M ASS S PECTROMETRY

3.4.3 M ASS S PECTROMETRY

3.4.3.1 Ionization The main prerequisite for MS is the introduction of ions in the gas-phase ions at

reduced pressures. The main challenge is to generate gas-phase ions from analytes in the LC eluent, while removing the solvent and maintaining adequate vacuum level in the mass spectrometer. This is achieved by evaporation, pressure reduction, and

ionization. Particle beam (PB) ionization 106 has the advantage of providing classical, library-searchable electron ionization spectra for compounds that were too thermally

labile or nonvolatile to be analyzed by GC-MS. The robustness and sensitivity of PB was limited and so, with the introduction of alternatives, it had limited application for routine trace analysis. Currently, the most popular technologies create ions at atmospheric pressure and then sample ions through so-called atmospheric pressure (AP) interfaces. Blakely and Vestal 107 introduced the TSP ionization source, which was capable of producing ions from an aqueous solution sprayed directly into the mass spectrometer using conventional analytical LC flow rates. There was much debate over the models proposed for the ionization mechanism, 108 which later contributed to the construction of models for ionization taking place in other API technologies. The use of TSP for small molecule LC-MS applications also facilitated familiarity with the concept of generation of ions through cation attachment (e.g.,

[M þ NH 4 ] þ ) and the use of negative ion mode for acidic compounds. TSP found tremendous use as an interface for higher flow-rate LC=MS, including for pesticide residue analysis. 109 Although its usage lessened as other API technologies become more popular, existing instruments could be relatively easily upgraded with the new,

more sensitive, and robust API devices. ES ionization 111 and APCI are now among the most commonly used techniques for creating ions from pesticides in

solution. The application of the more recent atmospheric pressure photoionization (APPI) to pesticide residue analysis is less common. 112 Although still at the devel-

opment stage, there has been growing interest in EI for LC-MS 113,114 to obtain library-matchable, readily interpretable, mass spectra, to aid the confirmation of

identity of targeted pesticides and, more importantly, for the characterization of unknowns such as metabolites or transformation products.

3.4.3.2 Electrospray Ionization The basic aspects of ES ionization and APCI are described in detail elsewhere 115 –120

but Willoughby et al. provide a good practical introduction to ionization used for LC-MS. 121 There have been considerable efforts directed at understanding the

Analysis of Pesticides by Chromatographic Techniques 79 mechanisms involved in ion production for electrospray; 122,123 but as practitioners,

we have come to accept that ES works and many might rather avoid considering the mechanisms involved. But, as Balogh argues, ‘‘understanding how ions are liberated from the liquid mobile phase in the gas-phase transition helps us understand and

diagnose issues such as lack of expected sensitivity and ion suppression.’’ 124 Two separate theories have been proposed, the charge residue mechanism and ion evap-

oration mechanism models, but Cole argues that both mechanisms might be working concurrently: the charge residue mechanism dominating at high mass and ion evaporation dominating for lower masses. 125 The ES probe, or device, is typically

a conductive capillary, usually made of stainless steel, through which the eluent from the LC flows. A voltage is applied between the probe tip and the sampling cone. In most instruments, the voltage is applied on the capillary, while the sampling cone is held at low voltage. The capillary, contained within a larger bore tube, allows a concentric nitrogen flow applied to the aerosol at its exit point so that capillary acts as a nebulizer. While this variant was initially called ‘‘pneumatically assisted elec- 126 trospray’’ or ‘‘ion spray,’’

this terminology appears to be replaced by the more generic ‘‘electrospray.’’ Aerosol droplet formation is enhanced by the added shear forces of the gas and heat transmitted from adjacent supplemental devices, direct heating of the gas itself or with the assistance of an additional heated desolvation gas. ES ionization takes place as a result of imparting the strong electrical field to the eluent flow as it emerges from the nebulizer, producing an aerosol of charged

