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  1551

( 2 )  See Table 2011.06A for the appropriate amount of the filtered extract to be added to each well. (Add in several portions. Well capacity is 2 mL.) ( 3 ) Elute at about 2 mL/min. Again, take care to avoid completely drying the sorbent for long periods. ( n )  Wash the SPE sorbent .—With the manifold’s valve 3/4 open, add 1 mL water to each well. Repeat two times with 1 mL water, eluting at approximately 3–5 mL/min. ( o )  Elute the analyte from the sorbent .—( 1 ) With the manifold valve completely open, remove the waste reservoir from the manifold and replace it with the receiving 96-well vessel. (Make sure the receiving vessel is aligned with the 96-well SPE plate.) ( 2 ) Load each well with 2 mL solvent for SPE elution and folates standard (methanol containing 10% formic and 1% ascorbic acid). ( 3 ) Allow gravity to elute the eluent into the receiving vessel. (Very low vacuum may be applied to facilitate elution, but do not exceed 1 mL/min flow.) ( p ) When elution is complete, turn off the low-vacuum pump and remove the 96-well receiving vessel from the manifold. ( q ) Transfer the eluent in the wells into vials for UPLC-MS/MS analysis ( take care to properly label the samples when tranferring into vials ). Label one vial “standard” and fill with folate analytical standard solution. Label one vial “blank” and fill with solvent for SPE elution and folates standard (methanol with 10% formic acid and 1% ascorbic acid). Prepare two vials for the RM (or NIST SRM 1846). The first vial is run at the start of the run and the second is a check at the end of the run. ( r )  Prepare the UPLC system and mass spectrometer for analysis .—( 1 ) Refer to supplemental information (preparing and running UPLC-MS/MS). ( 2 ) Load the autosampler in the UPLC autosampler with the vials from F ( q ). ( s ) Analyze the samples using the instrumental methods ( see Tables 2011.06B–E for details). ( t ) Check that all peaks are marked correctly and that the IS peak areas do not show interference (IS peak areas similar to IS peak areas of RM or NIST control samples). Rerun sample weighing up less sample if interference is suspected. G. Calculations ( a ) Determine response factors (RF) for folate vitamers vs IS ( 13 C-folic acid) on UPLC-MS/MS. RFs are determined for each daughter ion (two daughter ions per analyte). check at the end of the run. ( r ) Prepare the UPLC system and mass spectrometer for analysis .—( 1 ) Refer to supplemental information (preparing and running UPLC-MS/MS). ( 2 ) Load the autosampler in the UPLC autosampler with the vials from F ( q ). ( s ) Analyze the samples using the instrumental methods ( see Tables 2011.06B–E for details). ( t ) Check that all peaks are marked correctly and that the IS peak areas do not show interference (IS peak areas similar to IS peak areas of RM or NIST control samples). Rerun sample weighing up less sample if interference is suspected. G. Calculations ( a ) Determine response factors (RF) for folate vitamers vs IS ( 13 C-folic acid) on UPLC- S/ S. RFs are determined for each daughter ion (two daughter ions per analyte). Table 2011.06D. Gradient Time, min Flow rate, mL/ in % A % B Curve Initial 0.200 99.9 0.1 1.00 0.200 99.9 0.1 6 8.00 0.200 0 100 6 9.00 0.200 0 100 6 9.10 0.200 99.9 0.1 6 12.0 0.200 99.9 0.1 6 where F = RF for the analyte using a specic mass spectral daughter ion ( see Table 2011.06E ); PA 1 = peak area of IS in the standard solution; PA 2 = peak area of analyte daughter ion in the standard solution; C 1 = concentration of IS in the standard solution; C 2 = concentration of analyte in the standard solution; W = measured water fraction as determined by Karl Fisher, %; S = calcium fraction in Ca salt folate standard, % ( see Note ); and H i = vendor- stated dry weight basis of analyte i as stated on the Certicate of Analysis (% HPLC purity). Note : Only applicable for 5-formyltetrahydrofolate and 5-methyltetrahydrofolate. Percent Ca salt is 7.84 and 8.06%, respectively. % Ca = formula weight (FW) Ca  FW Ca folate salt × 100. ( b ) Determine the concentration (μg/100 g) of folate vitamer in the sample as follows (concentrations are calculated for each where F = RF for the analyte using a specific mass spectral daughter ion ( see Table 2011.06E ); PA 1 = peak area of IS in the standard solution; PA 2 = peak area of analyte daughter ion in the standard solution; C 1 = concentration of IS in the standard solution; C 2 = concentration of analyte in the standard solution; W = measured water fraction as determined by Karl Fisher, %; S = calcium fraction in Ca salt folate standard, % ( see Note ); nd H i = vendor-stated dry weight basis of analyte i as stated on the Certificate of Analysis (% HPLC purity). Note : Only li l f r 5-formyltetrahydrofolate and 5-methyltetrahydrofolate. Percent a i 7.84 and 8.06%, CAP 1 2 F   1( 2 1

