tory sam ple is the ma te rial sent to or re ceived by the lab o ra tory. The lab o ra tory re duces the lab o ra tory sam ple in size and fine ness to a test sam ple (or an a lyt i cal sam ple if only chem i cal or mi cro bi o log i cal anal y sis is in volved). A test (or an a lyt i cal) por tion Table 1. Nom i nal di men sions of stan dard test sieves

(USA stan dard se ries) Sieve designation

Nom i nal sieve opening, in.

Nom i nal wire di am e ter, mm In ter na tional

standard^a (ISO)

USA stan dard

12.5 mm^b ¹₂ in.^b 0.500 2.80

11.2 mm ⁷₁₆ in. 0.438 2.50

9.5 mm ³₈ in. 0.375 2.24

8.0 mm ⁵₁₆ in. 0.312 2.00

6.7 mm 0.265 in. 0.265 1.80

6.3 mm ¹₄ in.^b 0.250 1.80

5.6 mm No. 3 0.223 1.60

4.75 mm No. 4 0.187 1.60

4.00 mm No. 5 0.157 1.25

3.35 mm No. 6 0.132 1.00

2.80 mm No. 7 0.111 0.90

2.36 mm No. 8 0.0937 0.80

2.00 mm No. 10 0.0787 0.71

1.70 mm No. 12 0.0661 0.63

1.40 mm No. 14 0.0555 0.56

1.18 mm No. 16 0.0469 0.45

1.00 mm No. 18 0.0394 0.40

850 mm^c No. 20 0.0331 0.355

710 mm No. 25 0.0278 0.315

600 mm No. 30 0.0234 0.280

500 mm No. 35 0.0197 0.224

425 mm No. 40 0.0165 0.200

355 mm No. 45 0.0139 0.180

300 mm No. 50 0.0117 0.160

250 mm No. 60 0.0098 0.125

212 mm No. 70 0.0083 0.100

180 mm No. 80 0.0070 0.090

150 mm No. 100 0.0059 0.080

125 mm No. 120 0.0049 0.063

106 mm No. 140 0.0041 0.056

90 mm No. 170 0.0035 0.045

75 mm No. 200 0.0029 0.040

63 mm No. 230 0.0025

53 mm No. 270 0.0021

a These stan dard des ig na tions cor re spond to the val ues for test sieve ap er tures rec om mended by the In ter na tional Or ga ni za tion for Stan dard iza tion, Geneva, Swit zer land.

b These sieves are not in the stan dard se ries but they have been in cluded be cause they are in com mon us age.

c 1000 mm = 1 mm.

(17)

is re moved from the test sam ple for anal y sis. Once a test por tion is mea sured, by mass or vol ume, the term “sam ple” is no lon ger ap pro pri ate. Use “test” or “un known” as the mod i fier, e.g., “test so lu tion,” not “sam ple so lu tion.”

The op er a tion of ten called “prep a ra tion of sam ple” ap plies to the re duc tion of the lab o ra tory sam ple to the test sam ple, and not to the usual an a lyt i cal steps of so lu tion, sep a ra tion, pu ri fi ca tion, or iso la tion of the analyte.

The term “sam ple” will be used solely in the sta tis ti cal sense as a small por tion rep re sent ing a larger quan tity, such as a lot or a batch, where the po ten tial ex ists for a “sam pling er ror” due to the het er o ge ne ity of the par ent pop u la tion. Most sam ples are re moved from a static pop u la tion, such as a pile of fer til izer, a stack of cases, or a group of con tain ers. In a dy namic sit u a tion, how ever, where the pop u la tion changes with time as a flow ing river, cir cu lat ing blood, or a mov ing con veyor belt, the small por tion re moved should be called a “spec i men.” In these cases, the phe nom e non un der study and the sam pling er ror are con founded in such a way that they can not be sep a rated.

See Fig ure 1 [In ter na tional Un ion of Pure and Ap plied Chem is try,

“No men cla ture for Sam pling in An a lyt i cal Chem is try,” Pure &

Appl. Chem. 62, 1193(1990)].

