Recent developments in the application of liquid ... - Springer Link

Anal Bioanal Chem (2008) 390:253–256 DOI 10.1007/s00216-007-1674-4

TRENDS

Recent developments in the application of liquid chromatography–tandem mass spectrometry for the determination of organic residues and contaminants Rainer Schuhmacher & Michael Sulyok & Rudolf Krska

Published online: 31 October 2007 # Springer-Verlag 2007

Introduction Since the introduction of atmospheric pressure ionisation (API) liquid chromatography–mass spectrometry (LC-MS) for routine analysis in the early 1990s, large improvements in terms of sensitivity and robustness of the MS instruments have been achieved and new mass analysers such as the linear ion traps and a Fourier transformation (FT) ion trap MS (Orbitrap) have been designed for LC-MS/MS analysis of small molecules [1]. Improvements of LC-MS instrumentation, the establishment of maximum levels for e.g. pesticides [2, 3], mycotoxins [4] and pharmaceuticals [5] and the definition of strict analytical quality assurance criteria (e.g. [6, 7]) have greatly contributed to the increasing popularity of LC-MS/MS in recent years. Most applications of LC-MS/MS to the analysis of residues and contaminants (e.g. in food and environmental samples) focus on the accurate quantification or semi-quantitative screening of target compounds and the confirmation of their identity. Recent publications clearly indicate a trend towards multi-target methods covering up to more than 100 compounds in a single chromatographic run as well as towards screening of (mostly highly polar) transformation products of known residues and contaminants with the major goal to study their metabolisation or degradation during numerous processes. This article presents a short introduction to the variety of mass analyser configurations and recent developments of R. Schuhmacher (*) : M. Sulyok : R. Krska Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Str. 20, 3430 Tulln, Austria e-mail: [email protected]

high-performance liquid chromatography (HPLC) instrumentation. Additionally, three key topics of LC-MS/MS—matrix effects, multi-analyte methods and confirmatory analysis— are briefly discussed.

Instrumentation Mass spectrometry Electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) have evolved as standard techniques for the ionisation of most small molecules in trace analysis. In addition, atmospheric pressure photoionisation (APPI) has been developed to increase ionisation efficiencies of non-polar compounds such as polyaromatic hydrocarbons (PAH) or steroids [8]. The most frequently used tandem mass spectrometers for the determination of trace contaminants and residues are triple quadrupole instruments (QqQ), ion traps (IT), quadrupole–time-of-flight (Qq-TOF)- and QTrap instruments which can be used alternatively as QqQ or QqIT. Table 1 lists the tandem mass analysers together with their main fields of application and characteristics. QqQ and QTrap instruments are best suited for quantitative target analysis owing to their high sensitivity and wide linear range, when operated in the selected reaction monitoring (SRM) mode. The newest instruments enable the use of SRM dwell times as low as 10 ms without loss of sensitivity making multi-target methods possible. Ion trap instruments have also been further developed in the last few years. The latest generation of 2D and high capacity 3D ion traps (IT) show up to 80 times increased ion capacities compared to former 3D IT, reducing one of the major limiting factors for quantification. As stand-alone analysers, however, they still show limited capability for multi-target analysis,

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Anal Bioanal Chem (2008) 390:253–256

Table 1 Most frequently used tandem mass spectrometers in organic trace analysis Mass analyser

Preferred mode

Generally used for

Characteristics/comment

QqQ

SRM

Quantification and screening (target, multi-target) Confirmation by ratio of SRM transitions

High capacity IT (2D or 3D)

MS/MS

Quantification, target screening Confirmation by full MS/MS spectra

Qq-TOF

MS full scan MS/MS

QTrap (QqQ or Qq-IT)

SRM, MS/MS (Trap scan)

(Post)screening, multi-target screening Confirmation by full MS or MS/MS spectra Quantification, screening (target, multi-target) Confirmation by full MS/MS spectra

-High selectivity and sensitivity -Short SRM dwell times -Generally unit resolution, mass accuracy of 100–300 mDa -Low full scan sensitivity -High full scan sensitivity -Additional structure elucidation by MSn -Control of number of trapped ions to avoid space charge effects -Less suited for multi-target analysis -High resolution (10–15,000) and mass accuracy (1–10 ppm) -Very fast scanning capability -Limited full scan sensitivity & linearity -Full QqQ functionality plus sensitive IT modes -Unique MS/MS modes, e.g. EPI with precursor selection and fragmentation outside IT

