Mass spectrometer parameters such as Resolving Power, type of fragmentation, and mass calibration mode were optimized in the analysis of 24 pesticide metabolites in human urine using Ultra-High Pressure Liquid Chromatography coupled to Orbitrap High-Resolution Mass Spectrometer (UHPLC-HRMS). The best results were achieved with a Resolving Power of 25,000 FWHM and by applying Collision Induced Dissociation fragmentation mode (40 eV).
The ever growing number of chemicals being used, such as pesticides, care products, UV filters, parabens, and so on, has an impact on the environment and therefore on humans, especially in vulnerable populations [
Some pesticide metabolites are biomarkers of pesticide exposure. These metabolites are present in urine at concentrations of few ng mL−1 [
Over the last few years, liquid chromatography coupled to Orbitrap high-resolution mass spectrometry (LC-HRMS) has been applied in human biomonitoring studies [
In the existing literature on application of HRMS to food and feed contaminants [
How much Resolving Power is necessary to apply to a specific problem should be a frequent analytical question [
In order to obtain a suitable mass accuracy (< 5 ppm) in an Orbitrap spectrometer, a proper mass calibration must be employed. External mass calibration is performed previously to the analysis by direct infusion of a mix of compounds with known masses; the experimental m/z values obtained are then corrected with the theoretical m/z values in order to fit the accuracy of the analyzer. Until now, external mass calibration in Orbitrap has been widely employed in biological samples, food, and feed [
Unlike conventional quadrupole (QqQ) instruments, Orbitrap Exactive™ users do not implement a compound-specific fragmentation optimization. In Exactive™, two all ion fragmentation (AIF) modes are allowed: HCD and CID [
In a previous work [
This study has been developed in the framework of the DENAMIC project, which included all the required ethical approvals.
Solvents were specific for pesticide residue analysis and of analytical grade. Acetonitrile and methanol were supplied by Scharlab (Barcelona, Spain). Acetic acid (purity 98-100%),
Standards of pesticide metabolites (Table
Pesticide metabolites: diagnostic and fragment ions used for analysis.
Class | Compound | Metabolite | Acronym | Elemental composition | Diagnostic ion | Exact mass m/z diagnostic ion (Da) | Fragment elemental composition | m/z fragment ion (Da) |
---|---|---|---|---|---|---|---|---|
Organophosphate insecticides | Chlorpyrifos, chlorpyrifos-methyl | 3,5,6-Trichloro-2-pyridinol | TCPy | C5H2NOCl3 | [M-H]- | 195.91292 | - | - |
Parathion, methyl parathion | p-nitrophenol | PNP | C6H5NO3 | [M-H]- | 138.01966 | C6H4O2 | 108.02167 | |
Pirimiphos-methyl | 2-Diethylamino-6-methyl-6-hydroxypyrimidine | DEAMPY | C9H15N3O | [M+H]+ | 182.12879 | C7H12N3O | 154.09748 | |
Diazinon | 2-Isopropyl-4-methyl-6-hydroxypyrimidine | IMPY | C8H12N2O | [M+H]+ | 153.10224 | C4H6NO | 84.04439 | |
Coumaphos | 3-Chloro-7-hydroxy-4-methylcoumarin | CMHC | C10H7ClO3 | [M-H]- | 209.00109 | C9H5O2 | 145.02841 | |
Fenitrothion | 3-Methyl-4-nitrophenol | MNP | C7H7NO3 | [M-H]- | 152.03531 | C7H6O2 | 122.03733 | |
Dimethoate | Dimethoate | DIMET | C5H12NO3PS2 | [M+H]+ | 230.00690 | C2H6O2PS | 124.98206 | |
