AOAC Final Action Methods in 2019

AOAC INTERNATIONAL Official Methods Board (OMB)

Methods Book (Awards) Final Action in 2019 April 30, 2020

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( u )  Bottle top dispenser .—5 mL acid resistant (Brand, Essex, CT, USA or equivalent). ( v )  Desiccator .—Glass (VWR or equivalent.) Note : Nonspecific binding can occur with these analytes when using glassware, so plasticware should be used at all times for standard/sample preparation. All laboratory plasticware should be single-use whenever possible. Positive displacement pipets are also mandatory for pipeting to avoid contamination and for accuracy with organic solvents. C. Chemicals and Reagents ( a )  Water/ultra-pure water (UPW) .—OptimaMS grade (Thermo Fisher Scientific, Waltham, MA, USA or equivalent). ( b )  Acetonitrile .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( c )  Ammonium formate .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( d )  Formic acid .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( e )  Nitric acid .—70% (w/w), ACS grade (Avantor, Center Valley, PA, USA or equivalent). ( f )  Isopropanol .—Optima MS grade (Thermo Fisher Scientific or equivalent). ( g )  Desiccant .—VWR or equivalent. Note : All use of water in this method must be high-purity MS grade water. Perform all preparation steps with nitric acid in a fume hood, and wear necessary personal protective equipment when handling. D. Reference Standards ( a )  Reference standards .—( 1 )  L-Carnitine.— U.S. Pharmacopeia (Rockville, MD, USA) or equivalent). ( 2 )  Choline bitartrate.— TCI (Tokyo, Japan) or equivalent. ( b )  Reference internal standards .—( 1 )  L-Carnitine-d3 HCl.— CDN Isotopes (Pointe Claire, Québec, Canada) or equivalent. ( 2 )  Choline-1,1,2,2-d4 chloride.— CDN Isotopes or equivalent. E. Procedure ( a )  Reagent preparation .—( 1 )  Preparation of mobile phase.— ( a )  Mobile phase A.— 5 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid. Weigh 0.32 ± 0.01 g ammonium formate into a 1 L mobile phase bottle containing 500 mL UPW. Add a stir bar and then mix on a stir plate until dissolved. Add 500  L acetonitrile to the mobile phase container, along with 2.00 mL formic acid. Add a stir bar and mix on a stir plate until thoroughly mixed, typically for about 2 min. Solution is stable for 2 weeks when stored at room temperature. ( b )  Mobile phase B.— 30 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid. Weigh 1.89 ± 0.01 g ammonium formate into a 1 L mobile phase bottle containing 500 mL UPW. Add a stir bar and then mix on a stir plate until dissolved. Add 500 mL acetonitrile to the mobile phase container, along with 2.00 mL formic acid. Add a stir bar and mix on a stir plate until thoroughly mixed, typically for about 2 min. Solution is stable for 2 weeks when stored at room temperature. Note : Rinse all mobile phase bottles thoroughly prior to use with UPW and isopropanol. Use additional rinse with concentrated acid if high background signal is present. ( c )  Injector wash .—Mobile phase B is used as HPLC injector wash.

AOAC Official Method 2015.10 Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula Liquid Chromatography/Tandem Mass Spectrometry First Action 2015 Revised First Action 2016 Final Action 2019

[Applicable to the measurement of total or free carnitine and choline by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) in infant formula and adult/pediatric nutritional formula.] Caution : Refer to Material Safety Data Sheets (MSDS) for safety precautions when using chemicals. Use personal protective equipment recommended in MSDS. A. Principle Samples are extracted in water for free carnitine and choline. For total carnitine and choline, samples are digested with nitric acid by microwave. Free and total extracts are both diluted to volume with water, mixed with acetonitrile, and analyzed using LC-MS/ MS detection. B. Apparatus ( a )  LC system .—Prominence (Shimadzu, Kyoto, Japan) or equivalent. ( b )  MS/MS system .—API 4000 with electrospray ionization (ESI; AB Sciex, Framingham, MA, USA) or equivalent. ( c )  MS software .—Analyst (AB Sciex) or equivalent. ( d )  Analytical column .—Zorbax 300-SCX, 3.0 × 50 mm, 5 µm (Agilent, Santa Clara, CA, USA) or equivalent. ( e )  Microwave .—MARS6 (CEM, Mathews, NC, USA) or equivalent. ( f )  Microwave turntable, liner, and cap .—MARSXpress, 55 mL PFA Teflon®, 40 position (CEM or equivalent). ( g )  Vortex mixer .—VWR (West Chester, PA, USA) or equivalent. ( h )  Analytical balances .—Model CPA225D (Sartorius, Goettingen, Germany) or equivalent. ( i )  Horizontal shaker .—Model 6010 (Eberbach, Ann Arbor, MI, USA) or equivalent. ( j )  Magnetic stir plate .—Model PC-420D (Corning, Corning, NY, USA) or equivalent. ( k )  Positive displacement pipets .—Microman, various sizes (Gilson, Middleton, WI, USA) or equivalent. ( l )  Repeater positive displacement pipet .—Repeater Plus (Eppendorf, Hamburg, Germany) or equivalent. ( m )  Polypropylene tubes .—Digitube, assorted sizes (SCP Science, Montreal, Canada) or equivalent. ( n )  Mobile phase containers .—2 L glass (VWR or equivalent). ( o )  Syringe filters .—0.45 µm PTFE and GHP (Pall, Plano, TX, USA or equivalent). ( p )  Disposable syringes .—3 mL (BD Biosciences, Franklin Lakes, NJ, USA or equivalent). ( q )  Graduated cylinders .—Assorted sizes (VWR or equivalent). ( r )  Magnetic stir bars .—7.9 × 50 mm (VWR or equivalent). ( s )  Autosampler vials/caps .—1.5 mL silanized crimp top (VWR or equivalent). ( t )  Microcentrifuge tubes .—1.5 mL polypropylene (VWR or equivalent).

