AOAC OMA# 2011.06 Final Action Review-OMB

1886  B handari et al . : J ournal of AOAC I nternational V ol . 101, N o . 6, 2018

( g )  HPLC mobile phases (Mobile Phase A: 1% acetic acid; Mobile Phase B: methanol) .—For Mobile Phase A, thoroughly mix 500 mL water (LC/MS) and 5 mL glacial acetic acid. For Mobile Phase B, use methanol as is (LC/MS). E. Sample Preparation ( a )  Sample processing to make them homogenous .—All samples should be as uniform and representative of the product as possible. This should be accomplished by a thorough mixing or stirring of the sample before sampling. The dry samples are reconstituted to liquid samples, and the reconstituted liquid samples are used for analysis. ( b )  Reconstitution of powder sample into liquid .—Weigh 25.0 g ± 0.1 g of dry sample into a low-actinic glass 250 mL beaker or a bottle. Cover the regular bottle or beaker with aluminum foil if low-actinic bottles or beakers are not available. Note the weights. Add 200.0 g ± 0.1 g warm water (40 ± 5°C). Mix well until complete dissolution/suspension making sure there are no lumps. Record the final weight for each sample. Calculate weight (grams) of dry sample in the corresponding reconstituted liquid sample in every case. The dry samples are analyzed as reconstituted liquid samples and the ready-to-feed (RTF) samples are analyzed as is. The reconstituted samples as well as RTF samples are thoroughly mixed immediately before weighing the aliquot for the analysis. F. Procedure ( a )  Extraction .—( 1 ) Weigh 0.1000 to 2.000 g of sample, depending on anticipated total folate (in range of 20–500 ng), into a 50 mL centrifuge tube and record to 0.0001 g. ( 2 ) Prepare a method blank containing reagents only. This will be treated like a sample through the entire following method. ( 3 ) Add 10 mL extraction buffer (containing internal standards) and 50 μL of TCEP solution to each tube and mix with a vortex mixer. ( 4 ) Add 1mLprotease solution to each tube, fill the headspace with nitrogen, cover, and mix with a vortex mixer. Incubate for 3 h in a shaking water bath at 37°C at 60 rpm shaking speed. ( 5 ) Inactivate the protease by placing the tubes in a boiling water bath (100°C) for 5 min; shake the tubes every 1.5 min, and then remove from bath and cool to room temperature. ( 6 ) Add 1 mL α-amylase solution (charcoal treated) and 300 μL of rat plasma conjugase (charcoal treated) to each tube; fill the headspace with nitrogen, cover, and incubate for 16 h in a shaking water bath at 37°C at 60 rpm shaking speed. ( 7 ) Inactivate enzymes after 16 h incubation by placing the tubes in a boiling water bath (100°C) for 5 min. Shake tubes every 1.5 min, then remove from bath and cool to room temperature. ( 8 ) Centrifuge the tubes at 4 to 5°C for 10 min at 1400 × g . ( 9 ) Filter supernatant from each tube through a 0.45 μm PVDF syringe filter into separate labeled glass tubes (16 × 125 mm). ( b )  Extract purification .—( 1 ) Set up a 24-well SPE manifold. Set up one SPE tube for every sample and one for the method blank. ( 2 ) Condition each SPE tube as follows, without vacuum. First, condition with 2 mL methanol, and before the SPE sorbent is dry, repeat the first step with 2 mL water. ( 3 ) Load samples into corresponding activated SPE tubes over sorbent, without vacuum. First, load all of the

filtered supernatant (about 12 mL) for every sample into the corresponding SPE (no vacuum) tube. Then, wash each SPE tube with 3 mL water with no vacuum. Push out any residual water by gentle push with a pipette bulb. ( 4 ) Elute the analytes from the SPE sorbent. Empty the waste reservoir and place a clean microcentrifuge tube to collect the eluent corresponding to every SPE tube. Add 1 mL of the freshly prepared solvent for SPE elution to each SPE tube.Allow gravity to elute the effluent into the tubes and use a pipette bulb to push out any remaining solvent. Add 50 μL ammonium hydroxide (28–30%) to each tube and immediately mix with a vortex mixer for 20 s. Centrifuge the tubes in a microcentrifuge at 9500 × g for 5 min. Transfer supernatant from each tube into a corresponding HPLC vial for LC-MS/MS analysis. Prepare the LC and mass spectrometer for the analysis and load the vials in the autosampler. The sequence of the analysis includes the instrument blank, calibration standards (G through A; Table 2011.06A ), an instrument blank (neutralized SPE elution solvent), the method blank, and all of the samples. ( c )  Instrumental analysis .—Instrument blank, calibration standards G through A, as detailed in Table 2011.06A , are analyzed. These are followed by the analysis of an instrument blank, followed by the method blank and the sample extracts. The following are the LC conditions with Dionex Ultimate 3000 LC/Agilent 1290 LC: LC column, Waters Acquity UPLC HSS T3 (100Å), 1.8 μm, 2.1 × 50 mm; mobile phases, A = 1% acetic acid and B = 100% methanol; solvent composition, gradient (Table 2011.06B ); column temperature of 40°C; autosampler temperature of 5 ± 1°C; and injection volume of 2 μL. MS source conditions for some of the Sciex Triple Quads are provided in Table 2011.06C . Multiple reaction monitoring transitions for different folates are provided in Table 2011.06D .

Table 2011.06B. LC gradient

Mobile Phase B a , %

Time, min

Flow, mL/min

0.00 0.50 5.00 5.05 6.50 6.55 9.00

0 0

0.5 0.5 0.5 0.5 0.5 0.5

40

100 100

0 0

0.5 a  Mobile Phase B = Methanol. The Mobile Phase refers to 1% acetic acid in water.

Table 2011.06C. MS source conditions for some of the Sciex Triple Quads

Sciex Triple Quad

Source condition parameters

5500 6500 Qtrap 6500 plus

Curtain gas (nitrogen), psi

25

30

30

Collision-activated dissociation Medium Medium

8

Ion spray voltage, V

5500

5500

5500

Temperature, °C

700

400

400

Ion source gas 1 (zero air), psi Ion source gas 2 (zero air), psi

60 50

60 50

40 50