Introduction

Every year, more than 3.000 whole blood analyses are performed to detect deficiencies of thiamin (vitamin B₁), riboflavin (vitamin B₂) and pyridoxine (vitamin B₆). Several diseases are due to the lack of one of those vitamins. Insufficient intake of vitamin B could lead to Beriberi, or the lack of vitamin B₂ is known as Ariboflavinosis. These analysis use a HPLC-fluorescent quantitative method, which is time consuming and not efficient enough.
The laboratory wanted to develop another method by LC/MS, with few sample preparation steps and lower limits of detection.

Experimental conditions

The following method was conducted on an Agilent 1100 Series and an Agilent Electrospray Mass Spectrometry LC/MSD SL, with an HILIC column (stationary phase C₈/NH₄⁺ / 150x2.1mm / 5µm).
Mobile phase was : A Methanol/Water 50/50 (pH=5.50) ; B Methanol/Water 70/30 with 15mM ammonium acetate buffer (pH=5.50) and used at a flow rate of 0.22mL/min.
Vitamin detection was performed at specific m/z ratio : m/z=445 vitamin B₁ / m/z=786 vitamin B₂ / m/z=294 vitamin B₆.
Detections were performed on positive mode with fragmentor = 75V, V_cap = 3500V, P_neb = 60psig, T_gaz = 350°C.
Vitamin samples were of analytical grade and prepared at 25µM in Methanol/Water 70/30 pH=3.
These solutions were stored at -20°C.

Results

Figure 1 represents the optimal separation of vitamin B1, B2 and B6 with previous experimental conditions. Final solvent gradient was 0-0.5min (95%A and 5%B), 9.5min (0%A and 100%B) and 14.5-15min (95%A and 5%B). Gradient step was linear.
Retention times are 5.592min vitamin B₆, 7.029min vitamin B₂ and 10.889min vitamin B₁ : peaks are separated with an excellent resolution (R_min = 6.3) and total analysis time does not exceed 11min.

Conclusion

These results are promising : solvent gradient may be improved in order to reduce total analysis times, by reducing a little peaks’ resolution. The method can now continue to another step of development : quantification. One of the most important things that the developed method will have to be is to be sensitive enough.

Chromatogram of vitamins B at 25µM by LC/MS
Purple curve represents solvent gradient

Centre de Biologie Sud
Centre Hospitalier Lyon Sud
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Catherine BEKHALED