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Table 2015.10A. Preparation of working standard solutions a Standard solution Source solution ID Source concn, mg/mL

Source volume, mL

MS grade water, mL

Final volume, mL

Concn (µg/mL) extracted (ng/mL)

STD6 STD5 STD4 STD3 STD2 STD1

Stock solutions Stock solutions Stock solutions

25.0 25.0 25.0 5.00

2.00 each 1.60 each 0.800 each

6.00 6.80 8.40 9.00 9.60 9.80

10.0 10.0 10.0 10.0 10.0 10.0

5000 4000 2000

STD6 STD3 STD3

1.00

500

0.500 0.500

0.400 0.200

20 10

a  Alternate weights or volumes may be used to scale these preparations

( b )  Standard solutions preparation .—( 1 )  Preparation of choline/carnitine stock solutions .—( a )  Choline stock solution (~25 mg/mL) .—Weigh 0.5200 ± 0.01 g choline bitartrate into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL water. ( b )  Carnitine stock solution (~25 mg/mL) .—Due to the hygroscopic nature of L-carnitine, perform a Karl Fischer moisture analysis test to determine the water content of the material at the time of weighing.Weigh 0.2500 ± 0.01 g into a 20mLpolypropylene scintillation vial. Dissolve with 10.0 mL UPW. ( c )  L-Carnitine-d3 stock solution (~2.00 mg/mL; internal standard I; IS1) .—Weigh 0.02500 ± 0.001 g L-carnitine-d3 HCl into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL UPW. ( d )  Choline-1,1,2,2-d4 stock solution (~2.00 mg/mL; IS2) .— Weigh 0.03100 ± 0.001 g choline-1,1,2,2-d4 bromide into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL UPW. ( 2 )  Preparation of working standard solutions.— ( a ) Prepare the working standard solutions according to Table 2015.10A . ( b )  L-Carnitine-d3/choline-1,1,2,2-d4 (200 μ g/mL) IS working solution .—Transfer 10 mL IS1 solution and 10 mL IS2 solution into a 100 mL Digitube. Add 80 mL UPW to bring to a final volume of 100 mL. ( Note : Stock, intermediate, and working standards are stable for 6 months when stored in a refrigerator set to maintain 2 to 8°C. Protect standard solutions from actinic light. Alternate weights or volumes may be used to scale these preparations.) ( c )  Sample preparation .—( 1 )  Sample preparation for samples needing reconstitution.— ( a ) Weigh 10.00 ± 0.500 g sample in a suitable disposable cup or beaker. ( b ) Add UPW to bring the total weight (including the powder weight) to 100.00 ± 1.00 g. ( c ) Add a stir bar and stir as fast as possible without causing the sample to splatter. Stir for at least 10 min, but no longer than 30 min. Note : Adjust weights as needed to accommodate different powder types and levels of vitamins. ( 2 )  Free analysis of carnitine and choline.— ( a ) Weigh 0.1–1.0 g sample into a tared 50 mL Digitube depending on the concentration of each analyte (to fall within calibration curve). ( b ) A reagent blank, a reagent blank + IS, and working standards must be included for each analysis and treated the same as samples through the analysis. Add 50 μL of each working solution to separate 50 mL Digitubes. Final nominal concentrations for these after they have gone through the sample preparation (diluted

1000X) are listed in the Standard Solutions Preparation section to be used for construction of the calibration curve. ( c ) Add 50 μL intermediate mixed IS solution to each sample, internal working solution (IWS), and reagent blank + IS. ( d ) Dilute to 25 mL with MS grade water, cap, and thoroughly mix sample on a horizontal shaker. Sample extract is stable for 3 days when stored refrigerated and protected from light. ( e ) If dilutions are required, dilute samples to appropriate concentrations using the reagent blank + IS using microcentrifuge tubes to do the dilutions. ( f ) Aliquot 0.5 mL extract into a microcentrifuge tube, along with 0.5 mL acetonitrile. Mix well. ( g ) Filter samples through a 0.45 μm GHP syringe filter into a silanized injection vial. Standards do not need to be filtered through the syringe filter. Sample extract is stable for 3 days when stored refrigerated and protected from light. ( 3 )  Total analysis of carnitine and choline.— ( a ) Weigh 0.1– 1.0 g sample into a tared 55 mL MarsExpress vessel depending on the concentration of each analyte (to fall within calibration curve). For viscous (high-fat, high-protein) ready-to-feed samples, perform a predilution by weighing 1.0 g sample and adding MS grade water

Table 2015.10B. HPLC parameters Column Zorbax 300-SCX, 3.0 × 50 mm, 5 µm (Agilent, Santa Clara, CA, USA) Mobile phase A 5 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid Mobile phase B 30 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid Injection volume 5–10 μL Run time 4.2 min

Flow rate, mL/min

Phase B, %

Gradient program Time, min

0

1.00 1.00 1.00 1.00 1.00

0 0

1.0 1.5 2.5 3.0 4.2

100 100

0 0

1.00

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Table 2015.10C. LC-MS parameters

LC-MS settings a

LC-MS model

Sciex 4000

Sciex 6500

 Ionization mode  IonSpray voltage

MRM

MRM

1000 V 550 ° C

2000 V 550 ° C

 Turbo IonSpray temp.  Entrance potential (EP)  Collision gas (CAD)  Curtain gas (CUR)  Nebulizing gas (GS1)  Nebulizing gas (GS2)

10 V

10 V

Nitrogen, 5.0 Nitrogen, 20.0 Nitrogen, 60.0 Nitrogen, 60.0

Nitrogen, 5.0 Nitrogen, 20.0 Nitrogen, 60.0 Nitrogen, 60.0

 Needle position

Y = 5 mm, X = 5 mm

Y = 5 mm, X = 5 mm

LC-MS model

Agilent 6490

 Ionization mode

MRM 380 V

 Fragmentor

 Cell accelerator

5 V

 Gas temp.  Gas flow  Nebulizer

200 ° C

20 L/min

30 psi 225 ° C

 Sheath gas temp.  Sheath gas flow

11 L/min 1500 V

 Capillary +

 Nozzel voltage +  High-pressure RF +  Low-pressure RF +  Ionization mode  Capillary voltage  RF lens voltage  Source temp.  Nebulizer temp. LC-MS model