Omethoate | Omethoate | OMET | C5H12NO4PS | [M+H]+ | 214.02974 | C4H8O4PS | 182.98754 | |
Acephate | Acephate | AP | C4H10NO3PS | [M+H]+ | 184.01917 | C2H8O3PS | 142.99262 | |
Methamidophos | Methamidophos | MMP | C2H8NO2PS | [M+H]+ | 142.00861 | - | 112.01577 | |
Chlorethoxyphos, chlorpyrifos coumaphos, diazinon, disulfoton, ethion, parathion, phorate, phosalone, sulfotep, terbufos, azinphos-methyl, dichlorvos, dicrotophos, dimethoate, fenitrothion, fenthion, malathion, methyl parathion, trichlorfon, chlorpyrifos-methyl, methidathion, mevinphos, oxydemeton-methyl, phosmet, pirimiphos-methyl, temephos, tetrachlorvinphos, isazofos-methyl, naled | Diethyl phosphate | DEP | C4H11O4P | [M-H]- | 153.03221 | C2H6O4P | 125.00092 | |
Diethyl thiophosphate | DETP | C4H11O3PS | [M-H]- | 169.00937 | C2H6O3PS | 140.97807 | ||
Dimethyl thiophosphate | DMTP | C2H7O3PS | [M-H]- | 140.97807 | CH3O3PS | 125.95460 | ||
Dimethyl dithiophosphate | DMDTP | C2H7O2PS2 | [M-H]- | 156.95523 | CH3O2PS2 | 141.93174 | ||
| ||||||||
Phenoxy herbicides | 2,4-Dichlorophenoxyacetic acid | 2,4-Dichlorophenoxyacetic acid | 2,4-D | C8H6O3Cl2 | [M-H]- | 218.96212 | C6H3OCl2 | 160.95664 |
2,4,5-Trichlorophenoxyacetic acid | 2,4,5-Trichlorophenoxyacetic acid | 2,4,5-T | C8H5O3Cl3 | [M-H]- | 252.92315 | C6H2OCl3 | 194.91767 | |
| ||||||||
Chloroacetanilide herbicides | Atrazine | Atrazine mercapturate | ATZM | C13H22N6O3S | [M+H]+ | 343.15468 | C8H16N5S | 214.11209 |
Alachlor | Alachlor mercapturate | ALAM | C19H28N2O5S | [M+H]+ | 397.17916 | C5H8N O3 | 130.04987 | |
Metolachlor | Metolachlor mercapturate | METM | C20H30N2O5S | [M+H]+ | 411.19481 | C15H24N O2S | 282.15223 | |
| ||||||||
Pyrethroid insecticides | Commercial Pyrethroids | 3-Phenoxybenzoic acid | PBA | C13H10O3 | [M-H]- | 213.05571 | C12H9O | 169.06589 |
Cyfluthrin | 4-Fluoro-3-phenoxybenzoic acid | FPBA | C13H9FO3 | [M-H]- | 231.04629 | C12H8OF | 187.05647 | |
Permethrin, cypermethrin, cyfluthrin | cis-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid | cis-DCCA | C8H10O2Cl2 | [M-H]- | 206.99850 | - | - | |
trans-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid | trans-DCCA | C8H10O2Cl2 | [M-H]- | 206.99850 | - | - | ||
Deltamethrin | cis-(2,2-Dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid | DBCA | C8H10O2Br2 | [M-H]- | 294.89747 | - | - | |
| ||||||||
Internal Standards | PNP-D4 | C6HD4NO3 | [M-H]- | 142.04477 | - | - | ||
FPBA-13C6 | 13C6C7H9FO3 | [M-H]- | 237.06642 | - | - | |||
DCCA-13C2 | 13C2C6H9DCl2O2 | [M-H]- | 210.01149 | - | - | |||
ATZM-13C3 | 13C3C10H22N6O3S | [M+H]+ | 346.16475 | - | - | |||
2,4-D-D3 | C8H3Cl2O3D3 | [M-H]- | 221.98095 | - | - | |||
MMP-D6 | C2H2NO2PSD6 | [M+H]+ | 148.04627 | - | - | |||
DIMET-D6 | C5H6NO3PS2D6 | [M+H]+ | 236.04455 | - | - | |||
DBP | C8H19O4P | [M-H]- | 209.09481 | - | - |
A previously developed sample preparation was used [
Metabolites were extracted from the urine samples employing the dispersive solid phase extraction QuEChERS kits. In a 50 mL polypropylene tube, the urine was mixed with 10 mL of acetonitrile, a QuEChERS pouch, and 2 ceramic pieces. After centrifugation, the acetonitrile phase was transferred and evaporated to dryness in a water bath at 37°C under a nitrogen stream. Subsequently, 200
Chromatographic separation was performed on an ultra-high performance liquid chromatography (UHPLC) system Accela™ equipped with a Hypersil Gold column (100 mm x 2.1 mm, 1.9