using an analytical balance and accurately record the weight. Add one additional 50 mL centrifuge tube containing no sample as an enzyme blank. ( 1 ) Add 100 µL of the 50 µg/mL internal standard intermediate solution [to give 5 µg 13 C-folic acid (IS) in the sample] to each centrifuge tube. ( 2 ) Add 10 mL extraction buffer (10 mM phosphate buffer, pH 6.0, containing 1% ascorbic acid). Vortex to mix well. ( 3 ) Shake in a water bath set at 50°C for 30 min and then cool to room temperature. ( b ) To check the efficacy of the rat plasma conjugase, use 150 µL rat plasma test solution (20 µg/mL pteroyltri-γ- glutamate) in place of the homogenized sample in the 50 mL centrifuge tube. ( 1 ) Add 100 µL of the 50 µg/mL internal standard intermediate solution [to give 5 µg 13 C-folic acid (IS) in the sample]. Add 10 mL extraction buffer (10 mM phosphate buffer, pH 6.0, containing 1% ascorbic acid). Vortex to mix well. ( 2 ) Shake in a water bath set at 50°C for 30 min. Cool to room temperature. ( c ) Add 1 mL protease solution to each centrifuge tube, cover, vortex to mix, and incubate for 3 h in a water bath set at 37°C. ( d ) Inactivate the protease enzyme by placing the samples in a boiling water bath (100°C) for approximately 5 min with shaking. After inactivating the enzyme, cool the samples to room temperature. ( e ) Add 1 mL α -amylase solution to each centrifuge tube and incubate in water bath set at 37°C for 2 h. ( f ) Add 300 µL rat plasma conjugase to each centrifuge tube, cover, and incubate for 22 h in water bath set at 37°C. ( g ) After 22 h, inactivate the enzyme by placing the samples in a boiling water bath (100°C) for approximately 5 min. Cool to room temperature. ( h ) Centrifuge for a minimum of 5 min at room temperature. ( i ) Filter the supernatant through a 0.45 µm low-binding filter into a well of a 12-well reservoir. ( See Table 2011.06A for appropriate amount of supernatant to filter.) Repeat for each sample, taking care to filter samples into the wells in order. ( j )  Set up the 96-well SPE manifold .—( 1 ) Connect the manifold to the low-vacuum system. ( 2 ) Place a waste reservoir in the unit ( see instruction manual). ( k )  Condition the SPE sorbent .—( 1 ) Add 1 mL methanol to each well (use a 12-tip pipet or a robotic pipetting unit if available). ( 2 ) Turn on the low-vacuum unit and allow the conditioning solvent (methanol) to elute at a rate of approximately 2–5 mL/min. ( 3 ) Repeat with an additional 1 mL methanol. ( 4 ) Before the SPE sorbent is completely dry, repeat the process with water (2 mL in 1 mL portions). ( 5 ) Before the sorbent is completely dried of solvent, turn off the vacuum by completely opening the control valve on the manifold (refer to manifold’s manual). ( l ) Take care to avoid the sorbent being completely dry of solvent for a protracted period of time. Check and empty the waste reservoir frequently to avoid overflowing. ( m )  Load samples onto the SPE sorbent .—( 1 ) With the manifold’s valve completely open, add the filtered extracts to the wells. ( 2 ) See Table 2011.06A for the appropriate amount of the ltered extract to be added to each well. (Add in several portions. Well capacity is 2 mL.) ( 3 ) Elute at about 2 mL/min.Again, take care to avoid completely drying the sorbent for long periods. ( n ) Wash the SPE sorbent .—With the manifold’s valve 3/4 open, add 1 mL water to each well. Repeat two times with 1 mL water, eluting at approximately 3–5 mL/min. ( o ) Elute the analyte from the sorbent .—( 1 ) With the manifold valve completely open, remove the waste reservoir from the manifold and replace it with the receiving 96-well vessel. (Make sure the receiving vessel is aligned with the 96-well SPE plate.) ( 2 ) Load each well with 2 mL solvent for SPE elution and folates standard (methanol containing 10% formic and 1% ascorbic acid). ( 3 ) Allow gravity to elute the eluent into the receiving vessel. (Very low vacuum may be applied to facilitate elution, but do not exceed 1 mL/min ow.) ( p ) When elution is complete, turn off the low-vacuum pump and remove the 96-well receiving vessel from the manifold. ( q ) Transfer the eluent in the wells into vials for UPLC-MS/MS analysis ( take care to properly label the samples when tranferring into vials ). Label one vial “standard” and ll with folate analytical standard solution. Label one vial “blank” and ll with solvent for SPE elution and folates standard (methanol with 10% formic acid and 1% ascorbic acid). Prepare two vials for the RM (or NIST SRM 1846). The rst vial is run at the start of the run and the second is a Table 2011.06B. Inlet method Parameter Value Reservoir A1 10 mM Ammonium formate, pH 2.80 Reservoir B1 100% Methanol Pressure limits Lower = 0 psi; higher = 15000 p i Seal wash 5.0 min Column temperature 40°C Sample temperature 8°C Sample loop options P rtial loop with needle overll; loop ofine = 1.00 min Wash s l nts Weak = water (600 μL); strong = 50:50 water:methanol (1000 μL) Column HSS T3 UPLC column (1.8 μm; 2.1 × 150 mm) Injection volum 2 μL (Sample); 5 μL (blank)

i HSW CAP   )