Stan dard Op er a tions

(24) Op er a tions spec i fied as “wash (rinse, ex tract, etc.) with two (three, four, etc.) 10 mL (or other vol umes) por tions H₂O (or other sol vent)” mean that the op er a tion is to be per formed with in di cated vol ume of sol vent and re peated with same vol ume of sol vent un til num ber of por tions re quired have been used.

(25) Def i ni tions of terms used in meth ods in volv ing spectropho tom e try are those given in JAOAC 37, 54(1954). Most im por tant prin ci ples and def i ni tions are: (a) More ac cu rate in stru ment may be sub sti tuted for less ac cu rate in stru ment (e.g., spectrophotometer may re place colorimeter) where lat ter is spec i fied in method. Wave length spec i fied in method is un der stood to be that of max i mum absorbance (A), un less no peak is pres ent.

(b) Absorbance(s) (A): Neg a tive log a rithm to base 10 of the ra tio of trans mit tance (T) of test so lu tion to that of ref er ence or stan dard ma te rial. Other names that have been used for quan tity rep re sented by this term are op ti cal den sity, ex tinc tion, and ab sor bency.

(c) Ab sorp tivi ty(ies) (a): Absorbance per unit con cen tra tion and cell length.

a = A/bc

where b is in cm and c = g/L, or a = (A/bc ) ´ 1000, if c is mg/L. Other names that have been used for this or re lated quan ti ties are ex tinc tion co ef fi cient, spe cific ab sorp tion, absorbance in dex, and E_1cm^1%. (d) Trans mit tance(s) (T): Ra tio of ra di ant power trans mit ted by the test so lu tion to ra di ant power in ci dent on so lu tion, when both are mea sured at same spec tral po si tion and with same slit width.

Beam is un der stood to be par al lel ra di a tion and in ci dent at right an gles to plane par al lel sur face of test ma te rial. If test ma te rial is so lu tion, sol ute trans mit tance is quan tity usu ally de sired and is cal cu lated di rectly as ra tio of trans mit tance of so lu tion in cell to trans mit tance of sol vent in an equal cell. Other names that have been used for this quan tity are transmittancy and trans mis sion.

(e) Stan dard iza tion: Spectrophotometer may be checked for ac cu racy of wave length scale by re fer ring to Hg lines: 239.94, 248, 253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 404.66, 435.83, 546.07, 578.0, and 1014.0 nm. To check con sis tency of

absorbance scale, pre pare so lu tion of 0.0400 g K₂CrO₄/L 0.05M KOH and de ter mine absorbance at fol low ing wave lengths in 1 cm cell: 230 nm, 0.171; 275 nm, 0.757; 313.2 nm, 0.043; 375 nm, 0.991;

400 nm, 0.396. See NIST Spec. Pub. 378, “Ac cu racy in Spectrophotometry and Lu mi nes cence Mea sure ments,” 1973 (avail able from NIST, Of fice of Stan dard Ref er ence Ma te rials, B316, Chem is try, Gaithersburg, MD 20899, USA).

(26) Least square treat ment of data and cal cu la tion of re gres sion lines. This tech nique finds the best fit ting straight line for set of data such as stan dard curve. It cal cu lates that straight line for which the sum of squares of ver ti cal de vi a tions (usu ally A) of ob ser va tions from the line is smaller than cor re spond ing sum of squares of de vi a tion from any other line. Equa tion of straight line is:

Y = a + bX

where a is in ter cept at Y axis (X = 0), and b is slope of line.

Least square es ti mates of con stants are:

b X Y X Y n

X X n

i i i i

i i

= -

-

S S S

S S

( ) [( ) ]

[( ) ]

2 2

a = Y – bX

where S = “sum of” the n in di vid ual val ues of in di cated op er a tion, and X and Y are the av er ages of the X and Y points.

Ex am ple: To find “best” straight line re lat ing A(Y) to con cen tra tion (X):

Ob ser va tion No. (_i)

Con cen tra tion X_i

Absorbance

Y_i X_i² X_iY_i

1 80 1.270 6400 101.6

2 60 1.000 3600 60.0

3 40 0.700 1600 28.0

4 30 0.550 900 16.5

5 20 0.250 400 5.0

6 10 0.100 100 1.0

7 0 0.050 0 0.0

To tals:

n = 7 SX_i= 240 SY_i= 3.92 SX_i²= 1300 S(X_iY_i) = 212.1

X = SX_i/n = 240/7 = 34.29 Y = SY_i/n = 3.92/7 = 0.56 b = 2121 240 3 92 7

13000 240 7

77 7 4771 0 016

2

. [( )( . )]

[( ) ]

. .

