Overcoming Matrix Effects in Liquid Chromatography-Mass Spectrometry Achille Cappiello,* Giorgio Famiglini, Pierangela Palma, Elisabetta Pierini, Veronica Termopoli, and Helga Trufelli University of Urbino “Carlo Bo”, Istituto di Scienze Chimiche “F. Bruner” 61029 Urbino, Italy A major limitation in quantitative analysis with electro- spray ionization mass spectrometry (ESI-MS) is repre- sented by the so-called matrix effects in which the matrix coextracted with the analytes can alter the signal response, causing either suppression or enhancement, resulting in poor analytical accuracy, linearity, and reproducibility. In the direct electron ionization liquid chromatography-mass spectrometry (direct-EI LC-MS) interface the ionization process is based on electron impact ionization, and it occurs in the gas phase and is not influenced by coeluted matrix compounds. In this work we quantitatively evalu- ated matrix effects on enriched environmental and bio- logical samples, with different extraction procedures, using ESI and direct-EI LC-MS. As expected, the samples analyzed with direct-EI were not affected by matrix com- position, whereas with ESI we observed either signal suppression or enhancement, depending on the sample nature. The combination of liquid chromatography and mass spec- trometry into a single, well-integrated system (LC-MS) has revolutionized the approach to the quantitative determination of organic compounds in complex samples. Progress in life science research has been accomplished with the use of LC-MS, together with new applications of environmental, food safety, and homeland security interest. 1-4 LC-MS systems using an electrospray ion source coupled with a tandem mass analyzer (LC-ESI-MS/MS) have gained a great popularity because of their effectiveness for the quantitative determination at trace level of medium- and high- polarity analytes. The high selectivity and sensitivity of modern MS/MS systems have led to a growing trend of high-throughput analyses that entail little or no sample preparation and a minimal chromatographic retention. Thanks to these important aspects, LC-ESI-MS/MS has become a well-established standard analyti- cal technique in the pharmaceutical industry, clinical research, forensic analysis, environmental science, and in many other areas in which trace amounts of analytes in complex mixtures must be detected and characterized. 2,3,5-7 However, in the past few years the common perception that the high selectivity of LC-MS/MS guarantees an effective elimination of the interferences from endogenous impurities has been challenged. Many authors report that the presence of coextracted matrix can severely affect the quantification procedures based on ESI and atmospheric pressure chemical ionization (APCI) LC-MS methods. 8-12 This phenom- enon is called matrix effects (ME), and it is considered to be either an unexpected suppression or enhancement of the analytes response induced by the coeluting matrix. It can heavily affect reproducibility, linearity, and accuracy of the method leading to erroneous quantitation. 8,11 As a matter of fact, ME are becoming a major threat in the successful application of LC-ESI-MS, reducing the typical advantages of mass spectrometric detection in terms of selectivity and specificity, which is reminiscent of protocols used for less sophisticated detectors, such as ultraviolet or fluorescence. The exact mechanism of ME is still unknown. It is assumed that the coeluted matrix can influence signal intensity in a possible competition for the available charges and for the access to the droplet surface for gas-phase emission. 13-16 Postcolumn infusion and postextraction addition are the two main strategies proposed in literature to asses ME. In the first one a pump is used to deliver a constant flow of analyte into the LC eluent at a point after the chromatographic column and before the mass spectrometer. 17 A sample extract free of the analyte is then injected under the required chromatographic conditions, and the response from the infused analyte is recorded. Any endog- enous matrix component that elutes from the column and induces ME can be seen as a suppression or enhancement of the infused analyte signal. In the postextraction addition a sample extract with the analyte of interest added after the extraction (matrix-matched * Corresponding author. E-mail: achille.cappiello@uniurb.it. (1) Maurer, H. H. Anal. Bioanal. Chem. 2005, 381, 110–118. (2) Reemtsma, T. J. Chromatogr., A 2003, 1000, 477–501. (3) Prakash, C.; Shaffer, L.; Nedderman, A. Mass Spectrom. Rev. 2007, 26, 340–369. (4) Sforza, S.; Dall’Asta, C.; Marchelli, R. Mass Spectrom. Rev. 2006, 25, 54– 76. (5) Hopfgartner, G.; Bourgogne, E. Mass Spectrom. 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(16) Zhou, S.; Cook, K. D. J. Am. Soc. Mass Spectrom. 2001, 12, 206–214. (17) Bonfiglio, R.; King, R. C.; Olah, T. V.; Merkle, K. Rapid Commun. Mass Spectrom. 1999, 13, 1175–1185. Anal. Chem. 2008, 80, 9343–9348 10.1021/ac8018312 CCC: $40.75  2008 American Chemical Society 9343 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 Published on Web 10/30/2008