500 V 200 V 100 V

Waters TQD

MRM

2000 V 0.1 eV 150 ° C 450 ° C

 API gas

On

 Nebulizer gas  Desolvation gas

Nitrogen

900 L/h nitrogen 50 L/h nitrogen 0.25 mL/min argon

 Cone gas

 Collision gas

 API gas

On

 Nebulizer gas

Nitrogen

Transition monitored Q1/Q3 b

Analyte

Transition use/type

Dwell time, msec b

Collision energy, V b

Retention time, min

Carnitine

Quantitation

162.0/103.0 165.0/103.0 162.0/84.4 162.0/59.1 104.2/60.0 104.2/42.2 108.2/60.0

100 100 100 100 100 100 100

25 25 29 27 25 25 25

1.7 1.7 1.7 1.7 2.1 2.1

Carnitine-d3

Internal standard

Carnitine Carnitine Choline Choline

Qualifier Qualifier

Quantitation

Qualifier

Choline-d4 2.1 a  MS/MS settings may be modified except for ionization type and mode to obtain optimum chromatography and sensitivity. Exact mass ions may vary slightly from instrument to instrument because of unit resolution of quadrupole mass spectrometers. b  Settings listed are for Sciex series of LC-MS. Precursor/product masses and instrument settings may vary. Internal standard

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to a final weight of 5.0 g into a suitable plastic container. Mix well prior to weighing into the 55 mL MarsExpress vessel. ( b ) A reagent blank, a reagent blank + IS, and working standards must be included for each analysis and treated the same as samples through the analysis. Add 50 μL of each working solution to separate 50 mL Digitubes. The final nominal concentrations for these after they have gone through the sample preparation (diluted 1000X) are listed in the Standard Solutions Preparation section to be used for construction of the calibration curve. ( c ) Add 50 μL intermediate mixed IS solution to each sample, IWS, and reagent blank + IS. ( d ) Add 5 mL MS grade water and 2.5 mL of 70% (w/w) nitric acid with a bottle top dispenser. Cap tightly or utilize a capping station. Mix the sample by either vortexing or inverting. ( e ) Insert vessels into their appropriate sleeves and into the turntable. Microwave samples according to the following conditions: Power 1000 W; ramp to temperature 10 min; hold time 40 min; temperature 120°C. ( f ) Allow vessels to complete the cooling process in the microwave before removing caps to prevent loss of sample through pressure release. ( g ) Quantitatively transfer contents of the vessels into 50 mL Digitubes with MS grade water and dilute to a volume of 25 mL with MS grade water. ( h ) Filter samples through a 0.45 μm PTFE syringe filter into a microcentrifuge tube. Standards do not need to be filtered through the syringe filter. ( i ) If dilutions are required, dilute samples to appropriate concentrations using the reagent blank + IS utilizingmicrocentrifuge tubes to do the dilutions. ( j ) Aliquot 0.5 mL extract into a silanized injection vial, along with 0.5 mL acetonitrile. Cap and then mix well by shaking or

vortexing the vials. Prepared sample extracts in injection vials are stable for 24 h while stored 2–8°C and protected from light. ( d )  Instrument parameters .— See Tables 2015.10B and 2015.10C . F. Calculations Integrate the peak areas of the analytes and IS in both the sample and standard injections. Peak areas of reference standard analyte quantitation ions are divided by the corresponding IS peak areas to obtain relative analyte responses (normalized to IS). Relative responses of each analyte in the calibration standards are plotted on the y -axis against their corresponding concentrations on the x -axis to generate calibration curves with linear fit and 1/x2 weighting for all compounds. Analyte concentrations in the sample extracts are derived from the calibration curves and their concentrations in the samples are determined using the following equation: ( C × V × D ) / ( W × 1000) = mg/kg where C = analyte concentration in sample (ng/mL); V = final volume (mL); D = dilution (mL/mL); W = sample weight (g); and DOI: 10.5740/jaoacint.15-0144 (First Action) AOAC SMPR 2012.010 J. AOAC Int. 96 , 488(2013) DOI: 10.5740/jaoac.int.SMPR2012.010 AOAC SMPR 2012.013 J. AOAC Int. 96 , 492(2013) DOI: 10.5740/jaoac.int.SMPR2012.013 Posted: February 19, 2016 (First Action), August 2019 (Final Action) 1000 = conversion from ng/g into mg/kg. References: J. AOAC Int . 99 , 204(2016)

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AOAC Official Method 2015.14 Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 , and B 6 in Infant Formula and Related Nutritionals Enzymatic Digestion and LC-MS/MS First Action 2015 Revised First Action 2018 Final Action 2019

Table 2015.14B. Repeatability (RSD r ) summary statistics

No. fortified products Passed SMPR Failed SMPR

Avg. RSD r , %

SMPR for RSD r , %

Total vitamin B 1 (thiamine) B 2 (riboflavin)

2.3 4.2 2.7 2.2

14 11 14 14

0 3 0 0

≤5

B 3 (niacinamide) B 6 (pyridoxine)

[Applicable to determination of total vitamins B 1 , B 2 , B 3 , and B 6 in fortified milk powders, infant formulas, and adult/pediatric nutritional formulas with concentrations. Total B 1 , B 2 , B 3 , and B 6 can be quantified in ready-to-feed products with concentrations (mg/100 g) as low as 0.0062, 0.0043, 0.044, and 0.0056, respectively.] See Table 2015.14A for method performance requirements. See Tables 2015.14B and C for method performance information supporting acceptance of the method. A. Principle The method facilitates simultaneous quantitation of four water- soluble vitamins in infant formula and related nutritional products, including all AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs ® ) relevant forms of vitamins B 1 , B 2 , B 3 , and B 6 . Samples are prepared by enzymatic digestion with papain, α-amylase, and phosphatase to hydrolyze protein and complex carbohydrates and to free phosphorylated vitamin forms. Stable-isotope-labeled internal standards are incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. A series of six mixed working standard (MWS) solutions spanning 2 orders of magnitude in vitamin concentration are used to generate calibration curves based on the peak response ratio of the analyte to its stable-isotope-labeled internal standard. Prepared samples and working standard solutions are injected onto an ultra-high-pressure liquid chromatograph (UPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) for analysis. The MS/MS is configured to monitor molecular–fragment (precursor–fragment) ion pairs for each analyte and internal standard. This reaction forms the basis for method selectivity. Analytes are quantified by least-squares regression using the response ratio of the analyte to its internal standard. B. Apparatus and Materials (a) Control sample. —NIST SRM 1849a (U.S. National Institute of Standards and Technology, Gaithersburg, MD, USA), or current lot. Store at 4°C. (b) UPLC column. —Waters Acquity BEH C18, 2.1 × 100 mm, 1.7 μm (Part No. 186002352; Waters Corp., Milford, MA, USA). (c) UPLC system. —Waters Acquity Classic, or equivalent.

