Optimization of flow splitting and make-up flow conditions in liquid chromatography/electrospray ionization mass spectrometry.

Rationale: In liquid chromatography/mass spectrometry (LC/MS) the LC flow is often split prior to the mass spectrometer, for instance, when collecting fractions of the separated sample for other purposes or when less sensitive parallel detection is applied. The aim of this study is to optimize the actual split ratio and make-up flow composition.

Methods: Different types of splitters were evaluated in combination with a make-up flow. A home-made 1/10 T-piece splitter and commercial 1/10, 1/100 and 1/250 splitters were evaluated by continuous and accurate measurements of the actual split ratio throughout the LC gradient. The make-up flow composition was optimized for maximum electrospray ionization (ESI)-MS sensitivity in the positive mode using ESI efficiency measurements.

Results: Altogether 22 different solvent conditions were tested on 20 pharmaceutical compounds with a wide variety of functional groups and physicochemical properties (molecular weight, logP, and pK ). Methanol/10 mM formic acid in water (90/10) provided on average the best results.

Conclusions: Methanol/10 mM formic acid in water (90/10) proved to be the best make-up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.