Liquid Chromatography-ion Trap Mass Spectrometry Research Articles

Reactive oxidation products promote secondary organic aerosol formation from green leaf volatiles

Abstract. Green leaf volatiles (GLVs) are an important group of chemicals released by vegetation which have emission fluxes that can be significantly increased when plants are damaged or stressed. A series of simulation chamber experiments has been conducted at the European Photoreactor in Valencia, Spain, to investigate secondary organic aerosol (SOA) formation from the atmospheric oxidation of the major GLVs cis-3-hexenylacetate and cis-3-hexen-1-ol. Liquid chromatography-ion trap mass spectrometry was used to identify chemical species present in the SOA. Cis-3-hexen-1-ol proved to be a more efficient SOA precursor due to the high reactivity of its first generation oxidation product, 3-hydroxypropanal, which can hydrate and undergo further reactions with other aldehydes resulting in SOA dominated by higher molecular weight oligomers. The lower SOA yields produced from cis-3-hexenylacetate are attributed to the acetate functionality, which inhibits oligomer formation in the particle phase. Based on observed SOA yields and best estimates of global emissions, these compounds may be calculated to be a substantial unidentified global source of SOA, contributing 1–5 TgC yr−1, equivalent to around a third of that predicted from isoprene. Molecular characterization of the SOA, combined with organic mechanistic information, has provided evidence that the formation of organic aerosols from GLVs is closely related to the reactivity of their first generation atmospheric oxidation products, and indicates that this may be a simple parameter that could be used in assessing the aerosol formation potential for other unstudied organic compounds in the atmosphere.

Simultaneous Quantification of Methadone, Cocaine, Opiates, and Metabolites in Human Placenta by Liquid Chromatography-Mass Spectrometry

A validated method for quantifying methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, cocaine, benzoylecgonine, 6-acetylmorphine, morphine, and codeine in human placenta by liquid chromatography-ion trap mass spectrometry is described. Specimens (1 g) were homogenized and subjected to solid-phase extraction. Chromatographic separation was performed on a Synergi Polar RP column with a gradient of 0.1% formic acid and acetonitrile. The method was linear from 10 to 2000 ng/g for methadone and 2.5 to 500 ng/g for other analytes. Limits of detection were 0.25-2.5 ng/g, imprecisions < 9.1%CV, analytical recoveries 84.4-113.3%, extraction efficiencies > 46%, matrix effects -8.0-129.9%, and process efficiencies 24.2-201.0%. Method applicability was demonstrated by analysis of five placenta specimens from opioid-dependent women receiving methadone pharmacotherapy, with methadone doses ranging from 65 to 95 mg on the day of delivery. These are the first data on placenta concentrations of methadone and metabolites after controlled drug administration. Detection of other common drugs of abuse in placenta will also improve our knowledge of the usefulness of this matrix for detecting in utero drug exposure and studying disposition of drugs in the maternal-fetal dyad.

LC–MS characterization of metoclopramide photolysis products

The aim of this study was to investigate the photodegradation of the antiemetic drug metoclopramide. Metoclopramide aqueous solutions were photoirradiated at 254 nm under argon atmosphere. Irradiated metoclopramide solutions were analyzed by high performance liquid chromatography–ion trap mass spectrometry in order to characterize photolysis products. Rapid decrease in metoclopramide purity, following first-order kinetic, was observed following irradiation. The structures of 18 photolysis products were tentatively identified based on their mass spectra and fragmentation. The main degradation mechanism was scission of the chlorine which could be followed by polymerization of the resulting products since dimeric and trimeric products were observed.

Identification and quantification of glucosinolates in rapeseed using liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the speciation and quantification of glucosinolates in rapeseed is described. The method combines liquid chromatography (LC) with ion trap mass spectrometry (ITMS) detection. Electrospray ionization (ESI) has been chosen as the ionization technique for the on-line coupling of LC with ITMS. Glucosinolates are extracted from different rapeseeds with MeOH and the extracts are cleaned-up by solid phase extraction with Florisil cartridges. Aqueous extracts are injected into LC system coupled to an ITMS, leading to accurately quantify eight of the most important glucosinolates in rapeseed, by MS2 mode and confirming their structure by MS3 acquisition. All the glucosinolates found in rapeseeds provide good signals corresponding to the deprotonated precursor ion [M-H]-. The method is reliable and reproducible, and detection limits range from 0.5 nmol g(-1) to 3.7 nmol g(-1) when 200 mg of dried seeds of certified reference material are analyzed. Within-day and between-day RSD percentages range between 2.4-14.1% and 3.9-16.9%, respectively. The LC-ESI-ITMS-MS method described here allows for a rapid assessment of these metabolites in rapeseed without a desulfatation step. The overall process has been successfully applied to identify and quantify glucosinolates in rapeseed samples.

Characterization of Metabolites Formed During the Biotransformation of 17α-Ethinylestradiol by Nitrosomonas europaea in Batch and Continuous Flow Bioreactors

The biotransformation of 17alpha-ethinylestradiol (EE2) by an ammonia oxidizing bacteria, Nitrosomonas europaea, grown in batch (ammonia-rich) and continuous flow (chemostat, ammonia-limited) reactors was investigated. Both C-14 labeled EE2 (10 gammag/L) and unlabeled EE2 (1 mg/L) were used to facilitate metabolite identification under environmentally relevant physiological conditions. Whole cell ammonia monooxygenase (AMO) activity was not inhibited at the EE2 concentrations used in this study. Characterization of the primary metabolite formed during batch cultivation by liquid chromatography/ion-trap mass spectrometry (LC-ITMS) and nuclear magnetic resonance (NMR) spectroscopy showed modification at the ethinyl group and addition of a carboxyl group. This metabolite (M386) (revealed by m/z 385 in negative mode electrospray LC/ MS) was not formed in the abiotic control. In contrast, biotransformation of EE2 under continuous flow conditions showed formation of a monohydroxylated EE2 (revealed by m/z 311), but not M386. Furthermore, nitrated EE2 derivatives were formed in both batch and continuous flow cultures, as a result of abiotic transformation of EE2 in the presence of high concentrations of nitrite in the bioreactors. Results from this study underscore the importance of physiological state and growth conditions as critical variables that can dictate the metabolic pathway for EE2 biodegradation and the nature of byproducts formed.

Characterization of chemopreventive agents from the dichloromethane extract of Eurycorymbus cavaleriei by liquid chromatography–ion trap mass spectrometry

In the present study, we examined the potential chemopreventive activity of dichloromethane extract of Eurycorymbus cavaleriei by investigating the change of constitutions after incubation with glutathione (GSH). The major constitutions in the dichloromethane extract of E. cavaleriei were cumarin compounds and their cleavage pattern was examined by LC–MS-MS and the characteristic product ions at m/ z 206 and 207 were helpful to determine the substitutions of coumarinolignoid compounds. The mechanism of conjugations of 5′-demethylaquillochin and its isomer with GSH was discussed and validated through analysis of the conjugations of reference compound 6-hydroxy-7-methoxycoumarin with GSH by LC–MS-MS and NMR spectrum. The relative ability to induce the detoxification enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1) of nine coumarin compounds was tested which also showed 5′-demethylaquillochin exhibited the most potential chemopreventive ability. These observations suggest that 5′-demethylaquillochin and its isomer from the dichloromethane extract of E. cavaleriei have potential as chemopreventive agents through induction of detoxification enzymes.