(d) Tandem quadrupole mass spectrometer with ESI probe. — Waters Xevo TQ-S, or equivalent. (e) Analytical balances (3). —( 1 ) 6-Place balance, capable of accurately weighing 5.00 mg (for standards). ( 2 ) 5-Place balance (for samples). ( 3 ) Top-loading 2-place balance, capable of weighing to several hundred grams. (f) Water purifier. —Millipore Milli-Q Water Purification System, or equivalent (EMD Millipore, Billerica, MA, USA). (g) Water bath shaker.— Capable of maintaining 37°C (Lab- Line Orbit, or equivalent). (h) Bottle-top dispenser. —Capable of dispensing volumes of approximately 24 mL. (i) pH meter. —Capable of measuring a pH of 4.0–5.0. (j) Vortex mixer. (k) Multiposition magnetic stir plate. (l) Room light shields. —A.L.P. Protect-A-Lamp, UV cutoff at 460 nm, or equivalent (A.L.P Lighting Components, Inc., Chicago, IL, USA). (m) Graduated cylinders. —Various sizes, including 10, 100, 500, and 1000 mL. (n) Beakers. —Various sizes, including 100, 200, 400, 600, 1000, and 2000 mL. (o) Volumetric flasks. —Various sizes, including 10, 25, 50, 100, 250, and 2000 mL. (p) Mobile phase bottles. —Glass, various sizes, including 250, 500, 1000, and 2000 mL. (q) Pasteur pipets.— Disposable, plastic. (r) Amber bottles. —Volume capacity of 50 and 100 mL (for stock standard storage). (s) Weighing vessels. —Various, including disposable weighing boats and glass weighing funnels. (t) Positive displacement pipets. —Gilson Microman 10, 100, 250, and 1000 μL (Part Nos. F148501, F148504, F148505, and F148506, respectively; Gilson, Inc., Middleton, WI, USA).

Table 2015.14C. Reproducibility (RSD R ) summary statistics

Table 2015.14A. Method performance requirements

No. fortified products Passed SMPR Failed SMPR

Analytical range (μg/100 g) 20–2000 30–4000

Avg. RSD R , %

SMPR for RSD R , %

LOQ (μg/100 g) Rec. % RSD r , % RSD R , %

Total vitamin B 1 (thiamine) B 2 (riboflavin)

Total vitamin B 1 (thiamine) B 2 (riboflavin)

2.3 6.9 6.7 5.8

10 13 14 13

4 1 0 1

≤20 90–110 ≤5

≤10

≤30

≤10

B 3 (niacinamide) B 6 (pyridoxine)

B 3 (niacinamide) B 6 (pyridoxine)

200–10000 ≤200

10–2000

≤10

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(u) Positive displacement pipet tips. —Gilson Capillary Piston 10, 100, 250, and 1000 μL (Part Nos. F148312, F148314, F148014, and F148560, respectively; Gilson, Inc.). (v) Plastic syringes. —3 mL. (w) Syringe filters. —Polytetrafluoroethylene (PTFE) 0.45 μm, Acrodisc 25 mm, or equivalent (Pall Corp., Port Washington, NY, USA). (x) Plastic centrifuge tubes.— 50 mL, self-standing (Superior Scientific, Ltd., or equivalent). (y) Autosampler vials. —9 mm amber with screw top 12 × 32 mm presplit PTFE-silicon septa (Waters; Part No. 186000847C, or equivalent). ( z )  Stir bars .—Teflon-coated, magnetic. C. Reagents (a) Nicotinamide. —USP Reference Standard Official Lot (Cat. No. 1462006; U.S. Pharmacopeial Convention, Inc., Rockville, MD, USA). Store as indicated on label. (b) Niacin (nicotinic acid). —USP Reference Standard Official Lot (Cat. No. 1461003). Store as indicated on label. (c) Pyridoxine hydrochloride. —USP Reference Standard Official Lot (Cat. No. 1587001). Store in desiccator protected from white light. Dry according to manufacturer’s instructions before use. (d) Riboflavin. —USP Reference Standard Official Lot (Cat. No. 1603006). Store in desiccator protected from white light. Dry according to manufacturer’s instructions before use. (e) Thiamine hydrochloride. —USP Reference Standard Official Lot (Cat. No. 1656002). Store in desiccator protected fromwhite light. Measure moisture content of the powder before use. (f) Pyridoxamine dihydrochloride. —Fluka Analytical Standard (Cat. No. P9380; Sigma-Aldrich, St. Louis, MO, USA). (g) Pyridoxal hydrochloride. —Sigma, Cat. No. P9130 (Sigma- Aldrich). (h) 2 H 4 -nicotinamide. —Cat. No. D-3457 (CDN Isotopes, Pointe- Claire, Quebec, Canada). (i) 2 H 4 -nicotinic acid. —Cat. No. D-4368 (CDN Isotopes). (j) 13 C 4 -pyridoxine:pyricoxine.— HCl (4,5-bis(hydroxymethyl)- 13 C 4 ) (Cat. No. CLM-7563; Cambridge Isotope Laboratory, Tewksbury, MA, USA). (k) 2 H 3 -pyridoxal. —Cat. No. 7098 (IsoSciences, King of Prussia, PA, USA). (l) 2 H 3 -pyridoxamine. —Cat. No. 7099 (IsoSciences). (m) 13 C 4 -thiamine chloride. —Cat. No. 9209 (IsoSciences). (n) 13 C 4 , 15 N 2 -riboflavin. —Cat. No. 7072 (IsoSciences). (o) Acid phosphatase, type II from potato, 0.5–3.0 U/mg. —Cat. No. P3752 (Sigma-Aldrich). (p) Papain from Carica papaya, ≥3 U/mg. —Cat. No. 76220 (Sigma-Aldrich). (q) α - Amylase from Aspergillus oryzae, 150 U/mg. —Cat. No. A9857 (Sigma-Aldrich). (r) Hydrochloric acid, concentrated (12 M). —American Chemical Society (ACS) grade, or equivalent. (s) Ammonium formate.— For mass spectrometry (≥99.0%); Fluka 70221 or equivalent (Sigma-Aldrich). (t) Glacial acetic acid. —Sigma ACS reagent grade, or equivalent (Sigma-Aldrich). (u) Formic acid. —Sigma ACS reagent grade, or equivalent (Sigma-Aldrich). (v) Laboratory water. —18.0 MΩ, <10 ppb total organic carbon (TOC), or equivalent.

