AOAC 2017 First Action Methods

AOAC INTERNATIONAL Official Methods Board (OMB)

2017 Methods Book (Awards) First Action April 30, 2020

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(x)  Refrigerator.— Capable of maintaining 2–8°C, for storing the 3M MDA components. (y)  Computer.— Compatible with the 3M ™ Molecular Detection Instrument. (z)  Modified TSB.— Available from Mediabox™ Actero™ (Calgary, Alberta, Canada). (aa)  OctoMACS™.— Milteny Biotec (Gaithersburg, MD, USA). (bb)  Multistand to support OctoMACS.— Milteny Biotec. (cc)  MACS ® large cell separation columns.— Milteny Biotec. (dd)  Dynabeads ® anti-E. coli O157 .—Dynal Inc. (Lake Success, NY, USA). (ee)  Tryptic soy agar with 5% sheep blood (SBA). —TS-BD (Franklin Lakes, NJ, USA); sheep blood (Quad Five, Ryegate, MO, USA). (ff)  Rainbow®Agar O157.— Dehydrated (Biolog Inc., Hayward, CA, USA). (gg)  Novobiocin .—5.0 mg/L (Alfa Aesar, Tewksbury, MA, USA). (hh)  Cefixime .—0.05 mg/L (Sigma-Aldrich, St. Louis, MO, USA). (ii)  Potassium tellurite .—0.15 mg/L (Oxoid, Hampshire, UK). (jj)  HCl.— 1 N (Acros Organics, Morris, NJ, USA). (kk)  E. coli O157:H7 latex agglutination test kit .—RIM® E. coli O157:H7 Latex test kit (REMEL, Lenexa, KS, USA). (ll)  3M™ Petrifilm™ Rapid Aerobic Count.— Available from 3M Food Safety. (mm)  3M™ Rapid Plate Spreader.— Available from 3M Food Safety. C. General Instructions (a)  Store the 3M MDA 2 – E. coli O157 (including H7) at 2–8°C. Do not freeze. Keep kit away from light during storage. After opening the kit, check that foil pouch is undamaged. If pouch is damaged, do not use. After opening, unused reagent tubes should always be stored in the resealable pouch with the desiccant inside to maintain stability of the lyophilized reagents. Store resealed pouches at 2–8°C for no longer than 60 days. Do not use 3M MDA 2 – E. coli O157 (including H7) past the expiration date. (b)  Follow all instructions carefully. Failure to do so may lead to inaccurate results. The 3MMDA2 – E. coli O157 (including H7) is intended for use in a laboratory environment by professionals trained in laboratory techniques. 3M has not documented the use of this product in industries other than the food and beverage industries. For example, 3M has not documented this product for testing drinking water, pharmaceutical, cosmetics, clinical or veterinary samples. The 3M MDA 2 – E. coli O157 (including H7) has not been evaluated with all possible food products, food processes, testing protocols, or with all possible strains of bacteria. As with all test methods, the source of enrichment medium can influence the results. The 3M MDA 2 – E. coli O157 (including H7) has only been evaluated for use with the enrichment media specified in the Instructions for Use section. The 3M™ Molecular Detection instrument is intended for use with samples that have undergone heat treatment during the assay lysis step, which is designed to destroy organisms present in the sample. Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3M MDS instrument. The user should read, understand, and follow all safety information in the instructions for the 3M MDS and the 3M

AOAC Official Method 2017.01 Escherichia coli O157:H7 in Selected Foods 3M ™ Molecular Detection Assay (MDA) 2 – E. coli O157 (Including H7) Method First Action 2017 [Applicable to detection of Escherichia coli O157 (including H7) in raw ground beef (73% lean), frozen blueberries, fresh bean sprouts, and fresh baby spinach.] See Table 2017.01A for a summary of results of the interlaboratory study. See Table 2017.01B for detailed results of the interlaboratory study. A. Principle The 3M ™ MDA 2 – E. coli O157 (including H7) method is used with the 3M ™ Molecular Detection System (MDS) for the rapid and specific detection of E. coli O157 (including H7) in enriched food samples. The 3M MDA 2 – E. coli O157 (including H7) uses loop-mediated isothermal amplification of unique DNA target sequences with high specificity and sensitivity, combined with bioluminescence to detect the amplification. Presumptive positive results are reported in real-time while negative results are displayed after the assay is completed, 60 min. Samples are enriched in 3M ™ Buffered Peptone Water (BPW; ISO formulation). B. Apparatus and Reagents Items (a)–(g) are available as the 3M ™ MDA 2 – E. coli O157 (including H7) kit from 3M Food Safety (St. Paul, MN, USA). (a)  3M MDS.— MDS100. (b)  3M MDA 2 – E. coli O157 (including H7) .—Cat. No. MDA2ECO96, reagent tubes: 12 strips of eight tubes. (c)  Lysis solution (LS) tubes .—Twelve strips of eight tubes. (d)  Extra caps .—Twelve strips of eight caps. (e)  Reagent control .—Eight reagent tubes. (f)  Quick Start Guide. (g)  3M Molecular Detection Speed loader tray. (h)  3M Molecular Detection Chill Block insert.— Available from 3M Food Safety. (i)  3M Molecular Detection Heat Block insert.— Available from 3M Food Safety. (j)  3M Molecular Detection cap/decap tool for reagent tubes.— Available from 3M Food Safety. (k)  3M Molecular Detection cap/decap tool for lysis tubes.— Available from 3M Food Safety. (l)  Empty lysis tube rack.— Available from 3M Food Safety. (m)  Empty reagent tube rack.— Available from 3M Food Safety. (n)  BPW (ISO formulation) .—Formulation equivalent to ISO 6579:2002 Annex B or 3M equivalent. (o)  Disposable pipet .—Capable of 20 µL. (p) Multichannel (8-channel) pipet.— Capable of 20 µL. (q)  Pipet tips.— Sterile filter tip, capable of 20 µL. (r)  Filter Stomacher® bags.— Seward (Islandia, NY, USA) or equivalent . (s)  Smasher® XL bags.— Available from bioMérieux Industry (Hazelwood, MO, USA). (t)  Stomacher.— Seward or equivalent. (u)  Partial immersion thermometer.— Calibrated range to include 100 ± 1°C. (v)  Dry block heater unit.— Capable of maintaining 100 ± 1°C. (w)  Incubators.— Capable of maintaining 37 ± 1°C or 41.5 ± 1°C.

