Overcoming matrix effects in electrospray: Quantitation of β-agonists in complex matrices by isotope dilution liquid chromatography-mass spectrometry using singly ¹³C-labeled analogues

Abstract

In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of β2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different β2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC-ESI-MS in complex samples. In this procedure singly (13)C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of β2-agonists in human urine and bovine liver, even at the sub ngg(-1) and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97-114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of (13)C1-labeled standards and IPD is a reliable approach for accurate LC-MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry.