Method Submissions-5th Set of Nutrients_6-30-14 - page 61

(q) Acetic acid, glacial 100%anhydrous, GR for analysis
(r) Trifluoroacetic acid
(s) D-(-)-Fructose, Biochemika, >99%
(t) Acetonitrile
(u) Sodiummaleate buffer solution (100mM, pH 6.5) - Into a large beaker (>1000mL)
weigh11.6 gofmaleic acid (puriss., >99%) anddissolvewith900mLofwater. Adjust
the pH to 6.5 with 1 M sodium hydroxide solution(p).Transfer the solution to a
1000mL volumetric flaskandmakeup to themarkwithwater.
(v) Sodium acetate buffer solution (100mM, pH 4.5) - Into a large beaker (>1000mL)
containing900mL ofwater, pipette5.8mL of glacial acetic acid (q).Adjust topH4.5
with 1 M sodium hydroxide solution(p).Transfer the solution to a 1000 mL
volumetric flaskandmakeup to themarkwithwater.
(w) Sodium hydroxide solution (50mM) - Into a 100mL volumetric flask, weigh 0.2 g of
sodium hydroxide pellets and dissolve with 90 mL of water. Make up to the mark
withwater.
(x) Sodium borohydride (10mg/mL in 50mM sodium hydroxide) – accuratelyweigh 50
mg of sodium borohydride in to a polypropylene tube and record the weight.
Immediately before use dissolve in an appropriate volume of 50 mM sodium
hydroxide solution (w)togivea final concentrationof 10mg/mL.
(y) Acetic Acid (0.2M) – Into a 1000 mL volumetric flask containing 900 mL of water,
pipette11.6mLof glacial acetic acid (q).Makeup to themarkwithwater.
(z) FructanAssayKit, K-FRUC (Megazyme International Ireland Ltd,Wicklow, Ireland).
(aa) Sucrase (~4.5 U/mL) / α-amylase (~23 U/mL) / pullulanase (~4.5 U/mL) / maltase
(~45 U/mL) solution; dissolve the contents of the sucrase / amylase /pullulanase /
maltase vial from the fructanassay kit (z)in sodiummaleatebuffer (m) following the
instructions from the kit. Divide into aliquots of 2.0 mL (enough for 9 sample
determinations) and store frozenat -20°C inpolypropylene tubesuntil required.
(bb) Fructanase solution (exo-inulinase ~ 455 U/mL + endo-inulinase ~4.5 U/mL);
dissolve the contents of the fructanase vial from fructan assay kit(z)in sodium
acetate solution(v)following the instructions from the kit . Divide into aliquots of
2.0 mL (enough for 18 sample determinations) and store frozen at -20°C in
polypropylene tubes until required.
(cc)SPEwash solution - Into a100mL flask, add80mL of acetonitrile and100µL of TFA.
Makeup to themarkwithwater.
FOS-03
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