Daniel McMillan is with Waters Corporation.

Daniel McMillan

Monitoring a highly polar, small organic pesticide such as glyphosate in food and water sources presents a significant challenge. Polar pesticides are not amenable to standard extraction procedures, are frequently poor ionizers, and do not separate well. Current analysis methods rely on labor-intensive derivatization and cleanup steps. This study provides details of a robust high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the detection of glyphosate and its metabolites in complex food and water matrices, without the need for derivatization of samples prior to analysis. Polar pesticides are water soluble, and the extraction was therefore aqueous with methanol and formic acid added to improve efficiency. The large variety of interfering particulates in environmental and drinking water samples requires further sample preparation, and a simple filtration step was performed here before the MS/MS analysis. The food analytes were well separated from matrix interferences in most of the foods tested, and all water sample analytes were well retained after removal of interferences. This new method largely eliminated matrix effects and achieved unambiguous identification, reproducible retention times, and sensitivity; and attains the targeted limits of detection.

	Despite its association with various health risks, glyphosate remains the most commonly used herbicide worldwide. Glyphosate-based pesticide formulations are used as a simple solution to improve yields by reducing weed growth around glyphosate resistant crops. Almost inevitably, some level of glyphosate residue ends up in the food chain, and it has been detected in the urine of a range of animal species, including humans (1). Polar pesticides are frequently poor ionizers, may suffer from low extraction from the sample matrix, and demonstrate poor chromatographic separation. These pesticides, therefore, have historically required complex single residue methods to make them amenable to analysis, involving time-consuming derivatization steps and considerable clean up procedures.

	The analysis of glyphosate using high performance liquid chromatography with fluorescence detection (HPLC–FLD) in water is well known, with or without derivatization. Usually, glyphosate undergoes derivatization by the reaction of the native glyphosate with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before separation on a reversed-phase column. If the separation is performed on a polar chromatographic column, post-column derivatization before detection is required. The tedious derivatization step complicates the analysis, and the reproducibility is poor. Thus, there is a growing need for a method that can detect not only glyphosate and its major metabolite aminomethylphosphonic acid (AMPA), but also glufosinate and similar highly polar compounds, in their underivatized states.

	Modern triple quadrupole mass spectrometers that allow fragmentation analysis offer very low detection limits and high detection selectivity. This approach enables laboratories to rapidly screen samples for a variety of regulated pesticides using the European Union Reference Laboratories' (EURL) quick, easy, cheap, efficient, rugged, and safe (QuEChERS) sample extraction and cleanup method. Owing to this, multi-residue liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses have become the minimum standard for the quantification of many pesticides in food and water samples. Nevertheless, glyphosate and other highly polar pesticides are not suitable for this type of analysis, and remain challenging for routine screening.

	The EURL quick polar pesticides (QuPPe) method allows the simultaneous analysis of a number of highly polar pesticides that are not amenable to common multiresidue methods. However, in practice, the separation is not robust, and requires intense system maintenance. The method involves extraction with acidified methanol and LC–MS/MS measurement, using isotope labeled internal standards for accurate quantification. Ion chromatography is the preferred separation technique for polar ionic analytes, such as anions, cations or small polar analytes (metabolites), and sugars. To use an ion chromatographic approach for the analysis of polar pesticides offers the ability to include multiple analytes in a single injection, without derivatization or the use of an ion suppressor. Food testing laboratories would benefit greatly, in terms of both time and cost, from a methodology that avoids these complex analysis procedures. Here, we present a robust and sensitive method for the direct analysis of polar pesticides in food and environmental samples without derivatization (2).

	The EURL QuPPe method for extraction of polar pesticides from food samples of plant and animal origin is well developed (3). In this experiment, the QuPPe extraction was adjusted by using a little less methanol and more formic acid. Because polar pesticides are water soluble, the extraction was aqueous with methanol and formic acid added to improve efficiency. The addition of internal standards to matrices is considered essential in order to compensate for matrix effects and the shifting retention times observed in many chromatographic separation procedures. Dispersive solid-phase extraction (SPE) cleanup using C18 was also carried out as described in the QuPPe-AO3 method. A push-through method was finalized using two sorbents and SPE filters, because the extracts obtained from the QuPPe method require extensive cleanup for more stable chromatography and less pollution of the source.

