AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Table 2012.11A. Volumes required for working standard solutions

Table 2012.11C. Chromatographic parameters Instrument parameter

Waters Acquity UPLC

ISTD vol., µL a

SIIS vol., µL b

Vol. MeOH, µL

Vol. water, µL

Final vol., mL

Mobile phase A Mobile phase B

2 mM ammonium formate in water

Std

2 mM ammonium formate in methanol

WS1 WS2 WS3 WS4 WS5

45 90

75 75 75 75 75

1780 1740 1680 1326

100 100 100 100 100

2 2 2 2 2

Flow rate

500 µL/min 500 µL/min

150 500

Flow rate into MS

Column

Waters HSST 3 1.7 µm, 2.1 × 150 mm

1000

825

Column temperature

60°C 20 µL

a  ISTD = Vitamin D 2 3 mixed intermediate standard. b  SIIS = Stable isotope-labeled internal standard solution. /D

Injection volume

Injection type

Full loop Automatic Automatic

Presample air boundary Post-sample air boundary

( 1 ) Quantitatively transfer the entire contents each of an ampule of trideuterated D 2 and D 3 (1 mg) to a 200 mL volumetric flask using LC-MS-grade methanol to aid in dissolution of the standards and to wet the flask. Dilute to 200 mL with HPLC-grade methanol once the contents of both ampules have been completely transferred to the flask. ( f )  Working standards .—Prepare fresh on day of analysis. Expiration: 48 h. ( 1 ) Add 100 μL water, 75 μL SIIS mixture, the specified volume of native standard mix (ISTD), and methanol ( see Table 2012.11A ). ( 2 ) Cap and mix well. ( 3 ) Transfer to an amber autosampler vial and cap. F. Procedure—Sample Preparation ( a ) Accurately weigh designated sample size into a 50 mL centrifuge tube ( see Table 2012.11B ). ( b ) Add 75 μL SIIS and vortex for 10 s to mix. ( c )  Saponification .— ( 1 ) Add about 0.4 ± 0.1 g sodium ascorbate and vortex for a minimum of 10 s. ( 2 ) Add 6 ± 0.3 mL HPLC-grade methanol and vortex for 15 s. ( 3 ) Add 4 ± 0.3 mL of 45% potassium hydroxide solution and immediately vortex for a minimum of 20 s to thoroughly mix contents. ( 4 ) Place tubes in a preheated water bath at 75 ± 2°C for a minimum of 30 min making sure to vortex for approximately 5 s at approximately 10 and 20 min intervals. ( 5 ) After 30 min, remove the tubes from the water bath and promptly place into an ice bath for a minimum of 30 min to bring

Sample temperature

Ambient

Injector wash parameters

Weak wash solvent Weak wash volume Strong wash solvent Strong wash volume

Water

1000 µL Methanol

600 µL

Gradient method

Gradient detail

% A

% B

Curve

Time, min

0.0 3.0 6.0 8.0 8.5

10 10

90 90

1 6 7 6 6 6

0 0

100 100

10 10

90 90

12.0

( 1 ) Add 5 ± 0.3 mL acetonitrile to each tube, cap, and vortex at a moderate speed for a minimum of 5 s. ( 2 ) Add 22 ± 1 mL extraction solvent (20 + 80, ether–pentane) and shake in a wide, semicircular arc across the front of the body 20 times. Invert the tubes with each stroke. ( 3 ) Briefly centrifuge at moderate speed (approximately 300 × g for 1.5 min) to complete layer separation. ( 4 ) Draw off the clear ether–pentane and transfer the top layer to a 50 mL conical centrifuge tube, leaving behind a few milliliters of the ether–pentane mixture. ( 5 ) Evaporate to dryness the transferred ether–pentane layer in a 40 ± 4°C water bath, using a flow of nitrogen over the sample. ( 6 ) Remove the centrifuge tubes from the water bath as soon as evaporation is complete. Observe the extract, which should appear as a white or yellow film. Make sure the extract is completely dried before reconstitution. ( 7 ) Immediately reconstitute with 1.9 mL methanol and vortex for 10 s or until the solids have dissolved. ( 8 ) Add 100 μL laboratory water and vortex for an additional 5 s. ( 9 ) Filter reconstituted sample into HPLC autosampler vial using a PTFE syringe filter.

them rapidly to room temperature. ( d )  Liquid–liquid extraction .—

Table 2012.11B. Approximate sample weights Sample type

Sample size, g

Ready-to-feed

12

Concentrated liquids a

6

Powder products b 12 a  Reconstitute powder samples by weighing 25 g into 200 g laboratory water, and mix thoroughly to generate a homogenous mixture prior to sampling for analysis. b  Concentrated liquids are diluted 1:1 with laboratory water after weighing into sample tube.

© 2012 AOAC INTERNATIONAL