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J Biomol Tech. 2010 September; 21(3 Suppl): S37–S38.
PMCID: PMC2918137

Optimization of ESI at Capillary LC Flow Rates for Efficient Quantitative Biomarker Analysis

A. Berg
New Objective Inc., Woburn, MA, United States



Diverse requirements for small and large molecule analysis has driven the bifurcation of electrospray ionization (ESI) into high (mL/min) and ultra-low (nL/min) flow regimes. Small molecule analysis has predominantly relied on high flow rates using millimeter diameter liquid chromatography (LC) columns. Fast gradient elution on short (≤5 cm) columns with sample-to-sample injection times measured in 1-5 minutes (or better) dominates workflows. Large molecule (protein and peptide) analysis has relied on ultra-low flow ESI (nanospray) and the use of nanobore (≤75 um) LC columns. Workflows based on nanopsray/nanobore LC enable exceptional sensitivity and high separation power for complex mixtures. Long gradient elution LC with injection cycles of greater than 30-60 min (or more) dominates this workflow. Biomarker validation of proteins and peptides demands LC-MS/MS transition from qualitative to quantitative analysis. Small molecule workflows often lack sufficient sensitivity and selectivity required for biomarker validation. Large molecule workflows lack sufficient throughput for the quantification of large sample sets. Efficient ESI in an intermediate flow range, 1-20 uL/min, allows for the facile implementation of capillary scale (0.18 to 0.5 mm) columns. Capillary columns enable higher sensitivity than mm scale and deliver higher throughput, and robustness than nanobore LC. A novel approach to ESI based on high precision fused-silica components and microfluidic connectors enable the integration of concepts used in both high-flow ESI and nanospray. A precision emitter assembly enables stable ESI at flow rates from 1 to 20 uL/min at low (3%) and high (90%) organic mobile phase composition. Continuous flow experiments with 100 nM Buspirone and Angiotensin demonstrate relative standard deviation (RSD) of better than 10% on a 3D ion trap mass spectrometer. Robust performance is obtained on a single emitter assembly for more than 2,500 injections of analytical standard. Peak area ratios met RSD requirements for quantitative analysis.

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