droplets. Due to the solvent evaporation, the size of the droplet reduces, and, consequently, the density of charges at the droplet surface increases. The repulsion forces between the charges increase until there is an explosion of the droplet. These coulombic fissions continue until droplets containing a single analyte ion remain. The charge residue model suggests that a gas-phase ion forms only when solvent from the last droplet evaporates. In the ion evaporation model, the electric field strength at the surface of the droplet is thought to be high enough for solvated ions to attain sufficient charge density to be ejected from the surface of the droplet and transfer directly into the gas phase without evaporation of all the solvent. Ensuring that the compound of interest is ionized in solution critical for ES ionization, so mobile phases should have a pH such that the analytes will be ionized. Charging is usually accomplished by adding or removing protons but cation or anion attachment generating adduct ions is also common.

3.4.3.3 Atmospheric Pressure Chemical Ionization Although work that demonstrated APCI as an ionization technique for LC-MS was

published by Carroll 128 some time before Whitehouse’s work on electrospray, it was not widely adopted until the latter was commercialized. The complimentary

capability of APCI was marketed as enabling the analysis of compounds that resisted converting to gas-phase ions using ES, that is, the less polar and more volatile ones. In contrast to ES, APCI transfers neutral analytes into the gas phase by vaporizing the LC eluent contained within a nonconductive capillary inserted in a coaxial pneumatic nebulizer through which a gas is added to assist the ionization process. The mixture of gas and nebulized eluent passes through a heated zone that assists the

80 Analysis of Pesticides in Food and Environmental Samples solvent evaporation and the fine droplets are converted into desolvated molecules in

the gas phase. The desolvated analyte molecules are then ionized via chemical ionization; the transfer of charged species between a reagent ion and a target molecule to produce a target ion that can be mass analyzed. The corona-discharge needle in the APCI source produces a stream of electrons that ionizes the atmosphere surrounding the corona electrode, which consists mainly of nebulizer and drying gases (typically nitrogen and=or air), the vapor generated from the HPLC eluent, and the analyte molecules. The process starts by ionizing nitrogen and finishes with protonated water, water clusters, and solvent clusters as possible reagent ions. For successful APCI, the analyte must be volatile and thermally stable and the mobile phase must be suitable for gas-phase acid –base reactions. For example, when working in positive ion mode, the proton affinity of the analyte must be higher than the proton affinity of the eluent: that is, the analyte can acquire a proton from the protonated solvent. Since water cluster ions are a major source of reagent ions, the proton affinity of these clusters relative to analyte ions will have a profound 129 effect on sensitivity.

Similarly, the use of certain modifiers added to the mobile phase to enhance LC separation (e.g., triethylamine) can be the source of consider- able ion suppression in APCI. A strong base will receive protons from the reactant ions to form their protonated forms. Subsequent proton transfer will occur only if the analyte is more basic than the modifier. 130

Although the choice of the most appropriate interface as well as detection polarity are based on analyte polarity and LC operating conditions, many classes of compounds perform well using either technique and sometimes in both ion modes, whereas, for other compounds, the choice is more restricted. 131 Interfaces are selected based on individual preference derived from experience and available techniques as well as the magnitude of any matrix effects. Although there are a great number of examples of the use of APCI for pesticide residue analysis for both environmental and food applications, including some pioneering early work, 132 more recently the technique appears to be left in the wake of ES ionization’s overwhelming popularity. This may be related to the increasing number and wider range of pesticides currently sought but perhaps also reflects the improvements in source and probe design for ES not yet paralleled with APCI. The choice between ES and APCI is irrelevant when using the recently introduced multimode sources, which deliver simultaneous ES ionization and APCI. 133

3.4.3.4 Atmospheric Pressure Interfaces Common to all API sources for mass spectrometers is an ion inlet orifice that forms

an interface between the API region and the low-pressure region of the mass analyzer. This small orifice allows the vacuum system attached to the mass analyzer to maintain a satisfactory vacuum therein at a finite pumping speed. The API source of commercial LC-MS instruments is arranged orthogonally of the ion inlet orifice, providing improved tolerance to nonvolatile components in the LC eluent and a more

stable ion signal than axial predecessors. 134 Consequently, cone orifices can be larger than in previous designs. The combination of larger orifices and noise reduction largely compensates for transmission losses due to the orthogonal geometry, giving a