	The eluents' incompatibility with electrospray ionization sources requires the use of a suppressor, so as to lower the ion load for the electrospray ionization (ESI) source, and gain sensitivity. By employing a polyvinyl alcohol based column with quaternary ammonium groups, and using an ammonium bicarbonate buffer prior to detection by a highly sensitive quadrupole MS system, the need for a suppressor was removed.

	Environmental and drinking water samples vary widely in the degree of particle content, causing difficulties for LC injection and the reproducibility. SPE-type cleanup would add significant time, as well as financial cost, in a high throughput laboratory situation where minimal sample preparation is desirable. To overcome these challenges, and to be able to swiftly change between food and environmental samples, the same setup for food samples (except the 500 µL injection volume) was carried out. A simple filtration step, using Chromacol 17-SF-02 (RC) from 17-mm syringe filters, was performed when transferring the samples to the LC vials. Internal standards to a final concentration of 1 ppb were added to samples and standard solutions, and QC samples in tap water were prepared in a similar fashion. To minimize matrix interferences, standard additions to the samples were used.

	Separation was achieved using a Shimadzu Nexera ultrahigh-pressure liquid chromatography (UHPLC) system comprising LC-30AD pumps, a SIL-30AC autosampler fitted with a 500 µL loop, and a CTO-20A column oven. For chromatography, a polyvinyl alcohol column, with quaternary ammonium groups using a bicarbonate eluent between pH 8 and 10, was used.

	The finalized chromatographic method used 10 µL injections onto a 150 x 4 mm column, employing a 20 mm guard column of the same material, and a 0.5 µm filter, with a flow rate of 0.6 mL/min. Both columns were replaced every 250 samples to maintain performance, and to keep the MS source clean. Performance was shown to be robust and reproducible on a variety of food matrices used for method verification laid out in Table I, all subject to the cleanup method as described above.

	An injection volume of 500 µL was employed in a chromatographic method similar to that used for the food samples.

	Analyses were performed using a triple quadrupole linear ion trap mass spectrometer (Sciex QTRAP 6500+) in negative electrospray ionization mode (Table II, source parameters).

	At least two multiple reaction monitoring (MRM) transitions were optimized for each analyte, in order to quantify and confirm their concentration in all samples (Table III, analytes with MRM transitions employed).

	Data were acquired using Analyst 1.6.3 (Sciex) and processed for quantification and confirmation with reference to internal standards (IS), using MultiQuant 3.0.2 software (Sciex) (Table IV, summary for limits of detection).

	The stability and reproducibility of the method, in terms of retention time, peak width, peak area, and tailing factor, were found to be excellent, and over one thousand food samples from a variety of commodities were analyzed without system maintenance (Figure 1, reproducibility data for glyphosate internal standard [IS]).


	Figure 1: Reproducibility data for glyphosate internal standard (IS). The modified method for food samples tested over one hundred injections of extracts from the variety of food matrices presented in Table I. 

	The sensitivity of the method was demonstrated by considerably lower limit of detection (LOD) for every analyte and commodity combination than is required in most cases (Figure 3, example chromatography from surface water samples). Regulations are not expected to change significantly within the lifetime of the current generation of the most sensitive MS instruments. The linearity of the calibration curve was established in the concentration range of 15.6 to 1000 ng/L of glyphosate. By plotting the ratio of the peak area of the standard to that of the IS against the ratio of their concentrations at seven calibration levels, calibration curves were obtained (Figure 2, method sensitivity and linearity of glyphosate).


	Figure 2: Method sensitivity and linearity of glyphosate. Several measures of reproducibility based on the glyphosate internal standard (IS). Calibration standards were achieved using the modified method for water samples. Ion ratios were all within the specified ±20% tolerance.

	The method described is highly robust and sensitive, achieving the target limits of detection required to meet current and proposed regulations. The separation has also been found to minimize any matrix interferences in all but the most complex samples. Through incorporation of this high throughput method into the workflows of food testing laboratories, complex sample preparation can be eliminated, running costs can be kept low, and valuable time can be saved.


	Figure 3: Example chromatography from surface water samples using the modified water method. The graphs highlight that even at concentrations five times (5x) lower than the European Union (EU) water regulations requirement, the presence of glyphosate in real environmental samples was detected and confirmed by the ion ratio between primary and secondary multiple reaction monitoring (MRM) transitions.