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Table 2017.01A. Summary of results for the detection of E. coli O157:H7 in raw ground beef (325 g) Method a

3M MDA 2 – E. coli O157 (including H7)

Inoculation level

Uninoculated

Low

High

Candidate presumptive positive/total No. of samples analyzed

0/120

32/120

97/120

Candidate presumptive POD (CP)

0.00

0.27

0.81

(0.00, 0.03)

(0.19, 0.35)

(0.67, 0.93)

s r

0.00

0.45

0.37

b

(0.00, 0.17)

(0.40, 0.52)

(0.33, 0.42)

s L

0.00

0.00

0.15

c

(0.00, 0.17)

(0.00, 0.16)

(0.07, 0.32)

s R

0.00

0.45

0.40

d

(0.00, 0.24)

(0.40, 0.52)

(0.35, 0.50)

P value e

1.00

0.760

0.005

Candidate confirmed positive/total No. of samples analyzed

3/120 f

31/120

96/120

Candidate confirmed POD (CC) g

0.03

0.26

0.80

(0.01, 0.07)

(0.18, 0.34)

(0.69, 0.91)

s r

0.15

0.45

0.38

(0.14, 0.18)

(0.40, 0.52)

(0.34, 0.44)

s L

0.03

0.00

0.13

(0.00, 0.09)

(0.00, 0.13)

(0.04, 0.29)

s R

0.16

0.45

0.40

(0.14, 0.19)

(0.40, 0.51)

(0.36, 0.49)

P value

0.12

0.920

0.018

Candidate confirmed positive/total No. of samples analyzed Candidate presumptive positive that confirmed POD (C) h

0/120

29/120

92/120

0.00

0.24

0.77

(0.00, 0.03)

(0.16, 0.32)

(0.63, 0.91)

s r

0.00

0.44

0.39

(0.00, 0.17)

(0.39, 0.51)

(0.34, 0.45)

s L

0.00

0.00

0.18

(0.00, 0.17)

(0.00, 0.13)

(0.09, 0.37)

s R

0.00

0.44

0.43

(0.00, 0.24)

(0.39, 0.50)

(0.38, 0.52)

P value

1.00

0.908

0.002

Positive reference samples/total No. of samples analyzed

3/120 i

42/120

95/120

Reference POD

0.03

0.35

0.79

(0.01, 0.07)

(0.26, 0.44)

(0.67, 0.92)

s r

0.14

0.48

0.38

(0.00, 0.17)

(0.42, 0.52)

(0.34, 0.44)

s L

0.07

0.05

0.15

(0.03, 0.14)

(0.00, 0.23)

(0.07, 0.33)

s R

0.16

0.48

0.41

(0.00, 0.20)

(0.43, 0.52)

(0.37, 0.51)

P Value

0.001

0.342

0.01

© 2018 AOAC INTERNATIONAL

Table 2017.01A. ( continued ) Method a

3M MDA 2 – E. coli O157 (including H7)

Inoculation level

Uninoculated

Low

High

dLPOD (candidate vs. reference) j

–0.025

–0.11

–0.025

(–0.071, 0.010)

(–0.22, 0.01)

(–0.129, 0.080)

dLPOD (candidate presumptive vs. candidate confirmed) j

–0.025

0.01

0.008

(–0.071, 0.010)

(–0.10, 0.12)

(–0.092, 0.109)

a Results include 95% confidence intervals. b r = Repeatability standard deviation. c Among-laboratory standard deviation. d Reproducibility standard deviation. e  P value = Homogeneity test of laboratory PODs. f 16s rRNA identified one of the reported positives as a non- E. coli. g Confirmed positive (including false negatives). h Presumptive positives that confirmed (excluding false negatives). i 16s rRNA identified all three isolates as non- E. coli. j A confidence interval for dLPOD that does not contain the value 0 indicates a statistical significant difference between the two methods.

MDA 2 – E. coli O157 (including H7). Retain safety instructions for future reference. Periodically decontaminate laboratory benches and equipment (pipets, cap/decap tools, etc.) with a 1–5% (v/v in water) household bleach solution or DNAremoval solution. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the Safety Data Sheet for additional information and local regulations for disposal. To reduce risks associated with exposure to chemicals and biohazards, perform pathogen testing in a properly equipped laboratory under the control of trained personnel. Incubated enrichment media and equipment or surfaces that have come into contact with incubated enrichment media may contain pathogens at levels sufficient to cause risk to human health. Always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples. Avoid contact with the contents of the enrichment media and reagent tubes after amplification. Dispose of enriched samples according to current industry standards. To reduce the risks associated with environmental contamination, follow current industry standards for disposal of contaminated waste. D. Sample Enrichment: Foods (a)  Allow BPW ISO enrichment medium to pre-warm to 41.5 ± 1°C. See Table 2017.01C for matrix-specific enrichment protocols. (b)  Aseptically combine enrichment medium and sample. For all meat and highly particulate samples, the use of filter bags is recommended. (c)  Homogenize by stomacher, hand, or gentle agitation thoroughly for 2 ± 0.2 min. Incubate matrices according to the instructions provided in Table 2017.01C .