Identification of Quinone Methide Metabolites of Dauricine in Human Liver Microsomes and in Rat Bile

Dauricine is one type of the bisbenzyltetrahydroisoquinoline alkaloid derivative with antiarrhythmic effects. Severe liver toxicity was observed in experimental animals treated with analogues of dauricine, which may be caused by covalent binding of reactive metabolite(s) to critical macromolecules in tissues. The study described herein aimed at characterizing pathways of dauricine bioactivation and the CYP enzyme involved. In incubations of dauricine with NADPH- and GSH-supplemented human liver microsomes, four GSH conjugates with [M + H]+ ions at m/z 930, 916, 916, and 902, respectively, were detected by liquid chromatography-ion trap mass spectrometry. The structures of the four metabolites were determined to be GSH conjugates of dauricine, 2-N-demethyl dauricine, 2'-N-demethyl dauricine, and N-demethyl-O-demethyl dauricine. GSH conjugation took place with a strong preference at C-17, suggesting that the phenol moiety of dauricine and its metabolites underwent oxidation to quinone methide intermediates. The formation of the GSH conjugates was found to require the presence of NADPH. To identify the CYP isoforms that are responsible for bioactivation, dauricine was also incubated with recombinant human CYP450 enzymes. The formation of GSH was only observed with the incubation of CYP3A4. In addition, the level of these GSH conjugates in human microsomes was reduced upon the addition of a CYP3A4 inhibitor ketoconazole. The same GSH conjugates were also observed in rat bile following a single oral dose of 40 mg/kg dauricine. These studies suggest that the CYP3A4 mediated quinone methide formation was associated with dauricine bioactivation.

Prediction of breast cancer by profiling of urinary RNA metabolites using Support Vector Machine-based feature selection

BackgroundBreast cancer belongs to the most frequent and severe cancer types in human. Since excretion of modified nucleosides from increased RNA metabolism has been proposed as a potential target in pathogenesis of breast cancer, the aim of the present study was to elucidate the predictability of breast cancer by means of urinary excreted nucleosides.MethodsWe analyzed urine samples from 85 breast cancer women and respective healthy controls to assess the metabolic profiles of nucleosides by a comprehensive bioinformatic approach. All included nucleosides/ribosylated metabolites were isolated by cis-diol specific affinity chromatography and measured with liquid chromatography ion trap mass spectrometry (LC-ITMS). A valid set of urinary metabolites was selected by exclusion of all candidates with poor linearity and/or reproducibility in the analytical setting. The bioinformatic tool of Oscillating Search Algorithm for Feature Selection (OSAF) was applied to iteratively improve features for training of Support Vector Machines (SVM) to better predict breast cancer.ResultsAfter identification of 51 nucleosides/ribosylated metabolites in the urine of breast cancer women and/or controls by LC- ITMS coupling, a valid set of 35 candidates was selected for subsequent computational analyses. OSAF resulted in 44 pairwise ratios of metabolite features by iterative optimization. Based on this approach ultimately estimates for sensitivity and specificity of 83.5% and 90.6% were obtained for best prediction of breast cancer. The classification performance was dominated by metabolite pairs with SAH which highlights its importance for RNA methylation in cancer pathogenesis.ConclusionExtensive RNA-pathway analysis based on mass spectrometric analysis of metabolites and subsequent bioinformatic feature selection allowed for the identification of significant metabolic features related to breast cancer pathogenesis. The combination of mass spectrometric analysis and subsequent SVM-based feature selection represents a promising tool for the development of a non-invasive prediction system.

Identification and characterization of process related impurities in chloroquine and hydroxychloroquine by LC/IT/MS, LC/TOF/MS and NMR

The focus of this study is identification and characterization of major unknown impurities in chloroquine (CQ) and hydroxychloroquine (HCQ) bulk drug samples using liquid chromatography/ion trap mass spectrometry (LC/IT/MS) and liquid chromatography/time of flight mass spectrometry (LC/TOF/MS). The newly developed LC/MS method was employed for the analysis of both the drugs. The analysis revealed the presence of two impurities in each of the drugs. The impurities are designated as CQ-I, CQ-II (for chloroquine); HCQ-I and HCQ-II (for hydroxychloroquine). Three of the impurities, CQ-II, HCQ-I and HCQ-II were unknown have not been reported previously. Accurate masses of the impurities were determined by using Q-TOF mass spectrometer and fragmentation behavior was studied by an ion trap mass spectrometer. Based on the spectrometric data and synthetic specifics the structures of CQ-II, HCQ-I and HCQ-II were proposed as 1,4 pentanediamine, N 4(7-chloro-4-quinolinyl), N 4-chloromethyl, N 4-ethylamine; 2-(4-(7-chloroquinolin-4-ylamino) pentylamino) ethanol and [[4-[(7-chloro-4-quinolyl) amino] N-pentyl] N-chloromethyl- N-ethylamino] ethanol respectively. The impurities were isolated by semi-preparative HPLC and structures were confirmed by NMR spectroscopy. The formation and through characterization of known CQ-I impurity is also discussed.