Analysis of Pesticides by Chromatographic Techniques 81 large gain in sensitivity. The sampling of ions from atmospheric pressure into the

high vacuum region of the mass analyzer region requires significant pressure reduc- tion. A gas stream introduced into a vacuum system expands and cools down. When this gas stream contains ions and solvent vapors, the formation of ion –solvent clusters is observed. To obtain good sensitivities and high-quality spectra, one of the key roles of the interface is to prevent cluster formation. Declustering of analyte ions may be achieved using one or a combination of the following approaches; using

a countercurrent gas flow between interface and sampling plates, also known as ‘‘curtain gas,’’ using a heated transfer capillary between the API region and the nozzle –skimmer region, and=or using a drift voltage between the nozzle and skim- mer plates to promote intermolecular collisions between the analyte clusters and the background gas molecules. In APCI, nebulizer temperatures should be optimized to reduce cluster ion formation.

3.4.3.5 Characteristics of Atmospheric Pressure Ionization ES and APCI are soft methods of ionization as very little residual energy is retained

by the analyte on ionization. The major disadvantage of the techniques is that very little fragmentation is produced. The mass spectra generated by either technique are typically dominated by protonated or deprotonated molecules, [M þ H] þ or [M H] , depending on the ion mode used and adducts (e.g., [M þ Na] þ , [M þ

NH 3 ] þ , [M þ HCOO] ). This only provides information on molecular weight. This is very different from the information-rich spectra obtained with EI. For better selectivity through MS=MS or elucidation of structure, fragmentation is needed. Possible fragmentation techniques include ‘‘in-source’’ CID, CID in the collision cell of a tandem-type instrument and fragmentation in an ion trap.

One of the major problems encountered using LC-MS with ES is the presence of coeluting matrix compounds that alter the ionization of the target compounds, and which can reduce drastically the response affecting both quantification and detection of pesticide residues. This phenomenon is known as the matrix effect and, because it has an important impact on pesticide analysis, it has been the object of considerable study. 135,136 There are a number of ways matrix effects can be detected; the most straightforward way is the comparison of the response obtained from a standard solution with that from a standard solution prepared in a matrix extract. This approach can be extended to the comparison of calibration graphs obtained from the analysis of standards prepared in solution with those prepared in

matrix extracts. A third approach is the postcolumn infusion system, 137 in which continuous postcolumn infusion of the analyte of interest is performed while blank

extracts are injected into the LC column. This enables the evaluation of the absolute matrix effects on the analyte at different portions of the chromatogram, illustrating the need for change in the LC separation required to minimize the matrix effect. If matrix suppression cannot be eliminated by improved sample preparation or reoptimization of LC conditions, careful consideration of calibration strategy is needed to compensate as much as possible for matrix effects. Using matrix-matched calibrants, standard addition or stable isotope-labeled internal standards is recommended.

82 Analysis of Pesticides in Food and Environmental Samples

3.4.3.6 Tandem Mass Spectrometry Analyzers

A wide range of mass analyzers is used for pesticide residue LC-MS analysis. 138 Despite early successes, 139 single quadrupole instruments are rarely used now for pesticide residue analysis, as, when combined with API sources, the technique lacks the selectivity required for both detection in complex matrices and for confirmation of identity. Single quadrupole MS has been superseded by MS=MS. Originally, LC-MS=MS was mainly delivered on 3D QIT instruments, as they provided more cost-effective access to MS=MS than triple quadrupole instruments. 140 The applica- tion of wideband excitation (activation) and normalized collision energy leads to highly reproducible mass spectra without losses of sensitivity, which has enabled the

publication of searchable libraries. 141 An additional key feature of the ion-trap instrument is that it provides multiple stages of MS=MS (MS n ). Product ions of a

single m=z value produced by an MS=MS experiment are stored to the exclusion of product ions of all other m=z values to obtain a second iteration of MS=MS. This can

be carried out for several more iterations. Although most commercial 3D QIT mass spectrometers allow for MS n where n is 10 iterations, in practice the time it takes for

a typical HPLC peak to elute limits the experiment to MS 3 . Detailed studies of pesticide CID ion fragmentation processes and pathways have been reported using

