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Folic Acid by LCMS
With Minimized Sample Prep & Increased Sensitivity
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printable Application Sheet
Notes: Deficiency of folic acid (vitamin B9) is associated with several chronic diseases (for example
macrocytic anaemia, psychiatric disorders, neutral tube defects in newborns, cancer, Alzheimer’s disease
and cardiovascular disease in adults [1]; thus accurate assessment of folate status has become important
in the clinical setting, in epidemiological studies in clinical and research laboratories. FA cannot be
synthesized by mammalian cells and therefore must be supplied with diet. It is very important to establish
highly sensitive methods for the quantitative determination of trace amounts of this vitamin.
[1]. B.C. Nelson, K.E. Sharpless, L.C. Sander; J. Chromatogr. A, 1135 (2006) 203-211
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 10 mM ammonium acetate
B: 90% acetonitrile/10% DI water/10 mM ammonium acetate.
pH = 7 adjusted using ammonia |
| Gradient |
| Time (min) | %B |
| 0.00 | 100 |
| 5.00 | 90 |
| 9.00 | 50 |
| 10.00 | 100 |
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| Post time |
5 min. |
| Flow rate |
0.4 mL/min. |
| Samples |
Sample stock solution was made in Solvent A at a
concentration of2.0 mg/L. Sample for analysis was
made by diluting the stock with Solvent A, to which
0.1% ammonia was added. Sample final concentration
was 0.2 mg/L.
Please note that folic acid sample was prepared in solvent
A only as opposite to samples of other acids which are
prepared in 50% mixture of solvents A and B. When the
sample of folic acid was prepared in 50% A + 50% B the
MS signal was lower. |
| Detection |
ESI – neg - Agilent 6210 MSD TOF mass spectrometer. |
Discussion
A sensitive and reliable method was developed for the determination of folic acid (FA) and can be used in many
applications including the complex sample matrices without the solid-phase extraction clean-up. Since the method
uses LC-MS (negative-ion) it provides fantastic sensitivity as well as great selectivity. The use of chelating
agents or high concentration of salts in the mobile phase or ion pair reagents (non MS compatible) are not
required as in older methods for FA analysis. The LOQ is dramatically better than existing methods with UV
detection. When this methodology is used very fast equilibration between gradients is observed as well. Excellent
precision was achieved using the presented method. Twenty consecutive injections resulted in (%RSD = 0.26).
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