Helga Trufelli

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.

Electron ionization in LC-MS: recent developments and applications of the direct-EI LC-MS interface.

AbstractThe purpose of this article is to underline the possibility of efficiently using electron ionization (EI) in liquid chromatography (LC) and mass spectrometry (MS). From a historical perspective, EI accompanied the first attempts in LC-MS but, owing to several technical shortcomings, it was soon outshined by soft, atmospheric pressure ionization (API) techniques. Nowadays, two modern approaches, supersonic molecular beam LC-MS and direct-EI LC-MS, offer a valid alterative to API, and preserve the advantages of EI also in LC-MS applications. These advantages can be summarized in three crucial aspects: automated library identification; identification of unknown compounds, owing to EI extensive fragment information; inertness to coeluted matrix interferences owing to very unlikely ion–ion and ion–molecule interactions in the EI gas-phase environment. The direct-EI LC-MS interface is a simple and efficient solution able to produce high-quality, interpretable EI spectra from a wide range of low molecular weight molecules of different polarity. Because of the low operative flow rates, this interface relies on a nano-LC technology that helps in reducing the impact of the mobile phase on the gas-phase environment of EI. This review provides an extensive discussion on the role of EI in LC-MS interfacing, and presents in detail several performance aspects of the direct-EI LC-MS interface, especially in terms of response, mass-spectral quality, and matrix effects. In addition, several key applications are also reported. FigureRange of HPLC amenable compound classes by LC-EIMS

A new liquid chromatography-mass spectrometry approach for generic screening and quantitation of potential genotoxic alkylation compounds without derivatization

One of the crucial tasks of pharmaceutical industry is to quantify the potential genotoxic impurities (PGIs) coming from the process of drug production. The European Medicines Agency (EMEA) imposes analytical testing limits in the order of μg/g, depending on drug dosage and exposure period, that means the need of a sensitive and selective method of analysis. Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) has been demonstrated as the most versatile approach to detect PGIs in complex matrices. However, time consuming derivatization processes are needed to enhance sensitivity and selectivity, and to overcome matrix effects (ME) that may arise from active pharmaceutical ingredients (APIs) or excipients. We propose the use of the Direct-EI LC-MS as an alternative approach to detect and quantify PGIs in drug formulations. The Direct-EI LC-MS interface is based on electron ionization (EI) which is well suited for the detection of low molecular weight compounds of different polarity, without derivatization and with no sign of ME. The method has been successfully applied to the detection of PGIs belonging to the class of alkylation agents. Calibration experiments show satisfactory linearity and precision data. Recoveries in low enriched samples spanned from 55 to 82%, and were not affected by ME. The method limits of detection (LODs), varying from 0.13 to 1.5 μg/g, were satisfactory for the quantitation of the target PGIs at the level required by regulatory agencies.

Application of Liquid Chromatography-Direct-Electron Ionization-MS in an in Vitro Dermal Absorption Study: Quantitative Determination of trans-Cinnamaldehyde

We propose a new analytical approach, based on liquid chromatography (LC) coupled to electron ionization mass spectrometry (EI-MS), using a Direct-EI interface, for dermal absorption evaluation studies. Penetration through the skin of a given compound is evaluated by means of in vitro assays using diffusion cells. Currently, the most popular approach for the analysis of skin and fluid samples is LC coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). However, this technique is largely affected by sample matrix interferences that heavily affect quantitative evaluation. LC-Direct-EI-MS is not affected by matrix interference and produces accurate quantitative data in a wide range of concentrations. Trans-cinnamaldehyde was chosen as test substance and applied in a suitable dosing vehicle on dermatomed human skin sections. This compound was then quantified in aliquots of receptor solution, skin extract, cell wash, skin wash, carbon filter extract, cotton swab extract, and tape strip digest. On column limits of detection (LOD) and limits of quantitation (LOQ) of 0.1 and 0.5 ng/μL, respectively, were achieved. Calibration showed satisfactory linearity and precision for the concentration range of interest. Matrix effects (ME) were evaluated for all sample types, demonstrating the absence of both signal enhancement and signal suppression. The Direct-EI absorption profile was compared with that obtained with liquid scintillation counting (LSC), a recognized ME free approach. A good correlation was found with all samples and for the overall recovery of the dosed substance.

Determination of Natural Pyrethrins by Liquid Chromatography-Electron Ionisation-Mass Spectrometry

INTRODUCTION Pyrethrum extract is a mixture of six insecticidal compounds from the flower heads of Chrysanthemum cinerariaefolium L.. Since they only have low to moderate mammalian toxicity they can be used as natural insecticides in agriculture or to develop low cost and safe dermatological formulations. Because of the thermal instability of pyrethrins, analytical methods based on liquid chromatography (LC) are preferred over those based on gas chromatography (GC). A few applications using LC with mass spectrometry detection are presented in the literature. Current protocols for their characterisation by LC rely on the use of less sophisticated detectors such as UV detection. OBJECTIVE To develop the first liquid chromatography-electron ionisation-mass spectrometry (LC-EI-MS) method for pyrethrins detection and quantitation in pyrethrum extracts. METHODOLOGY A commercial pyrethrum extract and various samples of flower heads from C. cinerariaefolium L. were investigated using reversed-phase nano-liquid chromatography coupled to direct electron ionisation-mass spectrometry (nanoLC-direct EI-MS). The eluted compounds were identified through searches of the US National Institute of Standards and Technology (NIST) library, exploiting the direct EI capability to produce high quality EI mass spectra. RESULTS The method demonstrated satisfactory sensitivity (limit of detection (LOD) range: 0.04-0.38 mg/g), linearity (R² range: 0.9740-0.9983) and precision (RSD% range: 4-13%) for the quantitation of the natural pyrethrins in extracts from C. cinerariaefolium L. CONCLUSION The nanoLC-direct EI-MS technique can be a useful tool for the detection of pyrethrins.

Study on the maltooligosaccharide composition of mucilage samples collected along the northern Adriatic coast

The mucilage phenomenon, a sporadic but massive accumulation of gelatinous material, can cause serious damage to the tourism and fishing industries along the Adriatic coast. Mucilage is presently thought to be the result of the aggregation of dissolved organic matter (DOM) into particulate organic matter (POM). Three principal classes of compounds have been identified in organic matter by spectrometric determination: carbohydrates, proteins and lipids. Carbohydrates are suspected to play a role in the first steps of DOM aggregation. Despite its importance in understanding the processes leading to mucilage formation, our present knowledge of the composition of the mucilage carbohydrate fraction is incomplete. Due to its high sensitivity and specificity, liquid chromatography coupled with electrospray-ionization tandem mass spectrometry (LC-ESIMS/MS) is gaining an increasing importance as a powerful technique for carbohydrate purification and characterization in complex samples. In this work, LC-ESIMS/MS is proposed as a useful method for the investigation of the oligosaccharide content in mucilage samples. The approach was applied using 3-7 unit maltooligosaccharides as reference compounds. The composition of the investigated mucilage sample was further investigated combining LC-ESIMS/MS with classic approaches, such as spectroscopic techniques and liquid chromatography coupled with the refractory index LC-RI.