	Chromatographic performance using these modified methods for food and water samples achieved good separation between the analytes and from matrix interferences, and excellent repeatability in terms of peak profile and retention time. This cleanup is performed on all samples, not only for food from animal origin. The ion source was clean even after more than one hundred injections.

	In order to maintain peak shape and retention times, various chromatographic methods were investigated. Ion chromatography gave the most promising results, but the resulting eluents were not compatible with electrospray ionization sources and required a suppressor, which is detrimental to peak width. The need for a suppressor was removed by the use of ammonium buffers, a deviation from traditional LC buffers that enables detection of polar pesticides by MS/MS. The setup in this study allowed switching between polar and normal pesticide (reverse phase) suites without changing inlet, giving sharper, more intense peaks, and shorter run times. In busy food testing labs that primarily work with reversed-phase LC, this setup saves valuable time, eliminating the need to change inlet systems between analyses.

	Food samples analyzed using the modified method for QuPPe extracts achieved unambiguous separation between the analytes as well as excellent repeatability in terms of peak profile and retention times. Analytes were also well separated from matrix interferences in most of the foods tested. The EU maximum residue limits (MRLs) for pesticides in food commodities vary widely, depending on the pesticide and commodity in question. Although some matrix interference was present in the most complex food samples, LODs in both food and water samples were well below the concentration limits laid out in EU regulation. Over one thousand food samples from a variety of commodities were analyzed without maintenance of the system, and the stability in terms of retention time, peak width, peak area, and tailing factor was found to be excellent.

	This method largely eliminated matrix interferences in food sample extracts, further enhancing the reproducibility and robustness of the method. However, in the most complex food matrices, MRM ion ratios were observed outside of the standard ±20% tolerance. In future work, the QTRAP will be used to confirm positive results by their full scan MS/MS spectra, and the sample cleanup procedure will be further developed in order to remove background interferences.

	It is commonly known that LC–MS/MS, especially when working in ESI mode, is susceptible to matrix effects, affecting quantitative accuracy. This new methodology overcomes the necessity of pesticide derivatization. This was achieved by combining an ion chromatography separation with the high sensitivity of the mass spectrometer for analyte detection. Analytes are successfully separated using an LC column in a method switching reversed-phase system, with MS-amenable mobile phases at around pH 9. All analytes are well retained with reproducible retention times, allowing detection of the majority of background components. The method meets the Directorate-General for Health and Food Safety (DG SANTE) requirements of reproducibility (<20%) and recovery (80 to 110%), and the limit of detection (LOD) of the method is below 0.01 mg/kg. Excellent long-term stability and robustness were achieved throughout the validation of this method for food samples extracted by the QuPPe procedure.

	This modified LC–MS/MS analysis offers the possibility to analyze multiple polar pesticides in a single injection without derivatization. The ion chromatography based approach uses ammonium buffers, a deviation from traditional LC buffers that enables detection of polar pesticides by MS/MS. A sufficiently sensitive mass spectrometer allows the analysis to be performed without the need for an ion suppressor using a standard reversed-phase LC based system. This eliminates the need to change inlets between typical pesticide analyses.

	By removing the need for derivatization, sample preparation is greatly simplified, and the time required to analyze polar pesticides is reduced. As an additional advantage, the column lifetime is well over one thousand matrix samples, allowing high-throughput polar pesticide analysis with less system maintenance. This method was found to be considerably more robust and sensitive than other approaches, and has achieved the target limit of detection required to meet existing and proposed future regulations of glyphosate and similar polar pesticides.

	(1) M. Krüger, P. Schledron, W; Schrödl, H.W. Hoppe, W. Lutz, and A.A. Shehata, 

210 (2014). doi: 10.4172/2161-0525.1000210.

	(2) W. Broer, U. Chiuminato, J. Stahl-Zeng, D. McMillan and P. Taylor, "A Robust and Sensitive Method for the Direct Analysis of Polar Pesticides in Food and Environmental Samples Without Derivatization" (Nofalab Laboratories, Schiedam, the Netherlands; Sciex, Darmstadt, Denmark; Sciex, Warrington, United Kingdom, 2018).

