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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
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Effect of Methanol concentration in the injection solvent
The initial calibration work and the injection solvent for LC-MS analysis of the analytes used in the
AOAC proposed method contains methanol at 20% in a mixture with 80% of 1% acetic acid solution
along with 1% ascorbic acid (20:80:1 v/v/w). In the subsequent work we found a way to make the
method a little bit less time consuming and labor intensive and most important to improve analyte
stability which requires use of 90% methanol instead of 20% methanol. This experiment was
performed to make sure higher methanol does not affect the analytical response of the analytes . It has
been established earlier that injection of analytes in 90% methanol provides a satisfactory
chromatography. The response of analytes at 5 ppb and 500 ppb level was investigated with injection
solvent low in methanol (20%) and high in methanol (90%). The results are presented in Table 6. It can
be seen that higher methanol level provided a superior response in most cases or equivalent response.
Thus the standard solutions and extract for the analysis can be prepared in solutions containing 90%
methanol without any loss in the analytical response.
Table 6 : Comparison of response (peak area) of folate compound at two different concentrations in
different injection solvents differing in %MeOH
STD
Injection
Solvent*
Std Conc
ng/mL
FA
5-CH3-THF
5-CHO-
THF
THF
10-CH3-FA
90% MeOH
10:90:1, Formic,
MeOH, Ascorbic
(v/v/Wt.)
5
2.91E+03
9.28E+03
6.75E+03
2.19E+03
5.18E+03
20% MeOH
80:20,
1%Acetic,MeOH,
Ascorbic (v/v/Wt.)
5
2.18E+03
5.97E+03
5.77E+03
2.34E+03
2.45E+03
90% MeOH
10:90:1, Formic,
MeOH, Ascorbic
(v/v/Wt.)
500
3.34E+05
9.77E+05
7.09E+05
3.62E+05
5.54E+05
20% MeOH
80:20,
1%Acetic,MeOH,
Ascorbic (v/v/Wt.)
500
1.78E+05
7.56E+05
5.98E+05
2.94E+05
3.06E+05
*Injection Solvents: with 90% methanol is similar to the elution solvent of SPE; with 20% methanol some what similar to
the mobile phase
Evaluation of Solvent Blanks:
The solvent blank (methanol) did not provide any interference in the analysis of the targeted analytes
(Figure 5).
2011.06/Fol-22 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE