A simple and robust set-up for on-column sample preconcentration ? nano-liquid chromatography ? electrospray ionization mass spectrometry for the analysis of N-acylhomoserine lactones

Abstract

A simple method for the simultaneous, rapid and sensitive determination of N-acylhomoserine lactone signaling molecules in bacterial isolates, without prior sample preconcentration and with minimal sample cleanup, is presented. The analysis relies on the combination of analyte preconcentration and separation on a single device: a relatively large sample volume (1-5 microL) is directly loaded onto a laboratory-made, miniaturized (75 microm i. d.) reverse phase nano-liquid chromatography column, connected on-line to a microelectrospray-ionization ion trap mass spectrometer. In a first step the analyte is adsorbed (and so concentrated) at the beginning of the column, and is eluted and selectively separated in a second step by the organic mobile phase. Sample preconcentration follows the mechanisms of solid phase extraction on a nano-scale, while separation takes place according to classical liquid chromatography separation principles. The columns can be manufactured easily, are simply connected, and used with minimal solvent amounts; this makes this method extremely robust and cost-effective. The analytical setup was found to be routinely quantitative down to a concentration of 10 ng/mL (corresponding to a total analyte amount of 10 pg or ca. 50 fmol). The limit of detection was reached at 1 ng/mL (1 pg, ca. 5 fmol). Compared to the classical AHL analysis of bacterial cultures with biosensors, where selectivity and sensitivity is often limited, this rapid analytical technique is a substantial qualitative and quantitative improvement. Two unsubstituted N-acylhomoserine lactones could be identified and quantified from a Burkholderia cepacia culture supernatant in a chloroform extract.

Analysis of N‐acylhomoserine lactones after alkaline hydrolysis and anion‐exchange solid‐phase extraction by capillary zone electrophoresis‐mass spectrometry

“…More common approaches were developed recently for the direct assessment of HSLs with classical LC-MS [7] and with dedicated capillary techniques coupled to ESI-MS [8][9][10] …”

Specificity of Acyl-Homoserine Lactone Synthases Examined by Mass Spectrometry

“…Direct analysis of AHLs has been reported using GC-MS (6) and high-performance liquid chromatography (HPLC) coupled to ion trap mass spectrometry (LC-MS) (36,41,44,48,59). The addition of a preconcentration step has been reported to result in highly sensitive methods of AHL detection (13,14).…”

Development of a capillary electrophoresis‐mass spectrometry method for the determination of formaldehyde releasers as their hydrolysis products and amino alcohols from metal working fluids

“…The capillary was air-cooled at ambient temperature. Sheath liquid coupling was performed as described elsewhere [12], using conventional polyether-ether ketone fittings. Sheath liquid was delivered by a Hewlett-Packard 1100 (Waldbronn, Germany) HPLC pump with an on-line degasser operating at a flow rate of 4 mL/min.…”