MS n , but few LC-MS methods for pesticide residue analysis have been devel- oped making use of this extra selectivity. 143 Other restrictions of the commercial 3D

n 142

QIT are its inability to trap product ions below m=z 50, and the existence of a upper limit on the ratio between the precursor mass and the lowest trapped fragment ion mass that is ~0.3 (dependent on the q=z value). The fragment ions with masses in the lower third of the mass range will not be detected. When Soler et al. compared the use of 3D QIT and triple quadrupole for the determination of pesticide residues, they found that product ion scanning with either MS=MS or MS n could not provide sufficient sensitivity needed to monitor MRL compliance in oranges. 144 The future

of ion-trap technology may lie with the new linear ion traps (LIT), which can be used either as ion accumulation devices in combination with quadrupole (Q), TOF, and Fourier transform ion cyclotron resonance (FT-ICR) devices or as commercially available, stand-alone mass spectrometers with MS n capabilities. 145

LC-MS=MS with a triple quadrupole instrument in MRM mode is currently the most widely used technique for the quantification of target pesticides 146 –148 as it

delivers the sensitivity required for monitoring compliance with the legislation. The use of other modes, such as neutral loss or precursor ion scanning, is limited by poor sensitivity and specificity. Generally, a triple quadrupole instrument used in MRM mode provides an order of magnitude better limit of quantification than product ion scanning on a 3D QIT instrument. Due to the enhanced selectivity, interfering peaks from other pesticides or matrix are rarely observed. For confirmation of identity, the ratio of at least two MRM transitions is required to match that of a reference standard. One of the current challenges for LC-MS=MS is to be able to acquire sufficient data points for quantification while acquiring an ever increasing number of MRM transitions, with very short dwell times (e.g., 5 ms), especially when coupled with UPLC. Reports of using LC-MS=MS for determination of 50 pesticides or more are becoming more common. 149

Analysis of Pesticides by Chromatographic Techniques 83 The main limitation of triple quadrupoles in MRM mode is that confirmation of

identity is based on the ratio of one or more MRM transition rather than full MS=MS product ion spectra. The replacement of Q3 in a QqQ instrument with a scanning LIT enhances its sensitivity in product ion scanning mode. 150 Addition-

ally, the system has MS 3 capability and time-delayed fragmentation scans that aid structure elucidation. Quantitative (MRM) and qualitative (MS=MS or MS 3 product ion spectra) work can be performed concomitantly on the same instrument. Although reports of the use of the QqLIT instrument for pesticide residue analysis are currently limited to material from the vendor, 151,152 it is in routine use in some laboratories.

3.4.3.7 Time-of-Flight Analyzers As the lists of targeted compounds get longer, setting up time-segmented MRM

methods becomes more and more complicated. At the same time, interest in incorporating nontarget analysis into monitoring programs, especially for banned substances and unknown metabolites and transformation products, has grown considerably. Many of the new pesticides and their transformation products are readily analyzed by LC-MS. A screening and identification scheme for pesticides was reported, which employed searching a pesticides exact-mass library for the empirical formulas generated from accurate mass data acquired with high mass

resolution. 153 High resolution and accurate mass are available from four types of instruments: double-focusing magnetic sector, LIT-Orbitrap, FT-ICR, and TOF