-

- = = 3

a = 0.56 – 0.0163(34.29) = 0.001 @ 0 Best equa tion is then:

Y = 0.00 + 0.0163X

If for test sam ple, A = 0.82, cor re spond ing con cen tra tion (X) would be:

(18)

(19)

X = (Y – 0.00)/0.0163 = 0.82/0.0163 = 50.3

Many sci en tific and sta tis ti cal cal cu la tors are pro grammed to per form this cal cu la tion. It should be noted that the least square fit of a data set should not be the only cri te rion used in eval u at ing the va lid ity of a given data set.

High cor re la tion co ef fi cients (e.g., >0.99) do not nec es sar ily in di cate lin ear ity. This mis in for ma tion has been the sub ject of sev eral re ports from the An a lyt i cal Methods Com mit tee of the Royal So ci ety of Chem is try [An a lyst 113, 1469–1471 (1988); 114, 753(1989); 119, 2363–2366(1994)]. Sta tis tically, such a cor re la tion co ef fi cient ap plies only when both x and y are vari ables; a stan dard curve re quires that one pa ram e ter (con cen tra tion) be fixed (known).

When a high correlation is desired between the signal and concentration, use the symbol "r²" for the relationship as calculation by computer spreadsheet programs.

(27) Re cov ery (R) of analyte from for ti fied test ma te rial by a method of anal y sis. Frac tion of an analyte added to a test sam ple (for ti fied test sam ple) prior to anal y sis, which is mea sured (re cov ered) by the method. When the same an a lyt i cal method is used to an a lyze both the un for ti fied and for ti fied test sam ples, cal cu late per cent R as fol lows:

% Rec = C C C

F U

A

- ´100

where C_F = con cen tra tion of analyte mea sured in for ti fied test sam ple; C_U = con cen tra tion of analyte mea sured in un for ti fied test sam ple; C_A = con cen tra tion of analyte added to for ti fied test sam ple.

(Note: C_A is a cal cu lated value, not a value mea sured by the method be ing used.)

Con cen tra tion of added analyte should be no less than con cen tra tion of analyte in un for ti fied test sam ple. Sum of con cen tra tion of added analyte plus analyte pres ent be fore for ti fi ca tion should be in the same range as analyte con cen tra tion

sought in ac tual test sam ples. Ad di tion of analyte must not cause mea sur ing in stru ment to ex ceed lin ear dy namic range of stan dard curve. Both for ti fied and un for ti fied test sam ples must be treated iden ti cally dur ing anal y sis to min i mize ex per i men tal bias.

(28) Com mon safety pre cau tions are given in Ap pen dix B, Lab o ra tory Safety.

Re sults of Interlaboratory Study

(29) Users of methods should con sult the re port of the col lab o ra tive study (ref er ence given with the method) for de tails as to re sults of the interlaboratory study.

Ed i to rial Con ven tions

(30) For sim plic ity, the ab bre vi a tions Cl, H, I, N, and O are used rather than their di atomic forms. The charge may not be in di cated with the cor re spond ing ion where no am bi gu ity will re sult.

(31) Re agents and ap pa ra tus ref er enced with only a let ter, e.g., (c), will be found in the Re agent or Ap pa ra tus sec tion of the method.

(32) To con serve space, many ar ti cles and prep o si tions have been elim i nated.

Man u fac turers and Sup pliers

Many manufacturers and suppliers may be found by a search of the Internet. The same or equiv a lent prod ucts, in stru ments, sup plies, ap pa ra tus, or re agents avail able from man u fac tur ers and sup pli ers other than those named, or other brands from other sources, may serve equally well if proper val i da tion in di cates their use is sat is fac tory.

Ab bre vi a tions

(33) The fol low ing ab bre vi a tions, many of which con form with those of Chem i cal Ab stracts, are used. In gen eral, prin ci ple gov ern ing use of pe ri ods af ter ab bre vi a tions is that pe riod is used where fi nal let ter of ab bre vi a tion is not the same as fi nal let ter of word it rep re sents.

