AOAC OMA# 2011.06 Final Action Review-OMB

S zpylka et al .: J ournal of AOAC I nternational V ol . 95, N o . 6, 2012  1549

Table 2011.06B. Inlet method Parameter

Table 2011.06D. Gradient Time, min Flow rate, mL/min % A

Value

% B

Curve

Reservoir A1 Reservoir B1 Pressure limits

10 mM Ammonium formate, pH 2.80

Initial 1.00 8.00 9.00 9.10 12.0

0.200 0.200 0.200 0.200 0.200 0.200

99.9 99.9

0.1 0.1

100% Methanol

6 6 6 6 6

Lower = 0 psi; higher = 15000 psi

0 0

100 100

Seal wash

5.0 min

Column temperature Sample temperature Sample loop options

40°C

99.9 99.9

0.1 0.1

8°C

Partial loop with needle overfill; loop offline = 1.00 min

Concentration, µg/mL = [(standard, g × purity of analyte)] × (10 6 µg/g) ÷ (final volume, mL)

Wash solvents

Weak = water (600 μL); strong = 50:50 water:methanol (1000 μL)

Column

HSS T3 UPLC column (1.8 μm; 2.1 × 150 mm) 2 μL (Sample); 5 μL (blank)

( 5 ) Folic acid, 13 C-folic acid (IS), (6 S )-5-formyl-5,6,7,8- tetrahydrofolate, 10-methylfolic acid, and 10-formylfolic acid solutions are stable for up to 180 days (6 months) stored in –70°C freezer. ( 6 ) (6 R,S )-5-methyl-5,6,7,8-tetrahydrofolate and 5,6,7,8- tetrahydrofolate solutions are stable for only 90 days (3 months) stored in –70°C freezer. ( 7 ) Pteroyltri-γ-L-glutamate (FA-glu 3 ) is stable for 12 months (360 days) stored in –70°C freezer. ( 8 ) Other folate vitamers, such as 5,10-methylene-5,6,7,8- tetrahydrofolate and 7,8-dihydrofolic acid, are unstable and are oxidized to folic acid under these conditions. They were subsequently not used in the quantitation. ( e )  Folates intermediate standard solution.— 20 µg/mL. ( 1 ) Prepare a 100 mL folates intermediate solution such that each vitamer concentration is 20 µg/mL. The amount of each folate stock solution to add to the 100 mL low-actinic volumetric flask is determined using the following equation: Volume of folate vitamers stock solution to pipet into flask, mL = 100 mL × (20 µg/mL ÷ folate vitamer stock solution concentration, µg/mL) ( 2 ) Add approximately 50 mL folate intermediate standard solution solvent and sonicate in water bath set at 40°C for approximately 10 min to thoroughly mix. ( 3 ) Bring to volume with folate intermediate standard solution solvent and store at approximately –20°C (refrigerator freezer). This intermediate standard is stable for up to 30 days when stored at this condition. ( f )  Internal standard intermediate standard solution .—50 µg/mL. ( 1 ) Prepare a 100 mL internal standard solution such that the IS concentration is 50 µg/mL. The amount of internal standard stock solution to add to the 100 mL low-actinic volumetric flask is determined using the following equation: Volume of internal standard stock solution to pipet into flask, mL =100 mL × (50 µg/mL ÷ internal standard stock solution concn, µg/mL) ( 2 ) Bring to volume with the folate intermediate standard solution solvent and store at approximately –20°C until use. The solution is stable for up to 60 days when stored at this condition. ( g )  Folates analytical standard solution .—2 µg/mL. ( 1 ) Pipet 1 mL folates intermediate standard solution

Injection volume

( 4 ) Transfer the solution to a 1 L volumetric flask and bring to volume with DI water. ( c )  Solvent for SPE elution and folates standard.— Methanol with 10% formic acid and 1% ascorbic acid. ( 1 ) Into a 100 mL volumetric flask, transfer 10 mL formic acid and 1 g ascorbic acid. Add approximately 50 mL methanol. ( 2 ) Sonicate in a water bath set at approximately 25°C until complete dissolution. ( 3 ) Bring to volume with methanol. Use solution to elute folates from SPE sorbent after cleanup, to make analytical standard of folates, and as a blank in UPLC-MS/MS analysis. ( d )  Folates stock solution.— 500 µg/mL. ( 1 ) Accurately weigh 50 mg each of folic acid (FA), 13 C-folic acid (IS), 5,6,7,8-tetrahydrofolate (THF), (6 R,S )-5- methyl-5,6,7,8-tetrahydrofolate (5-CH 3 -THF), (6 S )-5-formyl- 5,6,7,8-tetrahydrofolate (5-CHO-THF), 10-methylfolic acid (10-CH 3 -FA), 10-formylfolic acid (10-CHO-FA), and pteroyltri- γ-glutamate (FA-glu 3 ) into separate 100 mL low-actinic volumetric flasks. ( 2 ) Add to each flask approximately 75 mL folates stock solution solvent and sonicate in a bath set at 40°C for about 20 min, or until complete dissolution. Add several drops of ammonium hydroxide to aid in dissolution if necessary. ( 3 ) Bring to volume with the folates stock solution solvent and transfer to freezer safe containers. ( 4 ) Calculate the concentration of the solutions:

Table 2011.06C. MS/MS tune method Parameter

Value

Capillary voltage, KV

3.00

Cone voltage, V

22

Extractor, V

5

RF lens, V

0.0

Source temperature, °C

120 300 700

Desolvation temperature, °C

Desolvation gas flow, L/h

Cone, L/h

60