(w) Methanol. —Fisher Scientific (Franklin, MA, USA) LC- MS/MS optima grade or EMD (EMD Millipore) Omni-Solve LC- MS grade. (x) Ethylenediaminetetracetic acid (EDTA), disodium salt dihydrate. —ACS grade (99–101%), or equivalent. (y) Potassium phosphate dibasic. —ACS grade (>98%), or equivalent. (z) Metaphosphoric acid. —ACS grade (33.5–36.5%), or equivalent. (aa) Buffer solutions.— pH 4.0, 7.0, and 10.0; for pH meter calibration. (bb) Phosphoric acid. —85%, ACS grade, or equivalent. (cc) Potassium hydroxide. —40%, ACS grade, or equivalent. D. Standard and Solution Preparation (a) Mobile phase A. —20 mM ammonium formate in water. Using a graduated cylinder, transfer 500 mL laboratory water to a mobile phase reservoir. Add 0.631 g ammonium formate and mix well. Scale as needed. Expiration: 3 days. (b) Mobile phase B. —Methanol. (c) Weak needle wash. —10% methanol in water. Expiration: 3 months. (d) Strong needle wash. —Methanol. (e) 50 mM ammonium formate. —Using a graduated cylinder, transfer 1400 mL laboratory water to an appropriate reservoir. Add 4.41 g ammonium formate and mix well. 1400 mL is adequate for six working standards and 32 samples. Scale as needed. Expiration: 3 days. (f) Enzyme cocktail. —( 1 ) Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir. Add 200 ± 10 mg acid phosphatase and 200 ± 10 mg papain. Mix for 10 min with magnetic stir plate and stir bar. Adjust to pH 4.0–4.5 with formic acid (approximately 100 μL). 200mL is adequate for sixworking standards and 32 samples. Scale as needed. Prepare fresh daily. ( 2 ) If a product contains extensive protein, or alpha-linked polysaccharides such as starch, use the following preparation: Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir. Add 200 ± 10 mg acid phosphatase, 80 ± 5 mg α-amylase, and 400 ± 10 mg papain. Mix for 10 min with magnetic stir plate and stir bar. Adjust to pH 4.0–4.5 with formic acid (approximately 100 μL). 200 mL is adequate for six working standards and 32 samples. Scale as needed. Prepare fresh daily. (g) Stable-isotope-labeled compounds. —Individual, internal standard stock solutions. Note : Internal standard stock solutions have an expiration of 6 months, however the following guidelines can be used to troubleshoot internal standards and extend the expiration dates indefinitely. The channel of the nonlabeled analyte of interest must be monitored to ensure the stable isotope-labeled internal standard does not contribute more than 20% of the area count of the lowest-level calibration standard. No response should be generated in any other channels being monitored in the method, as this is a sign of contamination, in which case fresh solution should be prepared or a fresh lot of material should be ordered. The area count of the internal standard should be at least 3× the area count of the analyte in the lowest-level calibration standard and the lowest level matrix-based QC sample. ( 1 )  2 H 4 -nicotinamide stock solution (approximate concentration: 560 μg/mL). —Weigh 14.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 25 mL volumetric flask with laboratory water and use quantity sufficient (QS) with laboratory water. Mix well and transfer to a 50 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination.

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( 2 )  2 H 4 -nicotinic acid stock solution (approximate concentration: 500 μg/mL) .—Weigh 12.5 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 25 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 50 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 3 )  13 C 4 -pyridoxine stock solution ( approximate concentration: 70 μg/mL ).—Weigh 7.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 4 )  2 H 3 -pyridoxal stock solution ( approximate concentration: 40 μg/mL ).—Weigh 4.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 5 )  2 H 3 -pyridoxamine stock solution ( approximate concentration: 40 μg/mL ).—Weigh 4.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 6 )  13 C 4 -thiamine chloride stock solution ( approximate concentration: 100  μg/mL ).—( a )  0.12  M HCl. —Add approximately 300 mL water to a 500 mL graduated cylinder. Add 5.0 ± 0.1 mL concentrated HCl and swirl to mix. Dilute to 500 mL with laboratory water and mix well. ( b ) Weigh 5.0 ± 0.1 mg 13 C 4 -thiamine into a tared weighing vessel. Quantitatively transfer to a 50 mL volumetric flask with 0.12 N HCl and QS with 0.12 N HCl. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 7 )  13 C 4 , 15 N 2 -riboflavin stock solution (approximate concentration: 73 μg/mL) .—( a )  1.0% Acetic acid in water. —Add approximately 30 mL water to a 500 mL graduated cylinder. Add 5.0 ± 0.1 mL glacial acetic acid and swirl to mix. Bring to 500 mL with laboratory water and mix well. ( b ) Weigh 7.3 ± 0.1 mg 13 C 4 , 15 N 2 -riboflavin into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with 1.0% acetic acid and QS with 1.0% acetic acid. Mix well and transfer to a 100 mL amber bottle and s tore refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. (h) Internal standard stock mixture (ISSM). —Combine 2500 μL of 50 mM ammonium formate with 250 μL 2 H 4 -nicotinamide stock, 250 μL 2 H 4 -nicotinic acid stock, 250 μL 13 C 4 -pyridoxine stock, 200 μL 2 H 3 -pyridoxal stock, 50 μL 2 H 3 -pyridoxamine stock, 250 μL 13 C 4 -thiamine stock, and 250 μL 13 C 4 , 15 N 2 -riboflavin acid stock. Volume provides sufficient ISSM for six working standards and 32 samples. Scale as needed. Prepare fresh daily. (i) Phosphate buffer solution, pH 5.0. —0.10 M potassium phosphate dibasic, 1% EDTA, 2% metaphosphoric acid. ( 1 ) Weigh 20.0 ± 0.2 g EDTA into a tared weighing vessel and quantitatively transfer to a 2000 mLbeaker containing approximately 1800 mL laboratory water and add a magnetic stir bar. ( 2 ) Weigh 34.8 ± 0.1 g potassium phosphate dibasic into a tared weighing vessel and quantitatively transfer to the 2000 mL beaker already containing approximately 1800 mL laboratory water and EDTA. Mix by stirring on a magnetic stir plate until both the EDTA and potassium phosphate dibasic are completely dissolved.