	(3) M. Anastassiades, D.I. Kolberg, A. Benkenstein, E. Eichhorn, S. Zechmann, D. Mack, C. Wildgrube, I. Sigalov, D. Dörk, and A. Barth, "Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of Plant Origin via LC–MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method)" Version 9.3 (EU Reference Laboratory for Pesticides Requiring Single Residue Methods [EURL-SRM], Fellbach, Germany, August 2017).

 are with Nofalab Laboratories in Schiedam, the Netherlands. 

 is with Sciex in Warrington, UK. Direct correspondence to: 

Articles

A new high-throughput LC–MS/MS method meets the challenge of eliminating matrix effects for monitoring, with high specificity,
polar organic pesticides such as glyphosate in food and water, while meeting targeted limits of detection.

A Robust and Sensitive Method for Detecting Glyphosate and Other Polar Pesticides in Food and Water: Multiple Analytes in a Single Injection without Derivatization

There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we know we don't know. But there are also unknown unknowns. There are things we don't know we don't know. 

—Donald Rumsfeld (US Secretary of Defense)

Traditionally, screening of targeted compounds in complex matrices involved the use of chromatographic techniques such as liquid chromatography (LC) and gas chromatography (GC) with detectors such as UV or flame-ionization detection (FID), which gave little selectivity. The sensitivity and selectivity of the analytical methods relied upon extensive sample extraction and cleanup procedures. Many laboratories have adopted the use of more selective detectors such as fluorescence for LC or electron capture, or nitrogen–phosphorus detectors in GC; however, methods using these approaches tend to be compound class specific and remained critically dependent upon sample preparation. The desire to analyze different compound classes and to reduce sample preparation has led many laboratories to use mass spectrometry (MS) for screening complex matrices. GC–MS and LC–MS can reduce the need for time-consuming sample preparation procedures and support the development of more generic extraction procedures that are a prerequisite for comprehensive multiresidue analysis.  

Selected ion recording (SIR) and multiple reaction monitoring (MRM) are common nominal mass (± 1 Da) MS and MS-MS methods that enable targeted screening of a finite number of compounds to be achieved (for example, in metabolite–impurity profiling and screening for pesticide residues or veterinary drug residues in food). While ideal for detection and quantitation, these approaches are impractical for comprehensive, generic screening and particularly for the identification of unknown or unexpected compounds. In contrast, time-of-flight (TOF) MS provides the ideal combination of speed, sensitivity, and selectivity. TOF-MS has the full-spectrum sensitivity and the spectral acquisition speed to take full advantage of the narrow peak widths from GC and ultrahigh performance LC. Another key advantage of orthogonal acceleration (oa) TOF-MS is in its ability to provide routine accurate mass measurement (typically <5 ppm root mean square). The importance of accurate mass measurement for the confident detection and identification of compounds from these data cannot be understated. Accurate mass provides the high level of specificity required for the component detection in the presence of multiple coeluted species and enables determination of elemental composition and can help structural elucidation of nontarget compounds. Recent advancements in software technology that utilize novel isotope modeling, results filtering, and accurate mass library searching routines have also made it possible to significantly simplify compound detection, identification, and confirmation from accurate mass data. 

Here we describe the feasibility of truly generic accurate mass ultrahigh performance LC–TOF-MS and GC–TOF-MS methods for the analysis of potentially unlimited numbers of compounds with minimal sample preparation. Results from the screening of veterinary drug residues and pesticides in food demonstrate the ability of this technology to screen for targeted compounds and to detect and aid the identification of unknown or unexpected components from complex matrices.  

Multiresidue Analysis of Pesticides in Food by GC–TOF-MS

The inappropriate or unlawful use of pesticides on agricultural produce can result in unacceptably high levels of residues in produce destined for human consumption. Produce that enters the food distribution chain that is to be used for this purpose must contain less than the statutory maximum residue limit (MRL) of any given residue. In the European Union, legislation sets and controls MRLs in food and feeding stuffs (1). One key feature of the recent legislation is that a default MRL of 0.01 mg/kg will apply to those commodities when no specific MRL is set. Given that there are over 800 compounds in use to control pests, it is more efficient to extract and determine as many of them as possible during a single analysis.  

To establish a suitable screening technique, a number of requirements must be met to effectively extract, separate, detect, and identify all components. These include minimal nonselective sample preparation for a wide range of compound groups with different polarities; simple high-resolution GC separation to minimize matrix interference while maintaining resolution of critical pairs; and automated peak detection and deconvolution of all components in the sample. 