(20)

a Ab sorp tivi ty(ies)

A Absorbance(s) through out (not re stricted to for mu las; not ab sorp tion). A¢ is used for stan dard; A₀ is used for blank; 3 digit sub script

nu mer als usu ally de note wave length in nm

A Am pere

Å Ang strom

AA Atomic ab sorp tion

AACC Amer i can As so ci a tion of Ce real Chemists ACS Amer i can Chem i cal So ci ety

amu Atomic mass unit

AOCS Amer i can Oil Chem ists’ So ci ety APHA Amer i can Pub lic Health As so ci a tion ASBC Amer i can So ci ety of Brewing Chemists ASTM Amer i can So ci ety of Testing and Ma te rials

atm. At mo sphere

AU Absorbance units

AUFS Absorbance units full scale BAM Bac te ri o log i cal An a lyt i cal Man ual

Bé De gree Baumé

bp Boil ing point

Bq Becquerel

C De gree Celsius (Centigrade) ca About, ap prox i mately Cat. No. Cat a log num ber

CDC Cen ters for Dis ease Con trol and Prevention cfu Col ony form ing unit(s)

Ch Chap ter

Ci Cu rie(s) (= 3.7 ´ 10¹⁰ Bq)

CI Color index

CIPAC Col lab o ra tive In ter na tional Pes ti cide An a lyt i cal Coun cil

cm Cen ti me ter(s)

concn Concentration

cP Centipoise

cpm Counts per min ute

CRM Cer tified ref er ence material

*cu in. Cu bic inch(es)

dc Di rect cur rent

DMF N,N-dimethylformamide

DMSO Dimethyl sulfoxide

EDTA Ethylenedinitrilotetraacetic acid (or -tetraacetate)

EIA Enzyme immunoassay

ELISA En zyme linked immunosorbent assay EPA U.S. En vi ron men tal Pro tec tion Agency

Exp Ex po nen tial

F De grees Fahr en heit [°C = (5/9) ´ (°F – 32)]

FAO Food and Ag ri cul ture Or ga ni za tion FDA U.S. Food and Drug Ad min is tra tion FEP Fluorinated ethylene propylene

*fl oz Fluid ounce (29.54 mL)

Ab bre vi a tion Word

fp Freez ing point

FSD Full scale deflection

*ft Foot (30.48 cm)

g Gram(s)

g Grav ity (in cen tri fug ing)

*gal. Gal lon(s) (3.785 L) gr. Grain(s) (1 grain = 64.8 mg) GC Gas chro ma tog ra phy

h Hour(s)

HorRat Horwitz ratio

HPLC High per for mance liq uid chromatography ICC In ter na tional As so ci a tion for Ce real Sci ence and

Technology id In ner di am e ter IgG Im mu no glob u lin G

*in. Inch(es) (2.54 cm)

IR In fra red

ISO In ter na tional Or ga ni za tion for Stan dard iza tion

kg Ki lo gram(s)

kPa Kilo pas cal

L Li ter(s)

LC Liq uid chro ma tog ra phy

*lb Pound(s) (453.6 g)

m Me ter(s); milli—as pre fix

m Molal

M Mo lar (as ap plied to con cen tra tion), not molal

mA Mil li am pere(s)

mW Megaohm

min Min utes

min. Minimum

mg Mil li gram(s)

mL Mil li li ter(s)

mm Mil li me ter(s)

mp Melt ing point

MS Mass spec trom e ter (spec trom e try) MSDS Material Safety Data Sheet

(www.cdc.gov/niosh/ipcs/nicstart.html) mm Mil li mi cron (10^–6 mm); use nanometer (nm)

(10^–9 m)

mV Mil li volt

MW Mo lec u lar weight (mo lar mass)

*N Nor mal (as ap plied to con cen tra tion; in equa tions, nor mal ity of ti trat ing re agent)

N New ton (10⁵ dynes)

n Re frac tive in dex NF Na tional For mu lary

NFPA Na tional Food Pro ces sors As so ci a tion NIST Na tional In sti tute of Stan dards and Tech nol ogy

ng Nanogram (10^–9g)

nm Nanometer (10^–9 m); for