Mass analysis was performed on the Orbitrap mass spectrometer Exactive™ analyzer (Thermo Scientific, Bremen, Germany). The system was equipped with a heat electrospray ionization interface (HESI-II). The ion-source parameters were previously optimized as follows: spray voltage: 3.5 kV (positive mode) and 2.5 kV (negative mode); sheath gas flow rate: 55; auxiliary gas flow rate: 10; skimmer voltage: 23 V; heater temperature: 300°C; capillary temperature: 150°C; capillary voltage: 45 V and tube lens voltage: 120 V. For more details of the HRMS analysis see Roca
The criteria for target compound identification were established following the SANTE/11813/2017 guideline [
To optimize the Resolving Power (R), the system operating in full-scan mode (50-800 m/z) was tested at the R of 10,000; 25,000; and 50,000 FWHM. 6 blank matrix urine aliquotes spiked with a mixed-standard solution of 24 target pesticide metabolites (see Table
The Resolving Power was evaluated measuring the peak area (signal intensity) and the mass accuracy (Δm) for the diagnostic and fragment ions of each metabolite. A scheme of the Resolving Power optimization study is detailed in Table
After the selection of the most suitable R, HCD and CID fragmentations were evaluated. Five spiked urine samples were injected with CID energies of 10, 20, 30, and 40 eV. We previously set the energy for HCD fragmentation to 20 eV. Once the CID energy was optimized, five different methods, in six spiked samples (50 ng·mL−1), were studied using or not HCD and CID fragmentations and using ESI+ and ESI- in the same or in different injections. The response was evaluated measuring the peak area of the fragment ions in (i) ESI+ with and without HCD (HCD= 20 eV); (ii) ESI- with and without HCD (HCD= 20 eV); (iii) ESI+ with and without CID; (iv) ESI- with and without CID; (v) ESI+ and ESI- in the same injection with and without CID. Fragmentation optimization data were acquired using the previously optimized R and external mass calibration.
With respect to mass calibration, both external and internal mass calibrations were evaluated. External calibration was performed using the mixtures ProteoMass™ LTQ/FT-Hybrid ESI Cal Mix in Pos an Neg Mode (Supelco, Bellefonte, PA, USA). Internal mass calibration was achieved introducing caffeine (M+H+ m/z = 195.08765 Da) in the mobile phase as a lock mass for positive ionization (ESI+).
In total, five spiked aliquotes were analyzed using internal and external mass calibration separately. The analytical response was evaluated measuring peak areas and mass accuracies (Δm) for the diagnostic and fragment ions. All mass calibration study data were acquired using the previously optimized R and fragmentation settings.
Data were processed using the TraceFinder™ 3.1 (Thermo Scientific, Bremen, Germany) and Xcalibur™ 2.2 (Thermo Scientific, Bremen, Germany) software.
In order to select the most appropriate R for the determination of pesticide metabolites in urine, the influence of this parameter on the signal and mass accuracy of the 24 compounds was investigated. Tables
Average peak area and coefficient of variation (CV, %) obtained at 3 different Resolving Powers (R) for pesticide metabolites diagnostic and fragment ions (n = 6).