Q linear quadrupole, q collision cell, IT ion trap, TOF time-of-flight analyser, SRM multiple reaction monitoring, MS/MS product ion scan, EPI enhanced product ion scan

since only a small number of analytes can be monitored simultaneously in the time window of a typical HPLC peak. Ion traps are particularly powerful for unequivocal confirmation or elucidation of the molecular structures of a limited number of compounds, since very fast and sensitive full scan modes (including MS2 and MSn) can be applied. Qq-TOF instruments have been improved in the last few years, but their sensitivity and linear dynamic range are still lower compared to QqQ, QTrap or IT instruments. Therefore, Qq-TOF instruments are mainly applied for screening and qualitative analysis instead of accurate and sensitive quantification of residues and contaminants. Their main advantages consist in the high resolving power (up to ca. m/Δm 15,000 FHWM) and high mass accuracy (e.g. 5–10 ppm). This makes Qq-TOF instruments well suited for confirmatory analysis [9]. If operated in the MS full scan mode, Qq-TOF instruments can also be used for post-target and general unknown screening by acquiring full MS spectra and subsequently extracting defined narrow m/z windows. At any time after the actual measurement, MS and MS/MS spectra can be inspected for the presence of selected m/z signals and the spectra can be compared to database entries [10]. Liquid chromatography In HPLC, the use of smaller stationary phase particles offers the most suitable way to achieve lower plate height values, allows for higher (and wider range of) linear velocities owing to a reduced mass transfer resistance and thus leads to enhanced chromatographic separation efficiencies. In the 1990s 3- to

3.5-μm particles were commercialised and allowed a 30–50% faster and more efficient separation compared to 5-μm particles [11]. In 2004, the so-called ultra-performance LC system (UPLC) using columns packed with 1.7-μm particles (C18functionalised, bridged ethylsiloxane-silica) with sufficient chemical (pH 1–12) and mechanical stability (up to 1,000 bar) was introduced to the market. Meanwhile, numerous manufacturers offer such HPLC systems and HPLC columns with particles ≤2 μm. These systems can either be used to shorten chromatographic run times without losing separation efficiency (short columns, high flow rates) or to improve separation of analytes and matrix components (optimum flow rate, longer columns). As the increased separation power leads to typical peak widths of a few seconds, MS analysers which have short cycle times such as QqQ in SRM mode and QqTOF instruments are best suited for hyphenation with this type of HPLC instrument. However, since such columns require frits of ≤1 μm, the danger of clogging by particulate matter might be a limiting factor for high-throughput routine applications [12]. The large number of polar target compounds to be analysed in complex mixtures has led to an increased use of stationary phases other than conventional reversed-phase C18 or C8. Besides employing polar embedded or polar end-capped stationary phases, hydrophilic interaction chromatography (HILIC) with eluents usually consisting of ca. 5–40% water in acetonitrile has been found to be particularly suited to mediate chromatographic retention of highly polar, noncharged compounds in combination with MS. The HILIC retention mechanism is based on partitioning of the analyte


between a water-enriched layer of stagnant eluent on a hydrophilic stationary phase (e.g. 2,3-dihydroxypropyl- or 3-aminopropyl-modified silica) and a relatively hydrophobic bulk eluent [13]. Eventually, mixed-mode RP columns offer further alternatives, especially for the separation of charged compounds such as organic acids or quaternary amines [14]. These silica-based columns are functionalised with an alkyl group also carrying an ion exchange group, thereby offering unique selectivity based on RP, ion exchange and HILIC mechanisms.

