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Reddy:

J

ournal of

AOAC I

nternational

V

ol.

98, N

o

. 4, 2015 

1137

(

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.

(

m

) 

EDC.

—0.25 M in 3% pyridine in ethanol.

(

1

) Weigh 1.20 ± 0.05 g EDC in weighing pan.

(

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.

(

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.

Table 2015.04A. Preparation of calibration standards

Standard

Working

standard

(1 + 10), μL

Working

standard, μL

Working

internal

standard, μL

Acetonitrile–water

(60 + 40, v/v), μL

1

10

40

950

2

20

40

940

3

50

40

910

4

10

40

950

5

50

40

910

6

100

40

860

Table 2015.04B. Instrumental conditions

Chromatography

Flow rate

300 µL/min

Column temperature

40

°

C

Injection volume

10 μL

Sample temperature

Ambient

Gradient program

Time

% A

% B

Curve

0.00

90

10

6

1.00

90

10

6

7.00

45

55

6

9.00

0

100

6

9.01

90

10

6

12.00

90

10

6

MS tune

Ionization mode

ESI-

Cone gas

250 L/h

Capillary

0.5 kV

Nebulizer

7.0 bar

Source offset

20.0 V

Collision gas flow

0.15 mL/min

Source temperature

150

°

C

Quad 1 resolution

Unit mass (0.75 Da FWHM)

Gas temperature

350

°

C

Quad 2 resolution

Unit mass (0.75 Da FWHM)

Desolvation gas flow

900 L/h

MS calibration range

50–2000 amu

MS/MS transitions

Compound

Parent mass,

m/z

Daughter mass,

m/z

Dwell, s

Cone (V)

Collision energy , V

MFA-2NPH

212

182

0.15

20

15

MFA-2NPH_IS

216

186

0.15

20

15

256