E. Preparation of the 3M™ Molecular Detection Speed Loader Tray (a)  Wet a cloth or paper towel with a 1–5% (v/v in water) household bleach solution and wipe the 3M™Molecular Detection Speed Loader Tray. (b)  Rinse the 3M Molecular Detection Speed Loader Tray with water. (c)  Use a disposable towel to wipe the 3M Molecular Detection Speed Loader Tray dry. (d)  Ensure the 3M Molecular Detection Speed Loader Tray is dry before use. F. Preparation of the 3M™ Molecular Detection Chill Block Insert Place the 3M Molecular Detection Chill Block Insert directly on the laboratory bench: The 3M Molecular Detection Chill Block Tray is not used. Use the block at ambient laboratory temperature (20–25°C). G. Preparation of the 3M Molecular Detection Heat Block Insert Place the 3M Molecular Detection Heat Block Insert in a dry double block heater unit. Turn on the dry block heater unit and set the temperature to allow the 3M Molecular Detection Heat Block Insert to reach and maintain a temperature of 100 ± 1°C. Note: Depending on the heater unit, allow approximately 30 min for the 3M Molecular Detection Heat Block Insert to reach temperature. Using an appropriate, calibrated thermometer (e.g., a partial immersion thermometer, digital thermocouple thermometer, not a total immersion thermometer) placed in the designated location, verify that the 3M Molecular Detection Heat Block Insert is at 100 ± 1°C. H. Preparation of the 3M Molecular Detection Instrument (a)  Launch the 3M™ Molecular Detection Software and log in. Contact your 3M Food Safety representative to ensure you have the most updated version of the software.

© 2018 AOAC INTERNATIONAL

Table 2017.01B. Comparative results for the detection of E. coli O157:H7in raw ground beef (73% lean) by the 3M MDA 2 – E. coli O157 (including H7) method vs USDA/FSIS MLG Chapter 5.09 in a collaborative study Uninoculated control Candidate presumptive (CP) Candidate confirmed (CC) a Candidate result (C) b Reference method (R) C vs R Statistic Matrix/inoculation level Lab N c X d POD (CP) N X POD (CC) N X POD (C) N X POD (R) dLPOD (C,R) Raw ground beef (73% lean)/uninoculated 1 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 2 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 3 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 12 0 0.00 NA 0 0.00 NA 0 0.00 12 0 0.00 0.00 0.00 5 12 0 0.00 12 2 0.167 12 0 0.00 12 0 0.00 0.00 0.00 6 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 dLPOD (CP,CC)

7 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 9 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00

10 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 11 12 0 0.00 12 1 f 0.08 12 0 0.00 12 0 0.00 0.00 -0.08 12 12 0 0.00 12 0 0.00 12 0 0.00 12 3 f 0.25 -0.25 0.00

13 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 14 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00

15 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Estimate All 120 0 0.00 120 3 0.03 120 0 0.00 120 3 0.03 –0.025 –0.025 LCL g 0.00 0.01 0.00 0.01 –0.071 –0.071 UCL h 0.03 0.07 0.03 0.07 0.010 0.010 s r i 0.00 0.15 0.00 0.14 LCL 0.00 0.14 0.00 0.13 UCL 0.17 0.18 0.17 0.17 s L j 0.00 0.03 0.00 0.07

LCL 0.00 0.00 0.00 0.03

UCL 0.17 0.09 0.17 0.14

s R k 0.00 0.16 0.00 0.16

UCL 0.00 0.14 0.00 0.14

LCL 0.24 0.19 0.24 0.20

P T l 1.00 0.122 1.00 0.001

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2 12 5 0.417 12 3 0.250 12 3 0.250 12 5 0.417 –0.167 0.167 3 a NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 12 2 0.167 NA 2 0.167 NA 2 0.167 12 4 0.333 –0.167 0.00

5 12 2 0.167 NA 2 0.167 NA 2 0.167 NA 4 0.333 –0.167 0.00 6 12 5 0.417 12 5 0.417 12 5 0.417 12 7 0.583 –0.167 0.00 7 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 12 3 0.250 12 3 0.250 12 3 0.250 12 1 0.083 0.167 0.00

9 12 3 0.250 12 3 0.250 12 3 0.250 12 4 0.333 –0.083 0.00 10 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

MPN/test portion 11 12 4 0.333 12 3 0.250 12 3 0.250 12 4 0.333 –0.083 0.083 12 12 4 0.333 12 4 0.333 12 4 0.333 12 5 0.417 –0.083 0.00 13 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

14 12 2 0.167 12 4 0.333 12 2 0.167 12 6 0.500 –0.333 –0.167 15 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA Estimate All 120 32 0.27 120 31 0.26 120 29 0.24 120 42 0.35 –0.11 0.01 LCL 0.29 0.19 0.18 0.16 0.26 –0.22 –0.10 UCL –0.09 0.35 0.34 0.32 0.44 0.01 0.12 s r 0.55 0.45 0.45 0.44 0.48 LCL 0.40 0.40 0.39 0.42 UCL 0.52 0.52 0.51 0.52 s L 0.00 0.00 0.00 0.05 LCL 0.00 0.00 0.00 0.00 UCL 0.16 0.13 0.13 0.23 s R 0.45 0.45 0.44 0.48 UCL 0.40 0.40 0.39 0.43 LCL 0.52 0.51 0.50 0.52 P T 0.760 0.920 0.908 0.342

Candidate presumptive (CP) Candidate confirmed (CC) a Candidate result (C) b Reference method (R) C vs R POD (CP) N X POD (CC) N X POD (C) N X POD (R) dLPOD (C,R) Raw ground beef (73% lean)/low 1 12 2 0.167 12 2 0.167 12 2 0.167 12 2 0.167 0.00 0.00 Matrix/inoculation level Lab N c X d dLPOD (CP,CC)

(Low inoculum level)

Table 2017.01B. ( continued )

Statistic

© 2018 AOAC INTERNATIONAL

3 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

5 12 8 0.667 12 8 0.667 12 6 0.500 12 10 0.833 –0.333 0.00 6 12 12 1.000 12 12 1.000 12 12 1.000 12 11 0.917 0.083 0.00 7 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

8 12 11 0.917 12 11 0.917 12 11 0.917 12 12 1.000 –0.083 0.00 9 12 11 0.917 12 11 0.917 12 11 0.917 12 11 0.917 0.00 0.00 10 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA

MPN/test portion 11 12 7 0.583 12 8 0.667 12 6 0.500 12 10 0.833 –0.333 –0.083 12 12 9 0.750 12 9 0.750 12 9 0.750 12 4 0.333 0.417 0.00 13 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA 14 12 9 0.750 12 8 0.667 12 8 0.667 12 9 0.750 –0.083 0.083 15 e NA NA NA NA NA NA NA NA NA NA NA NA NA NA Estimate All 120 97 0.81 120 96 0.80 120 92 0.77 120 95 0.79 –0.025 0.008 LCL 1.92 0.67 0.69 0.63 0.67 –0.129 –0.092 UCL –1.00 0.93 0.91 0.91 0.92 0.080 0.109 s r 3.42 0.37 0.38 0.39 0.38 LCL 0.33 0.34 0.34 0.34 UCL 0.42 0.44 0.45 0.44 s L 0.15 0.13 0.18 0.15 LCL 0.07 0.04 0.09 0.07 UCL 0.32 0.29 0.37 0.33 s R 0.40 0.40 0.43 0.41 UCL 0.35 0.36 0.38 0.37 LCL 0.50 0.49 0.52 0.51 P T 0.005 0.018 0.002 0.01

Raw ground beef (73% lean)/high 1 12 6 0.500 12 6 0.500 12 6 0.500 12 8 0.667 –0.167 0.00 2 12 12 0.917 12 11 0.917 11 11 0.917 12 11 0.917 0.00 0.083

4 12 12 1.000 12 12 1.000 12 12 1.000 12 9 0.750 0.250 0.00

dLPOD

(CP,CC)

Candidate presumptive (CP) Candidate confirmed (CC) a Candidate result (C) b Reference method (R) C vs R POD (CP) N X POD (CC) N X POD (C) N X POD (R) dLPOD (C,R)

High inoculum level

level Lab N c X d

Table 2017.01B. ( continued )

Matrix/inoculation

Statistic

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Table 2017.01C. 3M MDA 2 – E. coli O157 (including H7) enrichment protocols using prewarmed BPW ISO at 41.5 ± 1°C according to 2017.01 Sample matrix Sample size, g Enrichment broth volume, mL Enrichment time, h Homogenized Raw ground beef (73% lean) 325 975 10–18 Manually by hand or by stomaching Raw bagged spinach a 200 450 18–24 Gently agitated by hand for 5 min, do not stomach Fresh bean sprouts 25 225 18–24 Gently agitated by hand for 5 min, do not stomach Frozen blueberries a,b 25 225 18–24 Gently agitated by hand for 5 min, do not stomach

e  NA = Laboratory did not submit data for this matrix because of laboratory error. f  16S rRNA results identified isolates as non- E. coli . g  LCL = Lower confidence limit. h  UCL = Upper confidence limit. i  Repeatability standard deviation. j  Among-laboratory standard deviation. k  Reproducibility standard deviation. l  P T value = Homogeneity test of laboratory PODs.

Table 2017.01B. ( continued ) a CC = Number of confirmed positives (including false negatives).

b C = Presumptive positives that confirmed positive (excluding false negatives). c N = Samples analyzed. d X = Number of samples that obtained a positive.

a Leafy produce and fruit samples should be gently agitated by hand for 5 min. Do not blend or stomach. b Frozen samples should be equilibrated to 4-8°C before addition to enrichment broth.

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(b)  Turn on the 3M Molecular Detection Instrument. (c)  Create or edit a run with data for each sample. Refer to the 3M MDS User Manual for details. Note : The 3M Molecular Detection Instrument must reach and maintain temperature of 60°C before inserting the 3M Molecular Detection Speed Loader Tray with reaction tubes. This heating step takes approximately 20 min and is indicated by an ORANGE light on the instrument’s status bar. When the instrument is ready to start a run, the status bar will turn GREEN. I. Lysis (a)  Allow the LS tubes to warm up by setting the rack at room temperature (20–25°C) overnight (16–18 h). Alternatives to equilibrate the LS tubes to room temperature are to set the LS tubes on the laboratory bench for at least 2 h, incubate the LS tubes in a 37 ±1°C incubator for 1 h, or place them in a dry double block heater for 30 s at 100°C. (b)  Invert the capped tubes to mix. Proceed to next step within 4 h. (c)  Remove the enrichment broth from the incubator. (d)  One LS tube is required for each sample and the negative control (NC) sample (sterile enrichment medium). (1)  LS tube strips can be cut to desired LS tube number. Select the number of individual LS tubes or 8-tube strips needed. Place the LS tubes in an empty rack. (2)  To avoid cross-contamination, decap one LS tubes strip at a time and use a new pipet tip for each transfer step. (3)  Transfer enriched sample to LS tubes as follows: Transfer each enriched sample into individual LS tube first. Transfer the NC last. (4)  Use the 3M™Molecular Detection Cap/Decap Tool-Lysis to decap one LS tube strip, one strip at a time. (5)  Discard the LS tube cap. If lysate will be retained for retest, place the caps into a clean container for reapplication after lysis. (6)  Transfer 20 µL of sample into an LS tube unless otherwise indicated in the protocol table. (e)  Repeat step I(d) ( 2 ) until each individual sample has been added to a corresponding LS tube in the strip (Figure 2017.01A ). (f)  Repeat steps I(d) ( 1 ) – ( 6 ) as needed, for the number of samples to be tested. (g)  When all samples have been transferred, transfer 20 µL of NC (sterile enrichment medium, e.g., BPW) into an LS tube. Do not use water as an NC. (h)  Verify that the temperature of the 3M Molecular Detection Heat Block Insert is at 100 ± 1°C. (i)  Place the uncovered rack of LS tubes in the 3M Molecular Detection Heat Block Insert and heat for 15 ± 1 min. During heating, the LS solution will change from pink (cool) to yellow (hot). Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3M Molecular Detection Instrument. (j)  Remove the uncovered rack of LS tubes from the heating block and allow to cool in the 3M Molecular Detection Chill Block

