title: New approaches for lab-in-a-syringe systems
Creators: name: Mikhail, IE
Publisher: name: University of Tasmania
Year: 2020

description: The work presented in this thesis describes an innovative approach for biological sample preparation in a micro-volume syringe based on electrokinetic separation. For the first time, the embedded stainless steel syringe needle and plunger served as electrodes and the syringe barrel served as a separation channel. The advantages of doing this in a syringe is such that all the preparation of biological samples can be done in the device, such as sample dilution with the required buffer, enrichment of the targeted analytes and/or interfering molecules removal, and finally infusion to the detector. Furthermore, the online coupling of the in-syringe sample preparation with the electrospray ionization-mass spectrometry (ESI-MS) opens up new opportunities for ultra-sensitive and selective analysis of complex biological samples in an automated manner. Chapter 1 provides an insight into different sample clean-up and analyte enrichment strategies directly coupled with the ESI-MS and nano-ESI-MS for the analysis of biological fluids. The overview includes direct ionization methods (DIMs) along with the direct coupling of classical and innovative format of sample preparation techniques such as solid phase extraction (SPE), solid phase micro-extraction (SPME), liquid micro-extraction, and electrokinetic extraction (EkE) with ESI-MS and nano-ESI-MS. Chapter 2 introduces the first electroseparation-in-a-syringe system where a 25 μL analytical glass syringe was utilized for isoelectric focusing (IEF) of amphoteric compounds using the stainless-steel needle and plunger as electrodes. The mechanism relied on the generation of hydronium and hydroxyl ion fluxes from water electrolysis to form a neutralization reaction boundary (NRB) at which the amphoteric compounds can be focused within minutes. The focusing of different proteins such as hemoglobin, R-phycoerythrin, and bovine serum albumin was demonstrated by the developed IEF syringe. After optimization of the different experimental parameters affecting the IEF process and coupling of the IEF syringe with the ESI-MS, the IEF syringe-ESI-MS system was applied for the analysis of histidine in spiked urine samples as relevant for the diagnosis of histidinemia. Each 1 µL of urine was 10x diluted with the background electrolyte (BGE) via the IEF syringe itself and the final composition of the BGE was NH\(_4\) Ac, 1.0 mM, pH 4.0, in 70.0 % (v/v) acetonitrile. Voltages of −200 V (5 min) and −400 V were applied for the IEF step and the infusion step, respectively. The IEF syringe’s contents and the sheath liquid (0.2% (v/v) formic acid) were infused into the Agilent triple tube ESI sprayer at a flow rate of 4.0 μL/min. Furthermore, different systems for the clean-up of biological samples from the interfering proteins were developed. In Chapter 3, an EkE syringe is presented capable of the on-line removal of proteins from human serum samples within 320 seconds allowing the analysis of the weakly acidic compounds. The separation mechanism relies on the difference in the electrophoretic behavior of the serum proteins and the weakly acidic target analytes in a BGE composed of 50 mM formic acid (pH 2.5) in 30 % acetonitrile. 1 µL of spiked serum was 15x diluted with the BGE by the EkE syringe itself and the separation is accomplished by the application of a potential difference of 2000 V across the syringe for 320 seconds, utilizing the metallic syringe needle and plunger as electrodes, once the cationic proteins (pI &gt; 2.5) are concentrated and precipitated close to the plunger, the syringe drive of a syringe pump is actuated for infusion of the protein-depleted sample into the ESI-MS with a flow rate of 4.0 µL/min whilst applying 500 V. The syringe is interfaced with ESI-MS via a coaxial infusion of isopropyl alcohol 80.0 % (v/v) (flow rate, 10.0 µL/min) as a sheath liquid. The applicability of the EkE-ESI-MS method was demonstrated by the determination of naproxen and paracetamol in spiked serum using valproic acid as an internal standard (IS). Chapter 4 exhibits the designing of a bubble free in-syringe EkE system relying on the dissolution of the water electrolysis gases by increasing the pressure inside the syringe barrel. A sealed system was designed to pressurize the gases into the solution using a push-pull valve or a 3-port selector valve at the needle side and a gas-tight plunger. The developed design was applied to the electrokinetic elimination of human serum albumin from the samples using different BGE systems (acidic and basic) and different syringe volumes. Chapter 5 demonstrates the analysis of the weakly basic drugs in serum after the in-syringe EkE, the method is based on using NH\(_4\)OH (300 mM, pH 11.4) in 30 % acetonitrile as a BGE to negatively charge the serum proteins prior to their aggregation close to the plunger (the anode) by application of 800 V for 90 seconds on 10 µL of 5x diluted serum. Unlike the EkE system for acidic compounds analysis “Chapter 3”, a bubble-free EkE of the weakly basic compounds analysis was achieved by pressurizing the syringe as described in Chapter 4. After the EkE and valve switching, 8.0 µL of the syringe content were delivered to the ESI-MS at a flow rate of 5.0 µL/min. The sheath liquid was composed of methanol/water/formic acid, 75:24.5:0.5 and was infused with a flow rate of 5.0 µL/min. A reduced analysis time of 3.1 (1.5 min for the EkE and 1.6 min for the infusion) was achieved for the analysis of weakly basic drugs, 15 clomipramine, chlorphenamine, pindolol, and atenolol in spiked serum. Furthermore, a high sensitivity in ppb level was attained via coupling with ES-MS/MS and spiking the serum with the deuterated isotopes as ISs. In Chapter 6, a fully automated EkE-in-a-syringe platform is proposed, the system requirements, design, and workflow were recommended in details. Finally, Chapter 7 of the thesis summarizes all the developed EkE syringe ESI-MS systems and the different accomplished applications.