( 3 ) Weigh 40.0 ± 0.2 g metaphosphoric acid into a tared weighing vessel and quantitatively transfer to the 2000 mL beaker containing approximately 1800 mL laboratory water, EDTA, and potassium phosphate dibasic. Mix by stirring on a magnetic stir plate until the metaphosphoric acid is completely dissolved. ( 4 ) Adjust the pH of the solution to pH 5.00 ± 0.02 using 40% potassium hydroxide or 85% phosphoric acid. Quantitatively transfer solution to a 2000 mL volumetric flask and dilute to volume with laboratory water. Expiration: 48 h. (j) Native compounds. —Stock standard solutions. ( 1 )  Vitamin standard stock mixture (VSSM). —Accurately weigh the indicated amounts for the following standards using separate weighing funnels or other appropriate weighing vessel and quantitatively transfer to a 100 mL volumetric flask using phosphate buffer (pH 5): ( a )  Niacinamide. —70.5 ± 0.5 mg. ( b )  Thiamine hydrochloride. —10.5 ± 0.2 mg. Determine the moisture of the USP thiamine hydrochloride reference standard as directed on the container immediately before weighing. The percent moisture determined for the reference standard is used to calculate the concentration of thiamine in the VSSM. ( c )  Riboflavin. —7.0 ± 0.2 mg. Dry an appropriate amount of the USP riboflavin reference standard at 105 (±1)°C for 2 h (±10 min) before weighing. ( d )  Pyridoxine hydrochloride. —10.8 ± 0.2 mg. Dry an appropriate amount of the USP pyridoxine hydrochloride reference standard over indicating absorbent in vacuo for 4 h before weighing. QS to volume with phosphate buffer (pH 5) solution. Heat and slowly stir until the standards have completely dissolved (riboflavin dissolves more slowly) and the solution is clear. Do not heat the solution for more than 40 min and do not exceed 90°C. Store refrigerated (2–8°C). Expiration: 3 months. ( 2 )  Nicotinic acid stock solution. —550 µg/mL. Accurately weigh 13.7 ± 0.1 mg USP niacin (nicotinic acid, Cat. No. 1461003) reference standard. Quantitatively transfer nicotinic acid to a 25 mL volumetric flask. Add laboratory water to a total volume of about 20 mL and swirl until completely dissolved. Dilute to volume with laboratory water. Mix well. Expiration: 3 months. ( 3 )  Pyridoxal stock solution. —140 µg/mL. Accurately weigh 17.0 ± 0.5 mg pyridoxal dihydrochloride standard. Quantitatively transfer to a 100 mL volumetric flask. Add laboratory water to a total volume of about 70 mL and swirl until completely dissolved. Dilute to volume with laboratory water. Mix well. Expiration: 3 months. ( 4 )  Pyridoxamine stock solution. —160 µg/mL. Accurately weigh 23.0 ± 0.5 mg pyridoxamine hydrochloride standard. Quantitatively transfer to a 100 mL volumetric flask. Add laboratory water to a total volume of about 70 mL and swirl until completely dissolved. Dilute to volume with laboratory water. Mix well. Expiration: 3 months. (k) MWS. —Combine 500 μLVSSM, 25 μL pyridoxamine stock, 25 μL pyridoxal stock, and 65 μL nicotinic acid stock solutions in a 10 mL volumetric flask containing approximately 5 mL of 50 mM ammonium formate. Dilute to volume with 50 mM ammonium formate and mix well. Prepare fresh daily. (l) Working standard solution preparation. —( 1 )  WS1. —Add 20 μL MWS and 980 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. ( 2 )  WS2. —Add 50 μL MWS and 950 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily.

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desolvation gas flow of 800 L/h, cone gas flow of 150 L/h, nebulizer gas pressure of 7.00 bar, and collision gas flow of 0.15 mL/min with argon. Both quadrupoles are set to unit mass resolution. ( 3 )  Mass transitions. —Mass transitions for each vitamin and its corresponding internal standard are given in Table 2015.14D . Retention time windows are also given in the table. Like the tune parameters, these parameters may need adjustment based upon instrument model due to model-to-model mass variances. ( 4 )  UPLC-MS/MS equilibration. —The instrument should be held at initial conditions (withmobile phase flow on andMS at temperature) for 30–60 min before injection. Alternatively, 6–10 blank injections at the start of a sequence can be used for the same purpose. (d) Quality control .—( 1 ) Blanks of 50 mM ammonium formate need to bracket each calibration curve to enable a check for laboratory background and instrumental carryover. Background should be no more than 5% of the signal for the lowest working standard. ( 2 )  Calibration curve. —Calibration curves are set up to bracket the sample injections. Calibration residuals (relative error from known concentration) are expected to be ≤20% for pyridoxal and ≤8% for the other vitamins. A standard injection outside of this range can be excluded with evidence of a standard preparation error in a single calibration level leading to a high or low response for all vitamins or evidence of a one-off instrumental error, such as a missed injection. ( 3 )  Laboratory control. —NIST SRM 1849a, or current lot, serves as a control sample and should be prepared and analyzed with each sample set. The control result for each analyte must be within limits established by a control chart. By and large, the levels reported by this method are within the NIST certified range because of the minute concentration of phosphorylated vitamin forms in SRM 1849a. F. Calculations (a)  Vitamin stock solutions concentration calculation: where [Vit] Stk = vitamin standard stock solution concentration, in μg/mL; W S = weight of standard in mg; M = moisture content correction factor for the standard, if applicable; S = stoichiometric correction factor, if applicable, i.e., when reporting thiamin ion, use thiamine HCl MW of 337.27 and thiamine ion MW of 265.335 resulting in a correction factor of 0.7867; P = purity of standard as defined by the manufacturer; 1000 = units conversion factor, from mg to μg; and Vol = dissolution volume in mL. (b)  Calculation of vitamin concentrations in the MWS: [ ] Vol PSMW Vit 000 ,1 ××× × s Stk =