mass spectrometers. Reports of coupling magnetic sector instrument with API interfaces are limited. 154 While both LIT-Orbitrap and FTICR instruments, which combine high trapping capacity, MS n capabilities with excellent mass accuracy and resolving power, have considerable potential for analysis of pesticide res- idues, 155,156 neither of them are yet used for routine monitoring. The current trend in pesticide residue analysis is to use LC-TOF MS or LC-QTOF MS systems because they are easier and less expensive to operate compared with the other three mass spectrometers. 157 Although TOF-MS can record accurate full spectral infor- mation with good sensitivity, this is of limited use unless resolution and mass accuracy are enhanced, as the spectra generated using the soft ionization API inter- faces are usually characterized by a lack of fragment ions. LC-TOF instruments are capable of a resolving power of 10,000 –20,000 (FWHM) and an accuracy of 2–5 ppm and the rapid acquisition rate combines well with fast chromatography systems. High mass resolution allows the reduction of the mass window when extracting a specific mass, leading to a substantial reduction in chemical noise, facilitating the detection of the analyte in the extracted ion chromatogram. If the mass window is set too narrow, mass errors either from drift in instrument calibration or derived from coelution of isobaric interferences from the matrix can lead to errors in quantification or, false negatives if the compound falls out of the mass window. As a compromise between enhanced selectivity and prevention of reporting false negatives, a 50 mDa mass

window is recommended. 158 TOF was found to be around one order of magnitude less sensitive than a triple quadrupole instrument used in SRM mode 159 and so

sensitivity of LC-TOF may not always be sufficient for the intended application. The

84 Analysis of Pesticides in Food and Environmental Samples introduction of a concentration step may be required to meet required reporting

limits. Although acquisition rates have now improved (e.g., 20 spectra=s) and issues related to linear range still hamper the use of LC-TOF for quantification, some recent

success has been reported. 160 None of the commercial benchtop TOF systems currently available meet the definition of high resolution given in Decision

2002=657=EC 69 and so no added weight should be conferred for an accurate mass measurement. Currently, at least four ions would have to be measured earning four IPs for confirmation, whereas if one considers LC-TOF to be a high-resolution technique, then only two ions need to be measured as each ion earns two IPs. In-source fragmentation can be used to increase the number of measured ions but the origin of the fragment ions may not be unequivocal and the technique is prone to isobaric interference. An alternative approach for confirmation of identity using LC- TOF is to measure the mass error from the accurate mass measurement of the suspect positive and reference standard but the number of compounds sharing the same empirical formula and therefore the exact mass can be surprisingly high which makes

accurate mass measurements on the fragment ions necessary. 161 For elucidation of unknowns, the combination of accurate mass with accurate isotope composition and

the use of database searches may lead to a reduced number of potential candidates but rarely a single answer. In the absence of a list of possible candidates, the complimentary use of other techniques is normally required to obtain a molecular

formula and structure. 162 The development of the hybrid quadrupole –time-of-flight (QTOF) instruments

presents the analyst with all the advantages indicated for the LC-TOF with the additional capability of accurate mass product ion scans. Figure 3.5 shows a schematic of a QTOF instrument. QTOF allows the determination of elemental composition of all the product ions from an MS=MS experiment, a feature, which has been used for confirmation of identity for target compound analysis and elucidation of unknowns. 163 When confirming positive findings, both the exact

Detector Quadrupole

Collision cell

mass filter (Q) quadrupole (q) Orthogonal Orthogonal Orthogonal accelerator accelerator accelerator

Ion source

Ion guides

Reflectron TOF

FIGURE 3.5 Schematic overview of a QTOF mass spectrometer.

Analysis of Pesticides by Chromatographic Techniques 85 masses and the relative intensities of all the product ions can be compared with

those of the reference standard. The enhanced selectivity of MS=MS, when com- bined with high resolution and mass accuracy of the measurement of the product ions, provides low chemical background and hence improved quality of confirm- ation. Regardless of whether TOF is considered high resolution or not, the number of IPs possible with LC-QTOF is higher than for LC-TOF as the system is weighted for MS=MS. The LC-QTOF has been used for the elucidation of pesticide metabolites and transformation products, but the potential is more limited when no previous knowledge is available. In such cases, the most common approach is to search a database for the molecular formula. The accurate product ion mass spectrum provides additional structural information, which can be used for com- parative purposes with reference standards or to distinguish between isomers. The application of the database approach is limited by the absence of many compounds, including pesticide transformation products, which are not included in commercial databases.