Insert at least 5 min and a maximum of 10 min. The 3M Molecular Chill Block Insert, used at ambient temperature without the 3M Molecular Detection Chill Block Tray, should sit directly on the laboratory bench. When cool, the lysis solution will revert to a pink color. (k)  Remove the rack of LS tubes from the 3M Molecular Detection Chill Block Insert (Figure 2017.01B ). J. Amplification (a)  One reagent tube is required for each sample and the NC. (1)  Reagent tubes strips can be cut to desired tube number. Select the number of individual reagent tubes or 8-tube strips needed. ( 2 ) Place reagent tubes in an empty rack. (3)  Avoid disturbing the reagent pellets from the bottom of the tubes. (b)  Select one reagent control (RC) tube and place in rack. (c)  To avoid cross-contamination, decap one reagent tube strip at a time and use a new pipet tip for each transfer step. (d)  Transfer lysate to reagent tubes and RC tube as follows: Transfer each sample lysate into individual reagent tubes first followed by the NC. Hydrate the RC tube last. (1)  Use the 3M Molecular Detection Cap/Decap Tool-Reagent to decap the reagent tubes, one reagent tubes strip at a time. Discard cap. (2)  Transfer 20 µL of sample lysate from the upper half of the liquid (avoid precipitate) in the LS tube into corresponding reagent tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down five times. (3)  Repeat step J(d) (2) until individual sample lysate has been added to a corresponding reagent tube in the strip. (4)  Cover the reagent tubes with the provided extra cap and use the rounded side of the 3M Molecular Detection Cap/Decap Tool- Reagent to apply pressure in a back and forth motion ensuring that the cap is tightly applied. (5)  Repeat steps J(d) (1)–(4) as needed, for the number of samples to be tested. (6)  When all sample lysates have been transferred, repeat J(d) (1)–(4) to transfer 20 µL of NC lysate into a reagent tube. (7)  Transfer 20 µL of NC lysate into an RC tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down five times. (d)  Load capped tubes into a clean and decontaminated 3M Molecular Detection Speed Loader Tray. Close and latch the 3M Molecular Detection Speed Loader Tray lid (Figure 2017.01C ). (e)  Review and confirm the configured run in the 3M Molecular Detection Software. (f)  Click the Start button in the software and select instrument for use. The selected instrument’s lid automatically opens.

Figure 2017.01A

Figure 2017.01B

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K. Results and Interpretation An algorithm interprets the light output curve resulting from the detection of the nucleic acid amplification. Results are analyzed automatically by the software and are color-coded based on the result. A positive or negative result is determined by analysis of a number of unique curve parameters. Presumptive positive results are reported in real-time while negative and inspect results will be displayed after the run is completed. Presumptive positive samples should be confirmed as per the laboratory standard operating procedures or by following the appropriate reference method confirmation beginning with transfer from the primary BPW ISO enrichment to secondary enrichment broth(s), followed by subsequent plating and confirmation of isolates using appropriate biochemical and serological methods. Note : Even a negative sample will not give a zero reading as the system and 3MMDA2 – E. coli O157 (including H7) amplification reagents have a “background” relative light unit (RLU) reading. In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M recommends the user to repeat the assay for any inspect samples. If the result continues to be inspect, proceed to confirmation test using your preferred method or as specified by local regulations. For questions about specific applications or procedures, visit www.3M.com/foodsafety or contact your local 3M representative or distributor. Reference: J. AOAC Int. (future issue) Posted: March 2018

Figure 2017.01C

(g)  Place the 3M Molecular Detection Speed Loader Tray into the 3M MDS Instrument and close the lid to start the assay. Results are provided within 60 min, although positives may be detected sooner. (h)  After the assay is complete, remove the 3M Molecular Detection Speed Loader Tray from the 3M Molecular Detection Instrument and dispose of the tubes by soaking in a 1–5% (v/v in water) household bleach solution for 1 h and away from the assay preparation area. Note : To minimize the risk of false positives due to cross- contamination, never open reagent tubes containing amplified DNA. This includes RC, reagent, and matrix control tubes. Always dispose of sealed reagent tubes by soaking in a 1–5% (v/v in water) household bleach solution for 1 h and away from the assay preparation area.

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place in microwave. With power setting appropriate to microwave model and number of vessels used, ramp temperature from ambient to 200 ° C in 15 min. Hold at 200 ° C for 20 min. Cool vessels according to manufacturer’s directions, vent, and transfer digests to 100 mL volumetric flasks. Rinse each digestion vessel three times with approximately 10 mL water, C ( a ), and transfer rinse solution to the volumetric flask; dilute flask to volume with water, C ( a ), and mix. Filtering the digestate is optional, but necessary if problems with nebulizer clogging are experienced. Transfer to polypropylene, or other suitable, containers within 2 h, unless solutions are to be analyzed immediately. Dilute any digestates that are found to be above the standard curve range. Secondary dilutions require addition of appropriate amounts of HNO 3 and HCl to maintain the proportion of 9% HNO 3 and 3% HCl in the final solution to be analyzed. Detection Method E. Principle Digested test solution, or an appropriate dilution, is presented to the inductively coupled plasma-optical emission spectrometry (ICP- OES) instrument calibrated with acid matched standard calibrant solutions. An ionization buffer (cesium) is used to minimize easily ionized element (EIE) effects, and scandium and/or beryllium are used as internal standard(s). F. Instrumentation and Configuration ( a )  ICP optical emission spectrometer .—Capable of determining multiple wavelengths for each element of interest. A 3-channel peristaltic pump is desirable to avoid the necessity of having to manually add ionization buffer and internal standard to each test solution. Use a Meinhard or Seaspray nebulizer and Cyclonic spray chamber, or other components designed to optimize aerosol formation and maximize precision. Select sample and internal standard pump tubes, and peristaltic pump rotation speed, with regard to manufacturer’s recommendations, but try to keep sample and internal standard pump tubes of similar size, to maximize mixing accuracy, while maintaining needed detection levels. The analyst must compensate for EIE effects in the plasma since fertilizer materials can contain substantial concentrations of elements that provide a significant source of electrons to the plasma, such as K and Ca. The presence of an ionization buffer in all test solutions and standards will minimize the effect of varying concentrations of EIEs in the sample. Power settings and nebulizer gas flow should be optimized for robust plasma conditions. The analyst needs to ensure that the Mg 285.213:Mg 280.271 ratio (Mermet principle of robust plasma) demonstrates robust operating conditions in accordance with the ratio established by the instrument manufacturer. Two to three replicate readings with relatively long integration times are recommended to improve precision and detection capabilities. Properly matched test solution and calibration matrices and optimized instrument settings should result in internal standard ratios for most test solutions consistently in the range of 0.9 to 1.1. It is not typical to have the ratio lower than 0.8 over a very wide range of fertilizer material types. The occurrence of lower ratios is cause for troubleshooting. Select ionization buffer/internal standard solution, G ( i ), such that after mixing unknown and internal standard solutions using the instrument’s peristaltic pump, the combined solution presented to the nebulizer contains ≥2200 mg/kg cesium chloride; 0.75 to 1.0 mg/kg internal standard; and ≤7.2 mg/mL actual fertilizer material. [For example, these conditions would be met with a 1 g test portion digested and diluted to 100 mL; an ionization