( 3 )  WS3. —Add 100 μLMWS and 900 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. ( 4 )  WS4. —Add 200 μLMWS and 800 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. ( 5 )  WS5. —Add 500 μLMWS and 500 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. ( 6 )  WS6. —Add 1000 μL MWS to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. E. Procedure (a) Sample preparation .—( 1 )  For powdered products. —Using a tared beaker or low-density polyethylene cup, weigh 10.0 ± 0.3 g sample. Record weight to at least four significant figures. This is the powder weight. Add room temperature laboratory water to bring the total reconstituted sample weight (to include the product weight) to 100 ± 2 g. Record the weight to at least four significant figures. This is the reconstitution weight. Carefully add a stir bar so as not to splash the liquid from the beaker/cup and place it onto a stir plate. Set the stir plate to stir the sample as fast as possible without causing the sample to splatter or froth. Powder samples should stir for at least 10 min but not more than 30 min. ( 2 )  For reconstituted powders and liquid products. —Using a tared, 50 mL centrifuge tube, weigh the appropriate sample amount (1.000 ± 0.100 g for infant formula, 0.500 ± 0.050 g for pediatric formulas and theNISTSRM, and 0.250±0.050g for adult nutritionals). Record the weight to 0.0001 g. This is the sample weight. Add 100 μL of the ISSM via positive-displacement pipet. Vortex to mix. (b) Enzymatic digestion. —Add 5 mL of enzyme cocktail to all prepared samples and working standards. Cap and vortex immediately. Incubate at 37°C overnight with agitation in water bath shaker. Remove fromwater bath, and add 50 mM ammonium formate buffer to dilute to approximately 30 mL and vortex to mix. Filter approximately 2 mL aliquot of the sample extract into an appropriate size vial using a 0.45 μm PTFE syringe filter. Transfer 60 μL of filtrate to an autosampler vial with 940 μL of 50 mM ammonium formate buffer. Cap and vortex. The sample is ready for analysis. Samples have been determined to be stable for at least 48 h at room temperature. ( Note : The final 60:940 dilution can be changed depending on system sensitivity. This dilution is to reduce matrix effects of samples, but the dilution can be customized.) (c) LC-MS/MS analysis .—( 1 )  UPLC conditions. —Place freshly prepared mobile phases, weak needle wash, and strong needle wash onto the LC system. Purge old solvents from the solvent lines and needle washes. Injection volume is 10 μL and column temperature is 40°C. Mobile phase flow rate is 0.350 mL/min. Hold at 99% mobile phase A and 1.0% mobile phase B for 0.50 min, then ramp to 8.0% B over 2.00 min, ramp to 90% B over the next 2.50 min, and hold at 90% B for 1.00 min. Return to 99% mobile phase A and 1.0% mobile phase B over 0.10 min and hold for 1.9 min for reequilibration. Total gradient program is 8.00 min long. ( 2 )  MS tune conditions. —Clean the sample cone and MS source with 5% formic acid before analysis. Tune conditions can vary between instrument models, and appropriate balance must be struck to achieve adequate signal for each compound. Appropriate conditions must be determined experimentally for each instrument model. On a Waters TQ-S, ionization is performed by ESI+ at 2.5 kV. Additional tune conditions include source offset of 50 V, ion block temperature of 150°C, desolvation gas temperature of 500°C,

where [Vit] MWS = vitamin concentration in the MWS in ng/mL; [Vit] Stk = concentration of vitamin stock standard in μg/mL; and Vol = volume of stock solution added to MWS in μL. (c)  Calculation of working standard concentration:

] [ Vit

Vol

×

] [ Vit

=

MWS

MWS

WSx

500

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Table 2015.14D. Conditions for MS transitions on a Waters TQ-S along with retention time windows

Collision energy (V) Dwell time, s

Compound

Function No.

Start, min End, min

Molecular ion Fragment ion Cone voltage

Nicotinamide a Nicotinamide

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7

2.71 2.71 2.71 2.71 0.50 0.50 0.50 0.50 1.76 1.76 1.76 1.76 0.50 0.50 0.50 0.50 2.41 2.41 2.41 2.41 3.01 3.01 3.01 3.01 4.21 4.21 4.21 4.21

3.20 3.20 3.20 3.20 1.70 1.70 1.70 1.70 2.70 2.70 2.70 2.70 1.70 1.70 1.70 1.70 3.00 3.00 3.00 3.00 3.60 3.60 3.60 3.60 5.00 5.00 5.00 5.00

122.9 122.9 127.0 127.0 124.0 124.0 128.0 128.0 168.0 168.0 171.0 171.0 169.0 169.0 172.0 172.0 170.0 170.0 174.0 174.0 265.1 265.1 269.0 269.0 377.0 377.0 383.0 383.0

80.1 96.0 84.0

20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0

16.0 16.0 16.0 16.0 16.0 16.0 16.0 16.0 22.0 12.0 22.0 12.0 20.0 12.0 20.0 12.0 18.0 12.0 18.0 12.0 30.0 12.0 30.0 12.0 35.0 20.0 35.0 20.0

0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025 0.025

2 H 2 H

4 -nicotinamide a 4 -nicotinamide

100.0

Nicotinic acid a Nicotinic acid

80.0

106.0

2 H 2 H

4 -nicotinic acid a 4 -nicotinic acid

84.1

109.0

Pyridoxal Pyridoxal a

94.0

150.0

2 H 2 H

3 -pyridoxal 3 -pyridoxal a

97.0

153.0 134.0 152.0 136.0 155.0 134.0 152.0 138.0 156.0

Pyridoxamine Pyridoxamine a

2 H 2 H

3 -pyridoxamine 3 -pyridoxamine a

Pyridoxine a Pyridoxine

13 C 13 C

4 -pyridoxine a 4 -pyridoxine

Thiamine Thiamine a

81.0

122.0

13 C 13 C

4 -thiamine 4 -thiamine a

81.0

122.0 172.0 243.0 175.0 249.0

Riboflavin Riboflavin a

13 C 13 C

4 , 4 ,

15 N 15 N

2 -riboflavin 2 -riboflavin a

0.025 Although the mass transitions are expected to remain the same across instrument platforms, the other parameters may need to be adjusted to maximize sensitivity. a  Primary transition used in quantitation.

