Because of these limitations, there is currently a trend towards full scan MS experiments in residue analysis. Current screening approaches are performed using high performance ToF instruments, with mass accuracies of <5ppm and resolutions of about 15000, coupled to ultra high performance liquid chromatography (U-HPLC).
In complex sample matrices (eg food, feed, hair, honey) this limited resolution leads to inaccurate mass measurements caused by unresolved background matrix interferences. In this work a full scan screening approach using a novel single stage Thermo Scientific Orbitrap mass spectrometer coupled to U-HPLC is shown, capable of providing high mass accuracy at resolutions of up to 100000. Additionally two aspects of the analysis will be discussed which also greatly benefit from very high resolution: resolving co-eluting, isobaric target compounds; elemental composition determination.
A new non-hybrid benchtop LC-MS Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, Bremen, Germany) coupled to a U-HPLC chromatograph (Thermo Scientific Accela, Thermo Fisher Scientific, San Jose, USA) was used to evaluate a highly complex mixture of 116 pesticides, mycotoxins and plant toxins in different concentrations. A 12 min gradient was applied to a 50 x 2 mm RP C18 column (Thermo Scientific Hypersil GOLD 1.9um particles, Thermo Fisher Scientific, Madison, USA) with water/acetonitrile eluents. The method developed was evaluated with respect to sensitivity, selectivity and linearity in standard solutions and extracts from animal feed. Mass measurements were performed at different resolution settings (R = 15000 and R = 50000) to enable comparisons to data acquired by ToF instruments and to demonstrate the advantage of ultra high resolution. Orbitrap detection was carried out using automatic control of the number of ions entering the detector (AGC, target value = 10e6).
Resolution of Isobaric Pesticides
In cases where isobaric compounds co-elute, erroneous mass accuracy and elemental composition assignment will occur if the resolving power of the mass spectrometer is insufficient to separate these compounds. Fig.2 shows two pesticides Thiamethoxam (C8H10ClN5O3S) and Parathion (C10H14NO5PS), which have protonated molecular ions (MH+) at 292.02656 and 292.04031, respectively. A resolution higher than 40000 is needed to resolve the protontated molecular ion of these two compounds completely. This is a pre-requisite for analysis of low concentration compounds in the presence of higher abundant ones. The example in Fig.2 shows an approximate 1:3 mixture of both pesticides measured and simulated.
A limited resolution of 15 000 results in two major limitations. Firstly, the detection of unresolved doublets may result in significant mass errors which are outside the characteristic accuracy specification of the Exactive instrument.
As a consequence, at lower resolution settings the mass windows for elemental composition determination have to be increased, resulting in a much larger number of elemental composition proposals for the unknown or targeted compounds. This can be seen for the example (Fig.3) of Pirimicarb at m/z 239.1503. Due to the presense of an isobaric interference the peak at 239 shows a mass error of 6.5ppm. At a resolution of 15000 the underlying interference causes an apparent shift to higher mass, whereas at higher resolution (here 80000) the doublet is clearly resolved and the mass accuracy is well within instrument specifications.
In order to limit the number of candidate elemental compositions to a single confident assignment, a sophisticated software algorithm is used. It takes into account the peak height and mass accuracy of the monoisotopic peak and its isotopes.
However, in order to function correctly, all of the accurate mass values for the isotopic peaks must be within specified limits. The absence of interference peaks can only be assured by use of high resolution, (as can be seen in Fig.4). Here the fungicide Azoxystrobin is shown at resolutions of 15000 and 80000. The medium resolution spectrum shows very good mass accuracy for the monoisotopic peak, but gives unusually high mass errors for the A+1 and A+2 ions. This is due to an interference at m/z 405.1452, which cannot be resolved at medium resolution. Whereas the high resolution spectrum shows exellent mass accuracy for all three measured isotopes.
Determining elemental compositions using data acquired at ~15000 resolution will result in misleading or incorrect data.
Only sufficient high resolution allows the determination of the accurate mass of the complete molecular ion cluster and therefore allows automated assignment of an elemental formula with a high degree of confidence.
Analysing highly complex samples such as extracts from food or animal feeds, and the screening of regulated substances including pesticides, mycotoxins and veterinary drugs is a major analytical challenge for mass spectrometry.
On one hand, the methodology must have a high intra scan dynamic range in order to detect low concentrated compounds in presence of high abundant matrix ions, on the other hand high selectivity and high sensitivity is needed to avoid false positive, or even worse, false negative results.
In this procedure an extract from horse feed was analysed as an example of an extremely complex matrix, spiked with a mixture of 116 pesticides and mycotoxins. A dilution series ranging from 2 to 250ppb (for each compound) was measured in duplicates at two different resolution settings. In addition a 100ppb sample of the same mixture was analyzed at a resolution of 50000 in order to determine the maximum number of detectable substances for this method.
LC-MS analysis of the extracted spiked samples showed the presence of 95 out of 116 compounds at 100ppb in matrix. Fig.5 shows the overlaid ion chromatograms for all 116 compounds (3ppm window) at 50ppb (in matrix). The number of recovered pesticides in different concentrations is shown in Fig.5.
The data illustrates a greater number of detected compounds (higher sensitivity) with an extraction window of 3ppm at higher resolution setting. This is exemplified in Fig.6, where extracted ion chromatograms of Sulcotrion at 50ppb for R=15000 and R=50000 are shown. The higher resolved spectrum displays two peaks, of which the smaller one is Sulcotrion.
The lower resolved peak masks the pesticide signal completely. The only indication for the presence of Sulcotrion is a slightly broader peak or shoulder and a mass shift of the interfering ion towards higher masses. This would lead to a false negative result, if the analysis was only performed at a resolution of 15000 and is the major reason, why the number of identified components in the case of R=15000 decreases disproportionately to the measurements at higher resolving power (Fig.5).
For this reason, a dilution series was measured at higher resolution settings. One example (Metabenthiazuron) is shown in Fig.7. It demonstrates excellent linearity and sensitivity down to 2ppb level (2ng/mL).
- New Exactive mass spectrometer demonstrates superior mass resolving power compared to that obtained using TOF instruments.
- High resolving power (up to 100000) provides precise mass accuracy for complex sample analysis
- High resolving power provides excellent sensitivity, linearity and selectivity in multi-residue screening of complex matrices.
- Fast scan speed (10Hz) are fully compatible with the use of U-HPLC fast chromatography methods.
For the analysis of very complex samples it is advantageous to select the appropriate scan speed and resolution in order to avoid unresolved isobaric compounds (matrix ions from analyte ions) and still allow unambiguous detection of low abundant species.
- Markus Kellmann, Andreas Wieghaus, Helmut Muenster, Thermo Fisher Scientific, Bremen, Germany; Lester Taylor, Dipankar Ghosh, Thermo Fisher Scientific, San Jose, CA, USA. www.thermofisher.com. Acknowledgements: The authors would like to thank Paul Zomer and Hans Mol from the RIKILT Institute for Food Safety in Wageningen, The Netherlands, for providing the samples.