AOAC Official Method 2017.02 Arsenic, Cadmium, Calcium, Chromium, Cobalt, Copper, Iron, Lead, Magnesium, Manganese, Molybdenum, Nickel, Selenium, and Zinc in Fertilizers Microwave Acid Digestion and ICP-OES Detection First Action 2017 The method is a modification and extension of 2006.03 ( see 2.6.35). [Applicable to the determination of As, Cd, Co, Cr, Pb, Mo, Ni, and Se and to the determination of Ca, Cu, Fe, Mg, Mn, and Zn in all classes of fertilizers.] Caution : Observe standard precautions when handling concentrated acids and acid digests.When dispensing acid or venting vessels, use gloves, eye and face protection, and a laboratory coat. Never remove hot vessels from the microwave; wait until they are near room temperature. Keep microwave door closed while vessels are hot. The door is the primary safety device if a vessel vents. Digestion Method A. Principle Test portion is heated with either nitric acid (option 1) or with nitric and hydrochloric acids (option 2) in a closed vessel microwave digestion system at 200 ° C. B. Apparatus Microwave .—Commercial microwave designed for laboratory use at 200°C, with closed vessel system and controlled temperature ramping capability. It is recommended that a vessel design be selected that will withstand the maximum possible pressure, since some organic fertilizer products, and also carbonates if not given sufficient time to predigest, will generate significant pressure during digestion. (Vessels can reach 700 psi or more on occasion.) Vent according to manufacturer’s recommendation. ( Caution : Microwave operation involves hot pressurized acid solutions. Use appropriate face protection and laboratory clothing.) C. Reagents (Option 1 Applicable to Group A Metals Only): Nitric Acid Digestion ( a )  Water .—Use 18 Megaohm water throughout. ( b )  Concentrated HNO 3 .—Use trace metal grade HNO 3 throughout (nitric acid – HNO 3 , 67 – 70%, OmniTrace grade; EMD Chemicals, Darmstadt, Germany). D. Determination ( a )  Option 1 (applicable to Group Ametals only) .— See 2006.03D ( see 2.6.35). ( b )  Option 2 (applicable to Group A and Group B metals) .— Prepare solid materials according to 929.02 ( see 2.1.05). Accurately weigh 1.000 ± 0.10 g (0.500 g for organic matrixes) test portion and transfer to digestion vessel. Use a weighing paper insert to line the vessel walls during transfer, which will keep test portion from adhering to the sides of vessel. Fluid materials may be weighed directly after mixing. Add 9.0 ± 0.2 mL trace metal grade HNO 3 , G ( b ), allow samples to sit for approximately 20 min, and then add 3.0 ± 0.2 mL HCl, G ( c ). Loosely cap vessels without sealing, predigest at room temperature until vigorous foaming subsides, or overnight if time allows. Seal vessels according to manufacturer’s directions and

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and when an interferent is present in a particular line, the result for that line is omitted from the mean value reported. ( 2 ) Some ICP software has the capability of mathematically modeling potential interferents and deconvoluting the instrument response into an analytical element portion and an interferent portion. ( 3 ) Interelement correction is an alternative mathematical technique to use with instruments for which mathematic modeling is not available, or where direct spectral overlap negates use of the deconvolution technique. The following lines, if used, must utilize one of the correction techniques; corrections for other wavelengths may be applied as needed and appropriate: ( a ) As 188.980: Correct for Cr interference, or verify that Cr is not present at an interfering level in the test portion analyzed. ( b ) As 193.696: Fe affects the arsenic peak. Remove with an Fe model, or verify that Fe is not present at an interfering level in the test portion analyzed. ( c ) Cd 214.439 and 226.502: Fe, present in many fertilizers, interferes with both suggested Cd wavelengths. Mathematically correct instrument Cd response for the interference, or verify analytically that Fe is not present at an interfering level in the test portion analyzed. ( d ) Pb 220.353: Mathematically correct instrument Pb response for Fe interference, or verify that Fe is not present at an interfering level in the test portion analyzed. ( e ) Se 196.026: Mathematically correct instrument Se response for Fe interference, or verify that Fe is not present at an interfering level in the test portion analyzed. ( d )  ICP instrument calibration .—Prepare Group A working standard solutions from 1000 mg/L commercial stock standards. Custom blended multielement stock standard in an acid ratio (9% HNO 3 :3% HCl) is acceptable. Working standards should be prepared at concentrations listed in Table  2017.02B , if they fit the sensitivity of the available instrumentation. Calibration concentrations should be adjusted to match the sensitivity of an instrument. However, linear curves should have correlation coefficients of at least 0.999 and a standard error of no more than 10%. Quadratic calibrations should have a correlation coefficient of at least 0.999, a standard error of less than 10%, a curvature of no more than 25%, and an upward curvature of no more than 400%. G. Reagents (Option 2): Dual Acid Digestion ( a )  Water .—18 Megaohm water. ( b )  HNO 3 .—Trace metal grade HNO 3 (nitric acid – HNO 3 , 67 – 70%, OmniTrace grade; EMD Chemicals). ( c )  HCl .—Trace metal grade HCl (hydrochloric acid – HCl, 35 – 38%, trace metal grade; Cat. No. A508-500, Fisher Scientific, Pittsburgh, PA, USA ). ( d )  Triton X-100 solution .—Triton X-100 .— Octyl phenol ethoxylate (J.T. Baker Chemicals, Center Valley, PA, USA). ( e )  0.5% Triton X-100 solution .—Dilute 0.5 mL Triton X-100, G ( d ), to 100 mL with H 2 O, G ( a ). ( f )  Cesium chloride .—Formula weight 168.36, trace metal basis, purity >99.999%, Cat. No. 203025-50G (Sigma-Aldrich, St. Louis, MO, USA). ( g )  1000 mg/L Sc standard .—In 4%HNO 3 , Product No. 100048-1 (High Purity Standards, Charleston, SC, USA). ( h )  1000 mg/L Be standard .—In 4% HNO 3 , Product No. 1005-1 (High Purity Standards). ( i )  Ionization buffer/internal standard solution .—Weigh 8.0 g CsCl, G ( f ), into a 1000 mL acid-washed volumetric flask. Add 3 mL each of ICP grade scandium, G ( g ), and beryllium, G ( h ), 1000 mg/L stock solution, as internal standards. Also add 1 mL of 0.5% Triton X-100, G ( e ), dilute to volume, and mix. Store in a polypropylene bottle. ( Note : Reagent concentrations assume the use

