RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2004; 18: 141–148 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1293 Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices Masoud Zabet-Moghaddam, Elmar Heinzle and Andreas Tholey* Technische Biochemie, Universita ¨t des Saarlandes, 66123 Saarbru ¨cken, Germany Received 15 October 2003; Revised 1 November 2003; Accepted 2 November 2003 A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vita- mins was investigated. The ionic liquid matrices are composed of equimolar combinations of clas- sical MALDI matrices (sinapinic acid, a-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices. Copyright # 2003 John Wiley & Sons, Ltd. Due to its inherent robustness, low sample consumption and high sensitivity, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become one of the most commonly used analytical methods for biomolecules such as peptides, proteins and oligonucleotides. 1 It is now also widely accepted as a tool for the measurement of low mole- cular weight compounds of biological interest. 2 In contrast to the measurement of polymeric compounds, a matrix-to-ana- lyte ratio of 10:1 to 100:1 and a proper selection of matrices avoiding signal overlaps are the main requirements for the MALDI-based analysis of such compounds. 3 A number of publications have described the use of MALDI-MS for quantitative measurements, 4–9 for example, for the measurement of enzyme activities. 3,10,11 The inhomo- geneous distribution of analytes within sample spots is the most serious problem in the use of MALDI-MS for quanti- tative measurements. Inhomogeneity causes poor shot-to- shot and sample-to-sample reproducibility. 12 The use of internal standards can reduce these problems, provided that the molecular properties of the internal standard are similar to those of the analytes thus ensuring comparable crystal- lization behaviour on the target. The inhomogeneities can also be partially overcome by integral measurement over the preparation with an averaging of the collected signals, 3 but this procedure is time-consuming even in automated form. Several techniques have been developed to improve sample homogeneity, e.g., adding co-matrices like fucose to form a binary matrix, 13 fast evaporation methods using highly volatile solvents, 14,15 deposition of droplets by microdispenser, 16,17 electrospray-based sample deposition processes, 6 sol-gel-based systems, 18 aerospray sample deposition using CCA-thin layer preparation 19 or the use of solvent-free sample preparations. 20 The use of liquid matrices has been investigated by several groups. 21–23 Liquid matrices do not exhibit ‘hot spots’, refresh their surfaces continuously, 24 exhibit higher signal reproducibility, and are miscible with polar and nonpolar analytes. 2 However, the use of liquid matrices is still associated with low mass resolution, high chemical back- ground and poor ionization efficiency, leading to a restricted use in UV-MALDI-MS. In contrast, liquid matrices have been frequently used in IR-MALDI-MS. 23 Ionic liquids are a heterogeneous class of compounds. Regardless of their different physical and chemical properties they share three common features: (i) they consist of ions, (ii) have melting points below 1008C and are often liquid at room temperature, and (iii) possess only negligible vapour pressure at temperatures below their temperature of degra- dation. 25 Room temperature ionic liquids (RTILs) are typically formed by a heterocyclic cation based on substi- tuted imidazole or pyridine systems and an inorganic counter anion such as [AlCl 4 ]  , [BF 4 ]  or [PF 6 ]  . Recently, RTILs have been employed more and more as substitutes for traditional organic solvents in chemical and biocatalytical reactions due to their outstanding properties. 26,27 Armstrong and co-workers examined the use of RTILs as matrices for MALDI-MS analysis of peptides, proteins, technical polymers and DNA oligomers. 28,29 It was shown that this class of RTILs is not suitable for use as MALDI Copyright # 2003 John Wiley & Sons, Ltd. *Correspondence to: A. Tholey, Technische Biochemie, Universi- ta ¨t des Saarlandes, 66123 Saarbru ¨ cken, Germany. E-mail: a.tholey@mx.uni-saarland.de Contract/grant sponsor: Bundesministerium fu ¨ r Bildung und Forschung.