Generic extraction procedures have been reported for the analysis of a wide range of pesticides in fruits and vegetables (2) and fatty samples (3) typically using acetonitrile followed by dispersive solid-phase extraction (SPE) cleanup. 

We employed a GC–TOF-MS method for the targeted screening of pesticide residues in various food commodities at the legislated concentration of 0.01 mg/kg. The method is extended to include nontarget screening results using automatic peak detection, deconvolution, and library searching with accurate mass confirmation. The sample extraction, GC, and TOF-MS methods have been described previously (4). We used an oa-TOF instrument (GCT Premier, Waters Corporation) with electron ionization mode and spectra were obtained over the mass range 50–500 Da. We operated the instrument at over 7000 resolution (fwhm), and the instrument generated accurate mass spectra using a reference compound of known mass, which was introduced to the instrument throughout the analysis. 

Initially, four food commodities (cucumber, sweet pepper, grapefruit, and wheat flour) were screened for known pesticides to validate the approach. 0.01 mg/kg was chosen to be the reporting level because this is the target MRL for active substances in products for which no specific MRL is set (5). 

To illustrate the improvement in selectivity offered by accurate mass chromatograms, vinclozolin (0.01 mg/kg) was analyzed in cucumber, a relatively simple matrix. The extracted ion chromatogram at 

 212 in Figure 1(generated with a 1 Da window, as it is typically done when using a quadrupole or ion trap–type mass spectrometer) contains a number of intense peaks that could lead to interference when using automatic integration. In the accurate mass chromatogram (20 mDa, 

 212.0034), vinclozolin has little or no interference, improving the selectivity of the method. 

Improving the selectivity can lead to an increase in the signal-to-noise ratio (S/N), which can make the difference between detecting the compound and not detecting it (Figure 2). The nominal mass chromatogram (1 Da, 

 248) results in an S/N of 4:1. In the accurate mass chromatogram (20 mDa, 

 248.0397) the S/N has increased to 49:1. The mean difference between the nominal and accurate mass S/N was a factor of five for all residues. 

To test the quantitative capabilities of this approach, 30 standard compounds were prepared in the concentration range 5–500 pg/μL (equivalent to 0.005–0.5 mg/kg with PCB 138 used as an internal standard) and spiked into each matrix. The data from the subsequent GC–TOF-MS analysis were processed automatically using a dedicated software package for detection and quantification of target compounds (TargetLynx, Waters Corporation). In Figure 3, the quantitative results for one of the compounds in grapefruit, pirimiphos-methyl (at a spiked concentration of 0.01 mg/kg), are shown. All 30 residues could be screened using this method in all matrices to a concentration of 0.01 mg/kg, demonstrating the applicability of GC–TOF-MS in targeted screening and quantitation. 

Five matrices were used to further test the method — orange, cherry tomato, grapes, kiwi, and strawberry (a cucumber matrix–matched calibration curve was used for quantification purposes). Automatic quantification was carried out, with two accurate mass chromatograms (0.02 Da) processed for each residue. Figure 4 shows the quantification (0.067 mg/kg) of chlorpyrifos in grapes. This correlates well with a targeted tandem quadrupole MS-MS method (Quattro micro GC, Waters Corporation), in which the concentration was found to be 0.066 mg/kg (6). 

In an untargeted screening environment, TOF-MS generates an information-rich data set requiring considerable analysis to detect, deconvolute, and identify the compounds present, which is very time-consuming if performed manually. We chose to process the data using software  (ChromaLynx, Waters Corporation) that automatically plots the reconstructed ion chromatograms (RICs) of up to eight most intense ions at any point in the chromatogram. If a peak is found to satisfy a set of user-defined parameters, then the software will record this as a component peak and generate its deconvoluted mass spectrum. The spectrum can then be submitted to an automatic library search routine with the ability to test the confidence of library hits using accurate mass scoring.  

Processing of the 0.05-mg/kg spiked cucumber extract located 550 total components in the chromatogram, including 30 spiked residues. Etrimfos is an example of one of the untargeted compounds that we found (Figure 5), which was confirmed with three individual ions (within 1.1 mDa of their expected accurate mass). More than 90% of the spiked residues at 0.05 mg/kg were located and identified using this method, further demonstrating the applicability of GC–TOF-MS in nontarget screening. 

