54 / 363
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
Candidates for 2016 Method of the Year
53