Table 2017.02A. Recommended inductively coupled plasma- optical emission spectrometry wavelengths for Group A and B

metals Element

Wavelength(s)

As Ca Cd Co Cu Fe Mg Mn Mo Cr

188.980 a , 193.696 a

183.944, 318.127, 430.253 214.439 a , 226.502 a , 228.802 228.615, 230.786, 258.033 205.560, 267.716, 276.653

217.895, 222.778, 324.754, 327.395 234.350, 238.204, 240.489, 259.837 277.983, 278.297, 285.213, 383.829 260.568, 261.815, 263.817, 293.931

202.032, 204.598

Ni

216.555, 222.486, 231.604

Pb Se Zn

220.353 a 196.026 a

206.200, 213.857, 334.502, 472.215

a  Wavelengths with potential spectral interference.

buffer/internal standard solution of 8000 mg/kg cesium chloride and 3 mg/kg scandium and/or beryllium internal standard(s); and pump tubes of white/white (1.02 mm id) sample and orange/white (0.64 mm id) internal standard, the white/white contributing about 72%, and the orange/white contributing about 28%, to the final nebulized solution.] All analytical wavelengths should be corrected using an internal standard wavelength. However, best practice is to utilize similar transitions between analyte and internal standard. For example, the 188.980 wavelength is from arsenic in the atomic state, so the internal standard wavelength used for correction should also be from the atomic state, such as Sc 361.383. Conversely, match ionic sample lines with ionic internal standard lines. ( Note : Do not use yttrium as an internal standard, since it is found native at low levels in some phosphate ore sources.) ( b )  ICP wavelengths .—A number of wavelengths may be used for analysis of the elements of interest, depending on the capability of the analytical instrument used. At a minimum, select at least two wavelengths for each element of interest, and report the averaged value of closely agreeing results, with the exception of lead and selenium, for which there is only one reliable wavelength available. Table 2017.02A provides a list of suggested wavelengths, not in any order of preference, that have been found acceptable formost fertilizer materials. Other lines of appropriate sensitivity free of interferences or corrected for interferences may be just as acceptable. However, it is imperative that instrument response (both the wavelength peak scan and the calculated concentration) be reviewed for each test solution and element. Fertilizer materials are extremely variable in composition, and a wide concentration range of potential interfering elements is expected, so no single wavelength will work in every instance. Occasionally, data with an interference will inevitably be found and must be eliminated from inclusion in the mean calculation result for that particular element and sample. ( c )  Wavelength interference treatment.— Interelement interference can cause substantial error in analytical result. Error can be minimized by several techniques: ( 1 ) Three or more analytical lines may be used for a given element,

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Table 2017.02B. Preparation of working standards

Final working std concn, mg/L

Intermediate std, G ( l ), mL

Stock std, G ( m ), mL

HNO 3 a , G ( b ), mL

HCl a , G ( c ), mL

Std

Final vol., mL

Blank

0

0 5

0

0

90 45

30 15

1000

1

0.1 A/0.5 B

500

2

0.5 A/1 B

25

45

15

500

3

1 A/3 B

50

90

30

1000

4

5 A & B

5

90

30

1000

5

10 A & B

5

45

15

500

6

30 B

3

90

30

1000

7

50 B

5

90

30

1000

8

80 B

4

45

15

500

9

100 B

10

90

30

1000

10

200 B

10

45

15

500

11

300 B

15

45

15

500

12

400 B

20

40

15

500

13

600 B

30

40

15

500

14

10 A

5

45

15

a  Since commercial stock standards are often stored or preserved in acids, the volume of HNO include any contribution of HNO 3 and/or HCl from the commercial stock standard source.

3 and HCl added to the calibration standards should be adjusted to

of white/white, 1.02 mm id sample pump tube, and orange/white, 0.64 mm id internal standard pump tube. If the test solutions and internal standard solutions are mixed in different proportions by the instrument’s peristaltic pump, then adjust the reagent concentrations to meet concentration requirements of mixed solution nebulized by the instrument, as outlined in F . Note that sample and internal standard solution mixing ratio is proportional to pump tube flow rates, not proportional to pump tube IDs.) ( j )  Stock standard solutions, As, Cd, Co, Cr, Pb, Mo, Ni, and Se .—Working standards can be prepared from ICP grade 1000 mg/L commercial stock standard solutions for As, Cd, Co, Cr, Pb, Mo, Ni, and Se. A number of companies provide this stock standard service. ( k )  Stock standard solutions, Ca, Cu, Fe, Mg, Mn, and Zn .— Working standards can be prepared from 10 000 mg/L individual element ICP grade commercial stock standard solutions for Ca, Cu, Fe, Mg, Mn, and Zn. However, it is also acceptable to use commercially prepared custom blended stock standard mixtures containing some or all elements at stock concentrations. Anumber of companies provide this stock standard service. ( l )  10 mg/L intermediate standard solution for preparation of low-level working standards for As, Cd, Co, Cr, Pb, Mo, Ni, and Se .—Dilute 5.0 mL of stock 1000 mg/L standard solution, G ( j ), to 500 mL. Prepare fresh each time standards are prepared, and use immediately after preparation. ( m )  50 mg/L intermediate standard solution for preparation of low-level working standards for Ca, Cu, Fe, Mg, Mn, and Zn .— Dilute 5.0 mL of stock 10 000 mg/L standard solution to 1000 mL. Prepare fresh each time standards are prepared, and use immediately after preparation. ( n )  Working standard solutions (see Table 2017.02B ) for Ca, Cu, Fe, Mg, Mn, and Zn .—Standards should have the same acid concentration as digested test solutions. Date all calibration solutions whenmade, which are stable at roomtemperature for 60days.Monitor