Metabolite | Peak area | Diagnostic ion | Fragment ion | ||||
---|---|---|---|---|---|---|---|
R=10,000 | R=25,000 | R=50,000 | R=10,000 | R=25,000 | R=50,000 | ||
CMHC | Average | 908337 | 839333 | 570456 | 12927 | 26726 | 39492 |
CV (%) | 1.51 | 2.91 | 7.49 | 24.38 | 21.36 | 52.87 | |
DEAMPY | Average | 994268 | 880944 | 834834 | 63190 | 161187 | 175626 |
CV (%) | 17.15 | 28.51 | 10.49 | 22.31 | 16.20 | 40.94 | |
IMPY | Average | 1447089 | 1425080 | 1663104 | Not found | 47097 | 116409 |
CV (%) | 0.36 | 7.92 | 13.22 | Not found | 1.23 | 41.28 | |
PNP | Average | 6388647 | 6280026 | 6007343 | 3938342 | 3858484 | 4005830 |
CV (%) | 1.70 | 2.06 | 1.92 | 7.36 | 4.40 | 6.67 | |
TCPy | Average | 1090750 | 1148221 | 943686 | - | - | - |
CV (%) | 1.73 | 7.04 | 8.08 | - | - | - | |
MNP | Average | 3938892 | 3813784 | 3650660 | 503016 | 474896 | 634023 |
CV (%) | 3.10 | 3.85 | 4.18 | 11.40 | 13.27 | 19.46 | |
DMTP | Average | 76340 | 68339 | 88833 | 2644 | 6361 | 9973 |
CV (%) | 39.41 | 5.74 | 16.55 | 57.74 | 37.34 | 57.74 | |
DMDTP | Average | 111502 | 321392 | 247711 | 2679 | 16962 | 53675 |
CV (%) | 36.39 | 10.13 | 30.71 | 57.74 | 110.51 | 11.56 | |
DEP | Average | 1719737 | 1870997 | 1556112 | 196269 | 272546 | 272666 |
CV (%) | 4.09 | 4.33 | 5.83 | 21.00 | 12.49 | 6.12 | |
DETP | Average | 2224008 | 2154624 | 1819644 | 50934 | 141703 | 169339 |
CV (%) | 0.08 | 6.97 | 6.56 | 63.42 | 24.47 | 24.00 | |
AP | Average | 58729 | 74307 | 90733 | 157684 | 157729 | 206069 |
CV (%) | 8.34 | 33.62 | 17.63 | 7.21 | 4.59 | 13.43 | |
MMP | Average | 69818 | 178190 | 112470 | 19869 | 169131 | 69306 |
CV (%) | 7.38 | 2.89 | 4.58 | 25.93 | 3.05 | 7.43 | |
OMET | Average | 45484 | 226016 | 149173 | 13032 | 25505 | 32670 |
CV (%) | 69.04 | 23.35 | 17.68 | 35.04 | 22.09 | 32.95 | |
DIMET | Average | 380660 | 399855 | 329466 | 60934 | 83874 | 87121 |
CV (%) | 8.48 | 11.44 | 14.44 | 6.51 | 10.55 | 31.27 | |
PBA | Average | 648211 | 571829 | 363693 | 121191 | 138365 | 166373 |
CV (%) | 8.82 | 15.34 | 16.11 | 11.30 | 20.44 | 23.68 | |
FPBA | Average | 612579 | 519635 | 405204 | 73743 | 82755 | 112916 |
CV (%) | 13.94 | 23.07 | 11.99 | 16.68 | 13.95 | 31.57 | |
cis DCCA | Average | 171464 | 273263 | 194626 | - | - | - |
CV (%) | 28.60 | 16.39 | 13.55 | - | - | - | |
trans DCCA | Average | 453810 | 436816 | 314120 | - | - | - |
CV (%) | 6.12 | 6.49 | 13.04 | - | - | - | |
DBCA | Average | 6904 | 32503 | 32185 | - | - | - |
CV (%) | 31.56 | 40.91 | 16.74 | - | - | - | |
ATZM | Average | 273078 | 1179224 | 756085 | 1314830 | 1421843 | 1252716 |
CV (%) | 71.21 | 3.96 | 2.01 | 3.57 | 4.46 | 9.05 | |
ALAM | Average | 67659 | 687712 | 511658 | 831200 | 1255816 | 1242225 |
CV (%) | 12.30 | 15.92 | 7.77 | 7.02 | 2.35 | 5.40 | |
METM | Average | 268530 | 644807 | 446473 | 2762 | 5381 | 6657 |
CV (%) | 8.80 | 4.93 | 9.49 | 57.74 | 57.74 | 57.74 | |
2,4-D | Average | 1234998 | 1225331 | 907979 | 1130961 | 1117200 | 1224757 |
CV (%) | 6.27 | 4.39 | 5.67 | 3.85 | 8.45 | 3.68 | |
2,4,5-T | Average | 731224 | 786035 | 477627 | 677796 | 718415 | 777837 |
CV (%) | 10.79 | 10.86 | 10.65 | 7.95 | 12.15 | 13.05 |
Acquisition conditions: ESI+/ESI- in separated injections, HCD fragmentation 20 eV, and external mass calibration.
-: no fragment ions monitored.
Average and range of mass accuracies (∆m) (ppm) obtained at 3 different Resolving Powers (R) for pesticide metabolites diagnostic and fragment ions (n=6).
