AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

8.) Filter supernatant through 0.45um PVDF filter into disposable glass culture tubes (16x125mm) 9.) Set up 24-well manifold 10.) Condition the SPE as follows (no vacuum used): a.) Add 1mL methanol b.) Repeat with another 1mL methanol c.) Before SPE sorbent is dry repeat with water (2mL in 1mL portions) 11.) Loading Samples onto SPE sorbent (vacuum not used): a.) Add all of filtered supernatant to SPE sorbent (approx. 12mL). b.) Elute by gravity (no vacuum) c.) Wash with 1mL of water to each well. Repeat 2x’s with 1mL of water. d.) Use a syringe to push out last remnants of water 12.) Eluting the analyte from the sorbent: a.) Empty waste reservoir and add disposable 2mL microfuge tubes to collect eluent. b.) Load each well with 1mL of Solvent for SPE Elution and Folates Standard (methanol containing 10% formic and 1% ascorbic acid.) c.) Allow gravity to elute the eluent into the tubes. Use syringe to push out residual solvent d.) Add 50uLAmmonium hydroxide, vortex, centrifuge 12000rpm for 10min. 13.) Transfer eluent in the wells into vials for LC-MS/MS analysis 14.) Prepare HPLC and Mass Spectrometer for analysis. Standard Analysis: Calibration Curve: Calibration curve is drawn by preparing mixed working standard solutions of different concentrations containg all folate compounds at Concentrations of: 1, 2, 5, 20, 50, 200 and 500 ng/mL in 90% MeOH + 10% Formic acid + 1% ascorbic acid. Each level of standard also contains internal standard at concentration of 400 ng/mL. Single Level std. processed like sample: An aliquot mixed std. is taken containing about 50 ng of each of the folate compound. This is processed like sample. LC-MS Analysis of Folates: HPLC Conditions: Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 µm, 2.1 mm X 50 mm) with 1% acetic acid and methanol as mobile phase with a varied flow rate was used for HPLC of folate compounds. LC Conditions using Dionex 3000 Ultimate in front of APPI 5500 (Qtrap) detector:

Table 5: LC Conditions using Dionex 3000 Ultimate in front of APPI 5500 (Qtrap) detector

Time(min)

Flow (mL/min)

B%

0.00 0.50 5.00 5.05

0.500 0.500 0.500 0.500

0.0 0.0

40.0 100.0

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