In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry

Matrix effects can compromise ionization and hence reliability of electrospray ionisation mass spectrometry (ESI‐MS), making sample clean‐up including protein removal critical in bioanalysis. Automation of protein removal protocols is challenging. Here, an electrokinetic extraction (EkE) syringe is presented allowing for on‐line electrokinetic removal of serum proteins before ESI‐MS. The method relies on the electrophoretic migration of charged serum proteins away from neutral target analytes in an electric field, applied across the syringe barrel utilizing the metallic syringe needle and plunger as electrodes. Under acidic conditions, most proteins are cationic facilitating their accumulation at an cathodic plunger. Similarly, under basic conditions, the anionic proteins aggregate near the anodic plunger. Actuation of the plunger enables infusion of the deproteinated sample remaining in the barrel into the ESI‐MS for detection of acidic or basic molecules. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5x dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in‐syringe. Signal enhancements of 3.6‐32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE‐ESI‐MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng/mL for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs analysis, EkE‐ESI‐MS, the LOQs were 3.1 µg/mL and 2.9 µg/mL for naproxen and paracetamol, respectively. The EkE‐ESI‐MS and EkE‐ESI‐MS/MS methods showed good accuracy (%found of 81% to 120%), precision (< 20%), and linearity (r > 0.997) for all the studied drugs in spiked serum samples.