Metabolite | Δm (ppm) | Diagnostic ion | Fragment ion | ||||
---|---|---|---|---|---|---|---|
R=10,000 | R=25,000 | R=50,000 | R=10,000 | R=25,000 | R=50,000 | ||
CMHC | Average | 2.27 | 1.38 | 1.24 | 3.11 | 2.52 | 3.36 |
Range | 1.60 – 2.75 | 0.07 – 2.32 | 0.93 – 1.65 | 1.61 – 4.16 | 1.10 – 3.81 | 3.33 – 3.40 | |
DEAMPY | Average | 1.09 | 2.68 | 1.30 | 3.19 | 1.70 | 1.44 |
Range | 0.05 – 2.58 | 1.65 – 3.66 | 0.73 – 1.90 | 1.70 – 4.53 | 0.69 – 3.37 | 0.49 – 2.38 | |
IMPY | Average | 1.81 | 1.56 | 1.33 | > 5 | 1.61 | 1.99 |
Range | 1.25 – 2.14 | 1.36 – 1.76 | 1.26 – 1.36 | > 5 | 1.35 – 1.90 | 1.90 – 2.07 | |
PNP | Average | 2.69 | 1.94 | 1.63 | 3.64 | 2.18 | 1.96 |
Range | 2.31 – 3.08 | 0.77 – 2.87 | 1.20 – 1.86 | 2.92 – 4.99 | 1.09 – 3.13 | 1.48 – 2.28 | |
TCPy | Average | 2.23 | 2.01 | 1.83 | - | - | - |
Range | 0.28 – 3.01 | 0.42 – 3.12 | 1.05 – 3.38 | - | - | - | |
MNP | Average | 2.59 | 1.32 | 1.59 | 2.36 | 1.18 | 1.89 |
Range | 2.56 – 2.66 | 1.05 – 1.56 | 1.15 – 2.06 | 1.70 – 2.93 | 0.88 – 1.70 | 1.62 – 2.11 | |
DMTP | Average | 0.79 | 0.66 | 2.43 | 2.41 | 1.02 | 1.28 |
Range | 0.11 – 1.40 | 0.34 – 1.09 | 1.96 – 2.82 | 1.51 – 4.52 | 0.75 – 1.19 | 0.08 – 2.78 | |
DMDTP | Average | 2.47 | 1.49 | 2.14 | 1.96 | 2.67 | 1.31 |
Range | 1.26 – 3.21 | 0.87 – 2.52 | 1.65 – 2.43 | 0.25 – 4.03 | 1.67 – 4.26 | 0.49 – 2.10 | |
DEP | Average | 2.66 | 2.02 | 1.84 | 1.88 | 2.05 | 2.35 |
Range | 1.43 – 3.26 | 1.16 – 2.84 | 1.44 – 2.14 | 0.89 – 2.81 | 1.03 – 3.11 | 1.98 – 2.71 | |
DETP | Average | 3.46 | 2.02 | 1.96 | 2.26 | 3.13 | 2.10 |
Range | 3.28 – 4.99 | 1.27 – 2.63 | 1.30 – 2.29 | 0.61 – 4.22 | 2.30 – 4.22 | 1.31 – 3.04 | |
AP | Average | 1.66 | 1.41 | 1.39 | 1.91 | 2.26 | 1.79 |
Range | 0.42 – 4.80 | 0.60 – 2.10 | 0.48 – 2.57 | 0.06 – 4.00 | 1.30 – 3.16 | 1.13 – 2.11 | |
MMP | Average | 2.21 | 1.67 | 1.69 | 3.06 | 1.67 | 3.45 |
Range | 0.28 – 4.14 | 1.24 – 2.43 | 0.92 – 2.69 | 2.62 – 3.49 | 1.24 – 2.43 | 3.22 – 3.63 | |
OMET | Average | 0.86 | 1.92 | 2.09 | 1.55 | 3.36 | 1.90 |
Range | 0.03 – 1.39 | 1.11 – 2.61 | 1.86 – 2.38 | 0.47 – 2.39 | 2.17 – 4.98 | 1.19 – 2.99 | |
DIMET | Average | 2.79 | 2.55 | 2.13 | 2.38 | 2.34 | 2.67 |
Range | 1.18 – 4.62 | 2.18 – 2.92 | 1.14 – 3.10 | 0.38 – 4.06 | 1.10 – 3.90 | 2.20 – 3.11 | |
PBA | Average | 1.79 | 1.33 | 1.12 | 2.25 | 1.55 | 1.77 |
Range | 1.25 – 2.11 | 0.18 – 2.85 | 0.68 – 1.97 | 1.65 – 3.25 | 0.28 – 3.53 | 1.18 – 2.26 | |
