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Silliker

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

12

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