Matrix effects The high selectivity of MS instruments has initially led to the assumption that less effort is needed for sample preparation and chromatographic separation prior to MS detection. As a consequence, sample cleanup and chromatographic separation of analytes and co-eluting (but mostly invisible) matrix components sometimes was neglected. HPLC separations on columns of 3- to 5 cm length or even 1- to 2-cm pre-columns have been used to speed up the analysis. However, it was soon realized that co-eluting matrix components limit the accuracy of quantitative analytical methods by ion suppression/ enhancement in the MS ion source, e.g. through competition for the electrical charges or through affecting the evaporation of the ESI droplets and the analyte transfer to the gas phase [15]. Unfortunately, these effects are of unpredictable nature, as their extent depends on the type of matrix and analyte and frequently varies between different batches of a given matrix Fig. 1 Electrospray SRM chromatogram (positive mode) obtained for the simultaneous determination of a mixture of 100 different fungal secondary metabolites. For each compound two SRM transitions were monitored

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[16]. Ion suppression can easily be detected and distinguished from real loss of analyte by comparing the response of a standard prepared in neat solvent with spiked blank extracts or by monitoring the baseline of the post-column infusion of the target analyte during a chromatographic run of a blank matrix [15]. In the case of dedicated single-target (class) methods these effects can be reduced by improving the cleanup or chromatographic separation. For multi-target methods ion suppression effects should at least be evaluated during method development. The best way to compensate for matrix effects is the use of co-eluting stable-isotope-labelled internal standards, such as 2H- or 13C- labelled analytes, as the matrix may influence only very narrow sections of a chromatogram as it is itself subjected to the separation process [17]. Other approaches for correction of matrix effects include standard addition, matrix-matched calibration or the echo-peak technique [18].

Multi-target methods The need for monitoring of a huge number of regulated compounds in different matrices has led to the development of multi-target methods for the simultaneous detection of up to several hundreds of analytes in a single method, e.g. residues of pesticides [19], drugs [20] and fungal contaminants [21]. Figure 1 illustrates an example of an ESI-SRM chromatogram obtained for a mixture of 100 fungal secondary metabolites. Two SRM transitions were recorded for each compound to avoid false positive results (see section below).

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In (multi)-target analysis the wide range of physicochemical properties of the analytes makes proper sample cleanup and complete HPLC separation for each of the compounds of interest impossible. For this reason multi-target methods often require the latest generation of highly sensitive and robust MS instruments that allow the injection of crude sample extracts with minimum or no cleanup. This means, however, that signal suppression/enhancement effects and method selectivity have to be carefully evaluated during method development and validation.

Confirmation of positive results In an attempt to avoid false positive results and to standardize the criteria for confirmation of positive findings, the European Commission has established a system of identifications points (IP) [7] in confirmatory analysis. Depending on the residue/ contaminant three or four IP are required. One IP is awarded for each (precursor) ion and 1.5 points for each product ion in the case of low resolution MS/MS (e.g. QqQ and IT), whereas in the case of high resolution MS/MS two and 2.5 points are earned. (Note that a rather strict definition of high resolution, i.e., >10,000 for the entire mass range at 10% valley, is used in [7], which usually cannot be achieved by regular TOF instruments.) For example, in order to obtain 4.0 IP (as required for banned compounds) with a low resolution MS/ MS instrument two SRM transitions per analyte have to be monitored. In addition, criteria for the relative intensities of the recorded SRM transitions must fit to those of an authentic standard and the retention time must not deviate more than 2.5%. Although the European Commission decision 2002/ 657/EC is only mandatory for the analysis of animal tissue, these requirements are widely accepted and are therefore complied by most of the recently developed methods, yet there are still doubts whether they are sufficient. Most notably, the use of SRM transitions with a low diagnostic value (such as the loss of water or adducts deriving from the LC eluent) should be avoided [22, 23]. Therefore, it was suggested to acquire a whole product ion spectrum for confirmatory purposes, which is possible with the newest generation of sensitive and fast scanning instruments such as QTrap, IT or Qq-TOF. As co-eluting isobaric interferences could lead to false negative results in this approach, it was proposed to use mass resolutions ≥ 20,000 in order to be able to differentiate between MS/MS precursors of the analyte and isobaric nonresolved matrix constituents [22].

Outlook It can be expected that further improvements of LC-MS/ MS instrumentation (e.g. new combinations of mass


analysers and new software features) and its availability at lower price will further contribute to LC-MS/MS becoming the major tool for the analysis of multiresidues and contaminants in food, biological and environmental samples.

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