standard curve fit and intensity for signs of change and degradation over time. ( Note : Based on instrumentation, the calibration standards may be adjusted to fit the manufacturer guidelines regarding standard curve requirements. However, linear curves should have correlation coefficients of at least 0.999 and a standard error of no more than 10%. Quadratic calibrations should have a correlation coefficient of at least 0.999, a standard error of less than 10%, a curvature of no more than 25%, and an upward curvature of no more than 400%.) ( 1 )  10 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 100.0 mL intermediate standard solution, G ( m ), into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to acid-washed polypropylene bottle. ( 2 )  5 mg/L Ca, Cu, Fe, Mg, Mn, and Zn. —Pipet 100 mL of combined 50 mg/L element stock solution into a 1000 mL acid-washed volumetric flask. Add 90 mL trace metal grade HNO 3 and 30 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 3 )  1 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 50.0 mL of 10 mg/L intermediate stock solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 4 )  0.5 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 25.0 mL of 10 mg/L intermediate stock solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 5 )  0.1 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 5.0 mL of 10 mg/kg intermediate stock solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle.

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( 6 )  600 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 30.0 mL of single element or combined 10000 mg/Lmultielement stock standard solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to acid-washed polypropylene bottle. ( 7 )  400 mg/kg Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 20.0 mL of single element or combined 10 000 mg/Lmultielement stock solution into a 1 L acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 8 )  300 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 15.0 mL of single element or combined 10000 mg/L multielement stock standard solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 9 )  200 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 10.0 mL of single element or combined 10000 mg/L multielement stock standard solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 10 )  100 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 10.0 mL of single element or combined 10000 mg/L multielement stock standard solution into a 1000 mL acid-washed volumetric flask. Add 90 mL trace metal grade HNO 3 and 30 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 11 )  80 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 4.0 mL of single element or combined 10000 mg/L multielement stock standard solution into a 500 mL acid-washed volumetric flask. Add 45 mL trace metal grade HNO 3 and 15 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 12 )  50 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 5.0 mL of single element or combined 10000 mg/Lmultielement stock standard solution into a 1 L acid-washed volumetric flask. Add 90 mL trace metal grade HNO 3 and 30 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 13 )  30 mg/L Ca, Cu, Fe, Mg, Mn, and Zn .—Pipet 3.0 mL of single element or combined 10000 mg/Lmultielement stock standard solution into a 1 L acid-washed volumetric flask. Add 90 mL trace metal grade HNO 3 and 30 mL trace metal grade HCl, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( 14 )  0.0 mg/L all Ca, Cu, Fe, Mg, Mn, and Zn .—Add 45 mL trace metal grade HNO 3 and 30 mL trace metal grade HCl into a 500 mL volumetric flask, dilute to volume with H 2 O, mix, and transfer to an acid-washed polypropylene bottle. ( p )  Sampler wash solution, 9% HNO 3 :3% HCl .—Dilute 90 mL trace metal grade HNO 3 , G ( b ), and 30 mL trace metal grade HCl, G ( c ), to 1000 mL with H 2 O. H. Determination Analyze test solutions using an ICP-OES instrument calibrated with standard solutions. Insert a 10 mg/kg working standard or other suitable quality control solution every 10 test portions to monitor for instrument drift. For quality control, see section J .

I. Calculations , = � , � ∗ � 100 . , � ∗ � 1 1000 � ∗ � 1000 1 � where 100 mL assumes the microwave digest is diluted to 100 mL. Some of the Group A and B elements are routinely reported as percent concentrations. To convert a mg/kg result to percent, divide the mg/kg result by 10000 and change the unit frommg/kg to percent. J. Quality Control Each run should contain adequate quality control to monitor the analytical system. The following are recommended to be included with each batch prepared for digestion: ( a )  Accuracy check .—One or more digested reference materials of known concentration (e.g., NIST 695, Magruder Check Samples, AFPC Check Samples, etc.). ( b )  Precision check .—One of the unknowns should be duplicated to ensure that the process can repeat a similar result. ( c )  Method blank .—A digestion tube containing all reagents with no test portion that is processed identically to all others within the batch is recommended to ensure that no contamination of reagents, glassware, etc. has occurred. ( d )  Matrix spike recovery (optional).— One of the unknowns or reference materials can be spiked with a known concentration of all elements to ensure that the matrix does not significantly reduce or enhance the recovery of the desired analyte. ( e )  Continuing calibration verification (CCV).— A calibration standard run at periodic intervals (every 10th test solution) to verify the instrument is maintaining calibration. ( f )  Internal calibration verification (ICV).— An undigested reference solution from a source different from the calibration standards is run after the calibration to check the accuracy of the calibration. ( g ) For each element not reaching predetermined QC criteria, the instrument must be recalibrated and the impacted samples must be reanalyzed. ( h ) Limits of quantitation and detection (LODs and LOQs) should be determined for each element by each laboratory using the method. The author’s LODs and LOQs should be used only as a guide, but due to different instruments and configurations, these will vary from user to user. ( i ) A typical analytical sequence is as follows: ( 1 ) Instrument calibration standards; ( 2 ) ICV; ( 3 ) a series of test solutions based on digestion batch size, including digested QC, spike blanks, and QC duplicates and spikes; ( 4 ) CCV; ( 5 ) another group of test solutions and periodic CCV until finished; ( 6 ) final CCV and QC. It is recommended that one or more QC samples be included after every 10 digests. References: J. AOAC Int . 97 , 700(2014) DOI: 10.5740/jaoacint.13-408 (Original Publication) J. AOAC Int . 101 , 383(2018) DOI: https://doi.org/10.5740/jaoacint.17-0241 (First Action) Posted: July 20, 2017, May 2018

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