AOACSPIFANMethods-2017Awards

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Reddy: J ournal of AOAC I nternational V ol. 98, N o . 4, 2015  1137

( 2 ) Transfer to suitable storage vessel (minimum 25 mL capacity with a wide mouth). ( 3 ) Add 24 mL 3% pyridine in ethanol and mix well. Expiration 5 days. ( n )  Mobile phase buffer .—200 mM ammonium acetate in water. ( 1 ) Weigh 1.54 ± 0.05 g ammonium acetate in weighing pan. ( 2 ) Transfer to 100 mL volumetric flask with water. ( 3 ) Add 50 mL water and swirl until dissolved. ( 4 ) Fill to volume with water and mix well. Expiration 5 days. ( o )  Mobile phase A .—10 mM ammonium acetate in water. ( 1 ) Transfer 25 mL mobile phase buffer to 500 mL mobile phase bottle. ( 2 ) Add 475 mL water and mix well. ( 3 ) Sonicate for 5 min. Expiration 5 days. ( p )  Mobile phase B .—10 mM ammonium acetate in acetonitrile–water (95 + 5, v/v). ( 1 ) Transfer 25 mL mobile phase buffer to 500 mL mobile phase bottle.

Table 2015.04A. Preparation of calibration standards

Working standard (1 + 10), μL

Working internal standard, μL

Working standard, μL

Acetonitrile–water (60 + 40, v/v), μL

Standard

1 2 3 4 5 6

10 20 50

40 40 40 40 40 40

950 940 910 950 910 860

10 50

100

( 3 ) Add 50 mL ethanol and mix well. ( k )  Pyridine, 3% in ethanol. ( 1 ) Transfer 3 mL pyridine to 100 mL glass bottle. ( 2 ) Add 97 mL ethanol and mix well. ( l )  2-NPH .—Approximately 0.1 M in 0.1 N HCl–ethanol (50 + 50, v/v). ( 1 ) Weigh 0.375 ± 0.010 g wet powder into 25 mLvolumetric flask. ( 2 ) Add ~20 mL 0.1 N HCl–ethanol (50 + 50, v/v). ( 3 ) Sonicate and swirl until dissolved. ( 4 ) Fill to volume with 0.1 N HCl–ethanol (50 + 50, v/v). Mix well. Expiration 5 days.

( 2 ) Add 475 mL acetonitrile and mix well. ( 3 ) Sonicate for 5 min. Expiration 5 days. ( q )  Calibration standards (STD 1–6) .

( 1 ) Add volume aliquots (µL) listed in Table 2015.04A to glass centrifuge tubes. For method blank, use 1 mL acetonitrile– water (60 + 40, v/v). ( 2 ) Vortex.

( m )  EDC. —0.25 M in 3% pyridine in ethanol. ( 1 ) Weigh 1.20 ± 0.05 g EDC in weighing pan.

Table 2015.04B. Instrumental conditions

Chromatography

Flow rate

300 µL/min

Column temperature

40 ° C 10 μL

Injection volume

Sample temperature Gradient program

Ambient

Time 0.00 1.00 7.00 9.00 9.01 12.00

% A

% B

Curve

90 90 45

10 10 55

6 6 6 6 6 6

0

100

90 90

10 10

MS tune

Ionization mode

ESI-

Cone gas Nebulizer

250 L/h 7.0 bar

Capillary

0.5 kV 20.0 V 150 ° C 350 ° C 900 L/h

Source offset

Collision gas flow Quad 1 resolution Quad 2 resolution MS calibration range

0.15 mL/min

Source temperature Gas temperature Desolvation gas flow

Unit mass (0.75 Da FWHM) Unit mass (0.75 Da FWHM)

50–2000 amu

MS/MS transitions

Compound MFA-2NPH

Parent mass, m/z Daughter mass, m/z

Dwell, s

Cone (V)

Collision energy , V

212 216

182 186

0.15 0.15

20 20

15 15

MFA-2NPH_IS