FPBA | Average | 1.17 | 2.09 | 1.38 | 3.10 | 1.85 | 1.41 |
Range | 0.15 – 1.80 | 0.80 – 3.49 | 0.66 – 2.41 | 2.09 – 4.20 | 0.30 – 3.46 | 0.10 – 1.92 | |
cisDCCA | Average | 1.84 | 1.42 | 1.76 | - | - | - |
Range | 1.20 – 2.31 | 0.31 – 2.18 | 0.61 – 2.67 | - | - | - | |
transDCCA | Average | 2.37 | 1.82 | 1.41 | - | - | - |
Range | 1.05 – 3.47 | 0.23 – 3.04 | 0.31 – 2.45 | - | - | - | |
DBCA | Average | 2.92 | 2.07 | 1.78 | - | - | - |
Range | 1.80 – 4.10 | 1.08 – 2.74 | 0.36 – 3.16 | - | - | - | |
ATZM | Average | 3.61 | 2.29 | 2.98 | 1.18 | 1.84 | 1.85 |
Range | 2.29 – 4.97 | 1.43 – 2.87 | 2.17 – 4.12 | 0.83 – 1.63 | 1.42 – 2.32 | 1.56 – 2.27 | |
ALAM | Average | 3.41 | 2.57 | 3.29 | 2.30 | 2.14 | 1.96 |
Range | 1.24 – 4.76 | 1.69 – 2.85 | 2.85 – 4.76 | 1.38 – 3.25 | 1.61 – 3.38 | 1.61 – 2.43 | |
METM | Average | 2.37 | 2.68 | 2.77 | > 5 | 1.52 | 2.13 |
Range | 0.90 – 4.38 | 1.70 – 3.87 | 1.19 – 4.31 | 1.09 – >5 | 0.42 – 2.35 | 1.82 – 3.13 | |
2,4-D | Average | 2.65 | 1.92 | 1.72 | 3.21 | 2.19 | 1.89 |
Range | 1.98 – 3.62 | 0.97 – 2.56 | 0.74 – 2.77 | 2.82 – 3.87 | 1.08 – 3.00 | 1.76 – 2.14 | |
2,4,5-T | Average | 1.65 | 1.30 | 2.28 | 2.89 | 2.15 | 1.71 |
Range | 0.46 – 2.26 | 0.22 – 1.85 | 1.30 – 4.14 | 2.16 – 3.65 | 1.42 – 2.78 | 1.29 – 2.23 |
Acquisition conditions: ESI+/ESI- in separated injections, HCD fragmentation 20 eV, and external mass calibration.
-: no fragment ions monitored.
Regarding diagnostic ions, some specific metabolites such as DEAMPY, IMPY, PNP, TCPy, MNP, DEP, DETP, or DIMET presented similar areas at the three R checked (Table
For fragments, 17 out of 20 compounds presented higher intensities with R = 50,000 FWHM (Table
In addition, the R of 25,000 FWHM presented more data points (scans) per peak than R = 50,000 FWHM because in an Orbitrap analyser the scan speed decrease when R increase (i.e., 2 Hz at R = 50,000 FWHM; 4 Hz at R = 25,000 FWHM). The number of data points is important for peak shape and quantification.
Table
Number of ions (diagnostic and fragment ions) into the six mass accuracy ranges considered at 10,000, 25,000, and 50,000 FWHM.
Resolving Power | ∆m (ppm) | |||||
---|---|---|---|---|---|---|
≤ 1 | | | | | > 5 | |
10,000 | 2 | 12 | 19 | 9 | 0 | 2 |
25,000 | 1 | 22 | 19 | 2 | 0 | 0 |
50,000 | 0 | 30 | 11 | 3 | 0 | 0 |
Acquisition conditions: ESI+/ESI- in separated injections, HCD fragmentation 20 eV, and external mass calibration.
