Achille Cappiello

An overview of matrix effects in liquid chromatography–mass spectrometry

Matrix-dependent signal suppression or enhancement represents a major drawback in quantitative analysis with liquid chromatography coupled to atmospheric pressure ionization mass spectrometry (LC-API-MS). Because matrix effects (ME) might exert a detrimental impact on important method parameters (limit of detection, limit of quantification, linearity, accuracy, and precision), they have to be tested and evaluated during validation procedure. This review gives a detailed description on when these phenomena might be expected, and how they can be evaluated. The major sources of ME are discussed and illustrated with examples from bioanalytical, pharmaceutical, environmental, and food analysis. Because there is no universal solution for ME, the main strategies to overcome these phenomena are described in detail. Special emphasis is devoted to the sample-preparation procedures as well as to the recent improvements on chromatographic and mass spectrometric conditions. An overview of the main calibration techniques to compensate for ME is also presented. All these solutions can be used alone or in combination to retrieve the performance of the LC-MS for a particular matrix-analyte combination.

Overcoming Matrix Effects in Liquid Chromatography−Mass Spectrometry

A major limitation in quantitative analysis with electrospray ionization mass spectrometry (ESI-MS) is represented 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 evaluated matrix effects on enriched environmental and biological 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 composition, whereas with ESI we observed either signal suppression or enhancement, depending on the sample nature.

Organochlorine Pesticides by LC−MS

Contamination of water resources by organochlorine pesticides (OCPs) continues to receive widespread attention because of the increasing concern regarding their high persistence and bioaccumulation. These organic pollutants are not amenable by liquid chromatography (LC) coupled to atmospheric pressure ionization-mass spectrometry, which represents the method of choice for the characterization of pesticide residues in water. Gas chromatography-mass spectrometry provides excellent response for OCPs, but it falls short when complex, multiresidue analyses are required. As recently demonstrated, an efficient EI-based LC-MS interface can generate very good spectra for an extremely wide range of small-medium molecular weight molecules of different polarity and can represent a valid tool in solving the analytical challenge of analyzing OCPs by LC-MS. Based on this assumption, we present a new approach for the determination of 12 OCPs in water samples. The method requires a solid-phase extraction preconcentration step followed by nanoscale liquid chromatography coupled to a direct-electron ionization direct interface (Direct-EI). Direct-EI is a miniaturized interface for efficiently coupling a liquid chromatograph with an EI mass spectrometer. The capability to acquire high-quality EI spectra in a wide range of concentrations, and to operate in selected ion monitoring mode during analyses, allowed a precise quantification of the OCPs. Without sample injection enrichment, limits of detection of the method span from 0.044 to 0.33 microg/L, corresponding to an instrumental detection limit of 120-850 pg. In addition, a careful evaluation of the matrix effect showed that the response of the Direct-EI interface was never affected by sample interferences. From our knowledge, the proposed method represents the first application of LC-MS in the analysis of organochlorine pesticides.

MATRIX EFFECTS IN LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

Despite their enormous utility and diffusion, atmospheric pressure ionization mass spectrometry techniques are subjected to relevant drawbacks called matrix effects (ME). These effects could be summarized in matrix-dependent signal suppression or enhancement that could lead to erroneous quantitative results. The most important method parameters as well as linearity, precision, and accuracy could be modified due to interfering compounds present in the matrix. No validation methods could be accepted without a thorough evaluation of ME and possible strategies to minimize or to correct their influence should be addressed. In this article, we investigate mechanisms that lead to ME and discuss calibration techniques and other strategies to limit these phenomena. Significant examples from different fields of application are discussed as well. Sample preparation procedures, together with instrumental improvements and alternative calibration techniques used by many authors, are reported.

Single-step LC/MS method for the simultaneous determination of GC-amenable organochlorine and LC-amenable phenoxy acidic pesticides.

Water pollution by organochlorine pesticides (OCPs) is considered as an analytical challenge, since these persistent and nonbiodegradable pollutants are not amenable by liquid chromatography coupled to atmospheric pressure ionization mass spectrometry (LC/API-MS). This represents a significant constraint in multiresidue analysis of real samples, when high polar, poorly volatile compounds are present as well. This paper reports the development of an innovative single-step method for the simultaneous determination of OCPs and polar pesticides belonging to the class of phenoxy acids in water samples. The method is based on an off-line solid-phase extraction (SPE) procedure with Carbograph 4 followed by liquid chromatography coupled to a direct electron ionization mass spectrometer (LC/direct-EI-MS). The direct-EI capability of acquiring high-quality EI spectra and operation in selected ion monitoring mode allowed a precise quantification of OCPs and phenoxy acids in a single chromatographic run without derivatization. The instrumental response was characterized by excellent sensitivity, linearity, and precision. The SPE recovery rates in river water gave values equal or better than 80% for most of the compounds. The method limits of detection (LODs) span from 0.002 to 0.052 microg/L, allowing the detection of the selected pesticides at the limits required by the European Union (EU) legislation for drinking water.

Enhanced detection sensitivity by large volume injection in reversed-phase micro-high-performance liquid chromatography

Abstract High-performance liquid chromatography (HPLC) packed capillary columns have gained a great deal of interest for their role in conjunction with other analytical techniques, particularly mass spectrometry. However, the advantages offered by the low mobile phase flow-rate are often compromised by modest method detection limits due to sub-microliter injection volumes. This is particularly significant with a particle beam interface where the benefits obtained at very low solvent flow-rate (1 μ1) were outlined. In this paper, the effect of large injection volumes (up to 50 μl) on the chromatographic behaviour of a reversed-phase 250 μm I.D. packed capillary column was studied. Due to their differences in chemical properties and to their environmental impact, a selection of basic-neutral and acidic pesticides was found to be appropriate for this investigation. This study was focused on the peculiar sample-column relationship encountered in a micro-HPLC system. Loop shape and matrix composition were found to be the determinant factors for a correct solute displacement into the capillary column. Suitable conditions for the injection of large volumes of trace-level pesticides were described. Previously developed liquid chromatographic-mass spectrometric methods for the determination of several pesticides in water took great advantage from the larger sample availability.

Liquid chromatographic-mass spectrometric determination of phenolic compounds using a capillary-scale particle beam interface.

A capillary-scale particle beam interface was used to detect 18 phenolic compounds in red wine samples. This technique allows reproducible, library searchable electron ionization spectra at only 1 microliter/min mobile phase flow-rate for a sensitive detection of the analytes in complex matrices. The method makes use of a narrow bore, reversed-phase packed capillary column for sample separation. Detection limits were in the low picogram range for most compounds. Sensitivity and response linearity were evaluated for eight phenolic acids, which are often encountered in red wines. The phenolic compound composition was outlined in two red wines obtained using different aging processes.

Large volume injection of acidic pesticides by reversed-phase micro high-performance liquid chromatography

Modern liquid chromatography-mass spectrometry interfaces often require a limited intake of mobile phase to work properly. Packed capillary columns combine high chromatographic efficiency, reduced flow-rate and an appreciable reliability and can be successfully employed in such applications. Acidic pesticides, a group of important compounds widely used in agriculture and often encountered as environmental pollutants, are positively analyzed by reversed-phase HPLC. The combination of micro-HPLC and particle beam mass spectrometry offer a powerful tool for their determination at very low concentrations. However, any detection limit obtained by this instrumentation is impaired by the constraint of a very small injection volume compatible with a narrow-bore column. In this paper, a procedure based on large volume injection is presented. This procedure is capable of increasing up to 800 times the volume of sample that can be injected into a packed capillary column. Seventeen acidic pesticides were selected for this study representing most of the compound classes commonly found in several environmental matrices. This method differs from previous attempts because of the acidic dissociation in water which may interfere with a correct solute focusing. The larger sample availability consistently improved the method sensitivity based on a LC-MS technique.

A simple approach for coupling liquid chromatography and electron ionization mass spectrometry

A miniaturized, aerosol based interface for directly coupling a liquid chromatograph with a mass spectrometer is presented. The interface is entirely within the electron ionization (EI) source of the mass spectrometer and no additional, external devices are needed. This simple and effective approach exploits micro-flow nebulization technology providing a new interface suitable for a variety of applications of environmental and biological interest. The new interface provides necessary linearity, ruggedness, sensitivity, and reproducibility of response for trace level analysis, and readily interpretable mass spectra for unambiguous identification of unknown compounds of small to medium molecular weight.

Determination of aflatoxins in peanut meal by LC/MS with a particle beam interface

SummaryA new method for the analysis of aflatoxins in food extracts, based on liquid chromatography/mass spectrometry interfacing, is presented. The chromatographic separation was performed with a reversed phase packed capillary column coupled with a modified particle beam interface capable of handling microliter per minute flow rates. This system allows higher overall sensitivity and easier operation procedures. The method has proved to be particularly suitable for the analysis of the toxins in very complex matrices. The specificity of electron impact ionization allowed positive identification of the aflatoxins with an excellent response linearity for accurate quantitation.