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1396  Jing et al. : J ournal of AOAC I nternational Vol. 98, No. 5, 2015

( b )  Nitric acid. —69–70% reagent grade, J.T. Baker (Avantor Center Valley, PA) or equivalent. ( c )  Ammonium acetate. —Fluka (Sigma-Aldrich, St. Louis, MO) LC/MS additive grade or equivalent. ( d )  Ammonium hydroxide. —28–30% reagent grade [for adjusting pH in mobile phase also; check before use; MACRON (Avantor Center Valley, PA) or equivalent)]. ( e )  Deionized laboratory water.— ≥18 Mohm/cm (EMD D. Standards ( a )  L-Carnitine .—USP Reference Standard Cat. No. 1359903 or equivalent; store desiccated. ( b )  L-Carnitine-d 3 HCl (methyl-d 3 ) .—Cambridge Isotope Laboratories Inc. (Andover, MA) DLM-1871-0.1; store refrigerated. ( c )  Choline chloride .—USP Reference Standard Cat. No. 1133547 or equivalent. ( d )  Choline chloride-d 9 (trimethyl-d 9 98%) .—DLM-549-1 (Cambridge Isotope Laboratories Inc.). E. Preparation of Reagents/Standard Solutions ( a )  Mobile phase.— ( 1 ) Mobile phase A. —10 mMammonium acetate [water–acetonitrile (95 + 5, v/v)]. Quantitatively transfer 770 (±15) mg ammonium acetate into a 1 L glass bottle and dissolve with 950 mL laboratory water. Adjust pH up to 8.2–8.6 by ammonium hydroxide, C ( d ). Record the amount of ammonium hydroxide added. Add 50 mL acetonitrile to the bottle and mix well. Store at room temperature. Expiration: 1 week. ( 2 )  Mobile phase B. —10 mM ammonium acetate [water–acetonitrile (5 + 95, v/v)]. Weigh 770 (±15) mg ammonium acetate into a 100 mL beaker. Dissolve with 50 mL laboratory water. Add same amount of ammonium hydroxide as added into mobile phase A and then mix well. Quantitatively transfer the 50 mL solution into a 1 L glass bottle. Add 950 mL acetonitrile into the bottle and mix well. Store at room temperature. Expiration: 2 weeks. ( b )  Native stock solutions.— ( 1 ) Choline hydroxide (approximately 3000 µg/mL).— Store at 2–8°C. Expiration: 1 month (currently trying to extend to 1 year). ( a ) Dry approximately 200 mg choline chloride USPReference Standard Millipore Corp. Billerica, MA, or equivalent). ( f )  pH buffer solutions .—pH 4.0, 7.0, 10.0.

(BEH) HILIC 1.7 µ m, 2.1 × 100 mm (Waters Corp., Milford, MA) or equivalent. ( b )  Liquid chromatograph. —Waters Acquity UPLC Binary or equivalent. ( c )  Solvent manager. —Capable of 15 000 psi or equivalent. (Waters Corp.) ( d )  Detector. —Waters Acquity TQD or Xevo TQS triple quadrupole mass spectrometer (Waters Corp.) or equivalent. ( e )  Injector. —Waters Acquity sample manager with integrated column oven or equivalent. ( f )  Autosampler .—Waters Acquity with cooler and vials, or equivalent. ( g )  Nitrogen generator .—Peak Scientific (Billerica, MA) Model NM30LA or equivalent. ( h )  Data system. —Waters MassLynx, latest revision or equivalent. ( i )  Microwave digestion system. —MarsXpress (CEM, Matthews, NC) or equivalent. ( j )  Reaction vessel. —50 mL Teflon ® (CEM) or equivalent. ( k )  Centrifuge tubes. —Polypropylene, 50 mL capacity, disposable, or equivalent. ( l )  Vortex mixer with flat and cone top. ( m )  Balance 1.— Readable to at least 0.0001 g (Mettler- Toledo AT200, Columbus, OH) or equivalent. ( n )  Balance 2.— Readable to at least 0.001 mg (Mettler- Toledo XP 6) or equivalent. ( o )  Syringe.— 1 mL Luer tip. ( p )  Filters. —0.45 µm nylon membrane with syringe tip or equivalent. ( q )  Pipets.— 5–25, 50–250, and 100–1000 μL adjustable. ( r )  Pipet tips. ( s )  pH meter. ( t )  Volumetric flasks.— 10, 50, 100, 250 mL. C. Chemicals and Solvents ( a )  Acetonitrile. —Fisher Optimal (Fisher Scientific, Fair Lawn, NJ) LC/MS grade, or equivalent. Table 2014.04A. Volumes of each stock solution for IWS IWS Stock vol., µL MIX-IS, µL Final vol., mL 1 25 100 50 2 125 100 50 3 250 100 50

Table 2014.04B. Concentration of standards

µg/mL

Standard

Choline

Choline chloride-d 9

Carnitine

Carnitine chloride-d 3

Diluent

Final vol., mL

IWS 1 IWS 2 IWS 3

1.5 7.5

1.6 1.6 1.6

0.375 1.875 3.750

0.8 0.8 0.8

50 50 50 10 10 10

Water Water Water

15

ng/mL

90%

MeCN a MeCN MeCN

WS 1 WS 2 WS 3

30

32 32 32

7.5

16 16 16

150 300

37.5

75

a  MeCN = Acetonitrile.