Taking into account these results, a R = 25,000 FWHM was chosen because (i) it gave the highest signal (peak area) for more analytes; (ii) it presented good mass accuracy; and (iii) the scan time gave a suitable number of data points. An added advantage of using this intermediate R is that the instrument presents a sufficient speed to be able to use the detection of positive (ESI+) and negative (ESI-) ions in the same injection, increasing the speed of analysis and the throughput of the method. Martínez-Dominguez
Regarding the optimization of the CID energy, the highest fragment areas were obtained using a fragmentation energy of 40 eV. As an example, Figure
CID fragmentation of PBA (m/z = 169.06589). Variation of the fragment ion response (peak area) with the applied energy (eV). (1) Applying only ESI+; (2) using the polarity switching (ESI+, ESI- in the same injection). Acquisition conditions: R= 25,000 FWHM and external mass calibration.
Comparing the results obtained using CID and HCD, Figure
(a) Comparison between responses (peak area) of fragments obtained using CID (40 eV) and HCD (20 eV). (b) Comparison between CID fragmentation (40 eV) using the polarity switching function (ESI+ and ESI-) in the same injection and in two separate injections. Acquisition conditions: R= 25,000 FWHM and external mass calibration.
In general, the use of lock mass (internal calibration) improves mass accuracy. In addition, in the present method some fragments are below 138.06619 Da, which is the low mass in the ESI+ external calibration solution; consequently a continuous correction of the acquired masses (internal calibration) could avoid an excessive mass drift.
In order to check whether this general rule is applicable to this particular application, we studied the influence of the calibration mode on the mass accuracy of eight substances analyzed in ESI+ mode, using caffeine as internal standard. Table
Average mass accuracies (∆m) (ppm) and standard deviations (Std dev) using internal and external calibration for diagnostic and fragment ions in ESI positive mode (n=5).
Metabolite | Diagnostic ion | Fragment ion | ||||||
---|---|---|---|---|---|---|---|---|
Internal calibration | External calibration | Internal calibration | External calibration | |||||
∆m (ppm) | Std dev | ∆m (ppm) | Std dev | ∆m (ppm) | Std dev | ∆m (ppm) | Std dev | |
DEAMPY | 0.21 | 0.13 | 0.47 | 0.26 | 0.17 | 0.08 | 1.15 | 0.19 |
IMPY | 0.08 | 0.06 | 0.62 | 0.26 | 3.55 | 0.08 | 2.64 | 0.23 |
DIMET | 0.27 | 0.07 | 0.76 | 0.29 | 0.44 | 0.05 | 0.50 | 0.12 |
OMET | 0.14 | 0.10 | 0.53 | 0.39 | 0.12 | 0.11 | 0.90 | 0.33 |
AP | 0.12 | 0.10 | 0.41 | 0.21 | 0.15 | 0.08 | 0.99 | 0.27 |
ATZM | 0.49 | 0.15 | 0.99 | 0.41 | 0.19 | 0.04 | 0.91 | 0.36 |
ALAM | 0.24 | 0.27 | 1.14 | 0.55 | 0.17 | 0.14 | 0.59 | 0.17 |
METM | 1.52 | 1.38 | 2.23 | 1.07 | 0.77 | 0.45 | 1.96 | 0.39 |
Acquisition conditions: ESI+/ESI- in the same injection, R = 25,000 FWHM, and CID fragmentation 40 eV.
In general, external mass calibration is used in the literature for HRMS Orbitrap detector and internal mass calibration for HRMS QTOF detectors. External mass calibration of Orbitrap in urine, plasma, food, and feed has been widely performed for pesticides, parabens, veterinary drugs, biotoxins, mycotoxins, and other substances [
Mass spectrometry parameters such as Resolving Power, fragmentation mode, and type of mass calibration have been optimized for the analysis of 24 pesticide metabolites in urine. A Resolving Power of 25,000 FWHM, internal calibration, and CID fragmentation were selected as the best options to improve the signal intensity and the mass accuracy of diagnostic and fragment ions. This Resolving Power provides enough resolution to avoid isotopic interferences and allows the use of the polarity switching function (ESI+ and ESI– in the same injection), hence reducing the analysis time.
The optimized HRMS parameters allow the determination of pesticide metabolites in urine samples; however a further validation of the method is required to determine the LOD and other performance parameters.
We have not tested CID energies higher than 40 eV.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
The authors are very grateful to Marta Roca for her help in the experimental work. This study had the support of FP7-ENV-2011 DENAMIC Project (Code 282957).
Table SI.1: scheme of the Resolving Power optimization study. Figure SI.1: number of ions with high (