Introduction

This optimization was realised in the laboratory of Pharmacology and Toxicology in the Military Hospital Desgenettes. The laboratory has a variety of activities such as forensic, hospital toxicology, traffic controls or occupational medicine. During my placement, I had to optimize a method for the determination of metformin in plasma or urine. The latter were developed and validated for routine use several years ago. However, it turned out that many metformin analyzes was impossible to quantify. Thus, during my placement I tried to find the problem with this method in order to optimize and validate it.

Experimental conditions

In order to analyze metformin, it’s necessary to extract it from plasma with a solid-liquid extraction. After, we use a Liquid Chromatography with PhotoDiodes Array (PDA) detector. The extraction which was the problem begins with a conditioning of WCX cartridge with 2mL of methanol and 2mL of water. After putting 1mL of plasma or urine, there was a washing with water/NH₃ 5% and an other with methanol to eliminate impurities. Finally, the elution was carried out with methanol/HCl 2.5%. I tried to modify the two washings of extraction but without results. In a publication I found that methanol/HCl degraded metformin so I decided to replace methanol/HCl by methanol/acetic acid 3%.

Results

First, with the modification of each washing, there wasn’t good results. It was impossible to quantify metformin with these chromatograms. After replacing methanol/HCl by methanol/acetic acid 3%, I obtained a chromatogram with clearly separated peaks (figure 1). A new calibration line could be done with this new elution solvent (figure 2).

Conclusion

Finally, with the replacement of elution solvent, the new method could be validated and now it is used in routine analysis. The new method is more reliable and more robust than the other one.

Chromatogram with the new elution solvant

Calibration line of metformin in plasma

Hôpital d’Instruction des Armées Desgenettes
108, Boulevard Pinel
Laboratoire de Pharmacologie-Toxicologie
69003 Lyon

SECRETARY
Catherine BEKHALED