Multiresidue Analysis of Veterinary Drug Residues by Ultrahigh Performance LC–TOF-MS

A wide variety of veterinary drugs are used as antibiotics in livestock around the world. In many countries, including EU member states and the United States, testing for these compounds in animal-based food products is mandatory and the permissible concentration levels are strictly regulated. Due to the complexity of matrices (for example, muscle tissue, urine, and milk) and the large number of potential analytes, it is beneficial to use a generic analytical method applicable to a diverse range of samples, which means data can be reprocessed to search for new compounds if "food scares" arise. 

Traditional methods for analyzing veterinary drugs in biological matrices include microbiological or immunochemical techniques. While they are rapid and cost-effective, they are only suitable for one analyte or class, and have limited  quantitative capability. High performance LC with UV or fluorescence detection is commonly used for quantification and confirmation; however, extensive sample cleanup is needed to achieve the required sensitivity and selectivity of analysis.  

Here we demonstrate the feasibility of a truly generic LC–MS method for the untargeted analysis of potentially unlimited numbers of residues with minimal sample preparation. Milk samples were spiked with standard solutions containing 41 compounds from five classes (tetracyclins, sulfonamides, quinolones, macrolides, and benzimidazoles) at concentrations of 0.5, 1, 2, 5, 10, 25, 50, and 100 μg/L, respectively. These concentrations cover the relevant concentration range with respect to the MRL of the analytes. The experimental setup has been described previously (5), in which an ACQUITY UPLC System (Waters Corporation) was used with columns containing 1.7-μm size novel packing material and operated at high pressure to obtain maximum speed and chromatographic resolution. An oa-TOF-MS system (LCT Premier XE, Waters Corporation) was used with electrospray ionization (in positive ion mode) to acquire data over the mass range 100–1000 Da at high resolution (>10,000 fwhm). An independent reference compound was used over the duration of the analysis to enable generation of accurate mass data (<3 ppm).  

Initial manual interrogation of the data was performed to determine the sensitivity on spiked compounds in milk, and to investigate the quantitative capability of the ultrahigh performance LC-TOF system. Figure 6 shows that good linearity was observed and that the limit of detection/limit of quantitation (LoD/LoQ) is much lower than the MRL concentration. 

The Use of Isotope Ratio Models for Screening

Elucidation of structure for nontarget compounds using accurate mass requires the calculation of candidate elemental compositions. This type of analysis rarely results in a single candidate formula, and it is not unusual to have thousands of candidate formulas, even with high mass accuracy. As such, it is problematic to decipher which is the correct formula, so we employ i-FIT software (an accurate mass and isotope ratio correlation tool) to aid this process. The i-FIT software (Waters Corporation) is a two-stage process in which a mass spectral correlation algorithm orders the list of candidate formula by (i-FIT) score, followed by element prediction filters that remove formulas that are considered "incorrect" based on interpretation of the spectral data. The outcome is a "focused list" of potential candidate formulas. The element prediction filters have the potential to remove greater than 99% of the "incorrect" candidate formulas (7). 

Screening of compounds in this veterinary drug residue analysis study is carried out by a comparison of the experimental data to a potentially limitless library of experimental and theoretical spectra with accurate mass scoring providing increased confidence in compound library hits. A spectral library of standard solutions was acquired with retention time data (oa-TOF aperture voltage of 8 V), including spectra from collision-induced dissociation (CID) (aperture voltage of 30 V), which contained fragment ions to assist in compound characterization. These spectra were all quality checked by isotope interrogation software (i-FIT, Waters Corporation) (7). A library was created containing a normal MS spectrum and at least one fragmented spectrum for each compound (Mass-Lynx software, Waters Corporation). This was then converted to the National Institute of Standards and Technology (NIST) format (ChromaLynx software, Waters Corporation), which allows filtering of the matched spectra for screening purposes. A library search method specifies which libraries to use, how many ions to search against, maximum number of hits, and any screening parameters (including peak width, noise limits, retention time, polarity, and cone voltage). This method not only confirms a library hit at a glance, but any fragments formed by in-source CID also have an elemental composition based on that of the parent ion assigned to them, which makes structural elucidation much easier. 

