Ultrafast measurement of drugs using Probe Electrospray Ionization Mass Spectrometry (PESI-MS) and isotope dilution: An example with metformin

Abstract

Probe Electrospray Ionization (PESI) is a miniaturized electrospray (ESI) with a capillary of the nanoelectrospray converging on a solid needle with a tip of several hundred nanometers. This probe was developed to be coupled directly to a mass spectrometer (MS) without the need for chromatographic separation, allowing ultrafast analysis with minimal sample preparation. Metformin is the first-line treatment for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated metformin plasma concentrations. Therefore, a rapid measurement plays an important role in helping clinicians to diagnose overexposure to metformin. In the present study, our objective was to develop a PESI-MS method for an ultra-rapid determination of metformin in plasma by using an isotope dilution approach. Analysis was carried out with a PESI source installed on a triple quadrupole MS (LCMS 8060®, Shimadzu). A multiple reaction monitoring mode (MRM) with 2 transitions for metformin and metformin-D6 (internal standard) was used. To be able to apply an isotope dilution protocol, the main MS parameters were optimized to ensure that the m/z 130.15 > 71.10 (metformin) and m/z 135.95 > 77.15 (metformin-D6) transitions exhibited the same intensity. The method was fully validated with 6 concentration levels (0.5 to 50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested by analysing 6 plasmas spiked with a mix of 44 potential co-medications. Finally, the performance was compared with 2 classical LC-MS/MS and LC-DAD methods used in external labs, by analysing samples from 29 patients. The sample preparation consisted in mixing 10 μL of plasma with 1000 μL ethanol/ammonium formate buffer including metformin-D6 at a fixed concentration of 5 mg/L. Total run time was 0.31 min. The limit of detection and quantification was fixed at 0.5 mg/L. Isotope dilution gave satisfactory results in accuracy and precision for each concentration level (min-max: −12.1–15.8% and 1.0–7.1%, respectively). Thanks to the internal standard the matrix effect was fully corrected (bias less than 1%). The specificity study also reported satisfactory results. Finally, in the representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC-DAD and LC-MS/MS to PESI-MS (r2 > 0.99). The PESI-MS approach needs no sample preparation. Combined with a isotope dilution protocol, that requires no calibration curve building, offers the best possibility to design specific, sensitive, accurate and ultra-fast solutions. Our study presents a method which allows an accurate determination of metformin in about 30 seconds that will be helpful in a core lab when rapid diagnosis of LA is needed.