(e)  For vitamins B 3 and B 6 , the reported concentration of the directly measured forms are summed to report total. For example, concentration of nicotinamide and nicotinic acid are summed to report “Total B 3 ” and concentration of pyridoxal, pyridoxamine, and pyridoxine are summed to report “Total B 6 .” Thiamine and riboflavin do not require this step as their forms are hydrolyzed into a single directly measured form. References: J. AOAC Int . 99 , 776(2016)

where [Vit] WSx = vitamin concentration in the working standard in ng/mL; [Vit] MWS = concentration of vitamin in the MWS in ng/mL; Vol MWS = volume of the MWS in μL; and 500 = dilution factor. (d)  Vitamin concentration calculated in product from analytical result:

DOI: 10.5740/jaoacint.15-0315 (First Action) J. AOAC Int . (future issue) (Final Action) DOI: 10.1093/jaoacint/qsaa012 AOAC SMPR 2015.002 (Vitamin B 1 ) J. AOAC Int . 98 , 1094(2015) DOI: 10.5740/jaoac.int.SMPR2015.002

where [Vit] sample = vitamin concentration in product, μg/kg; [Vit] AS = vitamin mass in the analytical sample as calculated from calibration curve, ng/mL; RW = reconstitution weight (total), g, for direct weight (liquid) samples RW = 1; SW = analytical sample weight, g; PW = powder weight (for reconstituted samples), g, for liquid samples, this value is 1; and 500 = dilution factor.

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AOAC SMPR 2015.003 (Vitamin B 2 ) J. AOAC Int . 98 , 1096(2015) DOI: 10.5740/jaoac.int.SMPR2015.003 AOAC SMPR 2015.004 (Vitamin B 3 ) J. AOAC Int . 98 , 1098(2015) DOI: 10.5740/jaoac.int.SMPR2015.004 AOAC SMPR 2015.005 (Vitamin B 6 ) J. AOAC Int . 98 , 1100(2015) DOI: 10.5740/jaoac.int.SMPR2015.005

Posted: October 2018 (Revised First Action 2018 to include matrix extension to vitamin B 3 ); October 2019 (Final Action pre- publication); April 2020 (Final Action final publication)

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AOAC Official Method 2016.01 Salmonella spp. in Select Foods and Environmental Surfaces 3M™ Molecular Detection Assay (MDA) 2– Salmonella Method First Action 2016 [Applicable to detection of Salmonella spp. in raw ground beef (73% lean), raw ground chicken, chicken carcass rinse, chicken carcass sponge, pasteurized liquid whole egg, cooked breaded chicken, instant nonfat dry milk, black pepper, cocoa powder, raw whole shrimp, raw bagged spinach, creamy peanut butter, dry dog food, pasteurized processed American cheese, spent sprout irrigation water, and sealed concrete, stainless steel, and sealed ceramic tile environmental surfaces.] Caution : The 3M MDA 2– Salmonella 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– Salmonella 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– Salmonella has only been evaluated for use with the enrichment media specified in the manufacturers instructions for use. The 3MMDS 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 MDA 2– Salmonella . Retain the 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 DNA removal solution. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the Material Safety Data Sheet for additional information and local regulations for disposal. To reduce the risks associated with exposure to chemicals and biohazards, ( 1 ) perform pathogen testing in a properly equipped laboratory under the control of trained personnel; ( 2 ) always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples; ( 3 ) avoid contact with the contents of the enrichment media and reagent tubes

after amplification; and ( 4 ) 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. See Tables 2016.01A and 2016.01B for a summary of results of the interlaboratory study supporting acceptance of the method. A. Principle The 3M MDA 2– Salmonella method is used with the 3M MDS for the rapid and specific detection of Salmonella in enriched food, feed, and food-process environmental samples. The 3M MDA 2– Salmonella 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, whereas negative results are displayed after the assay is completed. Samples are preenriched in ISO BPW. B. Apparatus and Reagents Items ( b) –( g ) are available as the 3M MDA 2– Salmonella kit from 3M Food Safety (St. Paul, MN, USA). (a)  3M MDS .—MDS100 (3M Food Safety). (b)  3M MDA 2–Salmonella reagent tubes .—Twelve 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 .—3M Food Safety. (i)  3MMolecular Detection heat block insert .—3M Food Safety. (j)  3M Molecular Detection cap/decap tool for reagent tubes .— 3M Food Safety. (k)  3M Molecular Detection cap/decap tool for lysis tubes .— 3M Food Safety. (l)  Empty lysis tube rack .—3M Food Safety. (m)  Empty reagent tube rack .—3M Food Safety. (n)  ISO BPW .—3M Food Safety. Formulation equivalent to ISO 6579:2002 Annex B or 3M equivalent. (o)  Disposable pipet .—Capable of 20 μL. (p)  Multichannel (eight-channel) pipet .—Capable of 20 μL. (t)  Thermometer .—Calibrated range to include 100 ± 1°C. (u)  Dry block heater unit .—Capable of maintaining 100 ± 1°C. (v)  Incubators .—Capable of maintaining 37 ± 1°C or 41.5 ± 1°C. (w)  Refrigerator .—Capable of maintaining 2–8°C, for storing the 3M MDA components. (x)  Computer .—Compatible with the 3M MDS instrument. (y)  3M Enviroswab .—Hydrated with Letheen Broth (3M Food Safety, Banglore, Australia). (z)  3M hydrated sponge stick with 10 mL D/E .—3M Food Safety. C. General Instructions ( 1 ) Store the 3M MDA 2– Salmonella at 2–8°C. Do not freeze. Keep kit away from light during storage.After opening the kit, check (q)  Sterile filter-tip pipet tips .—Capable of 20 μL. (r)  Filter Stomacher ® bags .—Seward or equivalent. (s)  Stomacher .—Seward or equivalent.

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