Figure 7 shows the results for a milk sample spiked with several veterinary drugs at a concentration of 10 μg/L — equivalent to the lowest MRL of any of the 41 compounds. The two compounds required to be reported at this level are flubendazole and oxfendazole, both of which have been correctly assigned by reference to the library created from standards. 

Table I shows a summary of the MRLs (where applicable), the level at which ChromaLynx screening gives a positive (tentative in brackets) identification, and the LoDs for each of the spiked compounds. All library entries are correctly identified at or below their MRL. 

It is important to note that, while in this study a mass accuracy of <5 ppm has been considered confirmatory, there is currently no such consideration in EU legislation. A sample found to contain any of the analytes should therefore be subject to further analysis, typically on a tandem quadrupole–type MS-MS instrument, where product ion ratios are used for confirmation. Many groups are currently investigating and validating the use of accurate mass spectra for confirmatory purposes. However, at this early stage the authors recommend that two mass spectral peaks with <5 ppm accuracy, acquired with a mass resolution of at least 10,000 fwhm, should be considered for confirmation. Matching measured and theoretical isotope ratios has proven a reliable and conclusive method for the correct assignment of an analyte's identity and, hence, stringent criteria for this should be compulsory. 

Table I: AnalytesÃÂ´ MRL, screened concentration, and estimated LoD in milk			

GC–TOF-MS and ultrahigh performance LC–TOF-MS represent powerful solutions for the rapid, comprehensive, and confident screening of compounds in complex biological matrices. In the cases shown here, it has been possible to apply a generic, minimal sample preparation procedure while achieving sensitivity beyond the required MRLs for veterinary drug residues and pesticides in food and feedstuffs as defined in European legislation (1,5). This is possible due to the full-spectrum sensitivity provided by oa-TOF instrumentation and can be extended to large numbers of residues without the loss in sensitivity that would be observed using quadrupole or ion trap instrumentation. The increased chromatographic resolution from ultrahigh performance LC separations also helps minimize matrix effects and obtain analyte peaks suitable for quantification in the oa-TOF analyzer. These quantitative results have been shown to correlate with levels observed from analysis on tandem quadrupole instruments.  

Accurate mass measurement is a key metric for the successful screening of complex matrices. Its selectivity allows the analyst to confidently detect chromatographic peaks from complex mixtures in the presence of multiple coeluted compounds, while providing increases in S/N and limits of detection. Spectra with high mass accuracy and correct isotope ratios are also valuable aids for identifying contaminants, and might be appropriate for confirmatory analysis. 

With the development of specialized software tools, it is now possible to automatically detect peaks, deconvolute mixtures, perform library searching, and carry out accurate mass confirmation in a routine manner from complex LC–MS or GC–MS datasets. This provides the analyst with the capability to confidently quantify and confirm compounds in a targeted environment, or quantify and identify unexpected compounds in an untargeted environment. 

The authors would like to thank Hans van Rhijn from RIKILT Institute of Food Safety, Wageningen, The Netherlands, for both his collaboration in the veterinary drug residue analysis and his helpful comments in review of this article. We also thank Carmen Wauschkuhn, Diane Fuegel, and Michelangelo Anastassiades, CVUA Stuttgart, Fellbach 70736, Germany, for supplying sample extracts that were analyzed in the pesticide residue study. 

Peter Hancock, Daniel McMillan, Jeff Goshawk, Kirsten Hobby,

(1) Regulation (EC) NO 396/2005 of 23 February 2005, Off. J. of the European Union No. L70/1. 

Ultrahigh performance liquid chromatography (LC)–time-of-flight mass spectrometry –(TOF-MS) and gas chromatography (GC)–TOF-MS are powerful approaches for screening target compounds and identifying or characterizing nontarget compounds in complex mixtures. The combination of accurate mass data and newly developed software enables truly generic screening methods with TOF-MS, and the confident detection, identification, and confirmation of small molecules in a range of application areas.

Daniel McMillan

Articles

A Robust and Sensitive Method for Detecting Glyphosate and Other Polar Pesticides in Food and Water: Multiple Analytes in a Single Injection without Derivatization

An Expanding Role: The Use of Ultrahigh Performance LC–TOF-MS, GC–TOF-MS, Accurate Mass, and Isotope Modeling for Screening Complex Mixtures