Novel Automated Biomarker Discovery Work Flow for Urinary Peptidomics

Urine is potentially a rich source of peptide biomarkers, but reproducible, high-throughput peptidomic analysis is often hampered by the inherent variability in factors such as pH and salt concentration. Our goal was to develop a generally applicable, rapid, and robust method for screening large numbers of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. Peptide samples were trapped, desalted, pH-normalized, and fractionated on a miniaturized automatic reverse-phase strong cation exchange (RP-SCX) cartridge system. We analyzed eluted peptides using MALDI-TOF, Fourier transform ion cyclotron resonance, and liquid chromatography-iontrap mass spectrometry. We determined qualitative and quantitative reproducibility of the system and robustness of the method using BSA digests and urine samples, and we used a selected set of urine samples from Schistosoma haematobium-infected individuals to evaluate clinical applicability. The automated RP-SCX sample cleanup and fractionation system exhibits a high qualitative and quantitative reproducibility, with both BSA standards and urine samples. Because of the relatively high cartridge binding capacity (1-2 mL urine), eluted peptides can be measured with high sensitivity using multiple mass spectrometric techniques. As proof of principle, hemoglobin-derived peptides were identified in urine samples from S. haematobium-infected individuals, even when the microhematuria test was negative. We present a practical, step-by-step method for screening and identification of urinary peptides. Alongside the analytical method evaluation on standard samples, we demonstrate its feasibility with actual clinical material.

Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography–ion trap mass spectrometry

Liquid chromatography-ion trap mass spectrometry was used for the detection and structural characterization of metabolites of the anti-obesity drug sibutramine. Metabolites were profiled from incubations of sibutramine in primary cultures of rat hepatocytes. In addition, enantioselectivity of sibutramine metabolism was investigated by carrying out separate incubations with (R)- and (S)-sibutramine. As a result, biotransformation profile for sibutramine with rat hepatocytes is proposed. Nineteen metabolites and several of their isomers formed via demethylation, hydroxylation, dehydrogenation, acetylation, attachment of CO(2), and glucuronidation were identified in MS(2) and MS(3) experiments, though the exact position of the functionality, mostly hydroxylation, could not always be determined from the mass spectrometric information. However, clear enantioselective formation was observed for two hydroxyl derivatives and two glucuronide conjugates, indicating that the hydroxyl/glucuronic acid moiety in those structures is close to the chiral center. Most of the metabolites found in this study are new metabolites of sibutramine, which were not previously reported.

Metabolic Kinetics of tert‐Butylhydroquinone and Its Metabolites in Rat Serum after Oral Administration by LC/ITMS

Liquid chromatography-ion trap mass spectrometry (LC/ITMS) was employed for a metabolic kinetics study of tert-butylhydroquinone (TBHQ) and its metabolites in rat serum after oral administration. The mean serum concentration-time data of TBHQ and its four metabolites (M1-M4) in both male and female rats after oral administration of TBHQ at dosages of 7, 350 and 700 mg/kg were all best-fitted by a two-compartment model with first-order absorption. TBHQ was rapidly absorbed, distributed and metabolized. The metabolites were also distributed rapidly except M2, but eliminated more slowly than TBHQ. At the doses of 350 and 700 mg/kg, the total concentrations of TBHQ and its metabolites in serum were much higher for male than female rats during the metabolic process (P < 0.01). In addition, the serum concentrations of M1, M3 and M4 were all much higher for males than for females (P < 0.01) during the metabolic process, while no significant differences between male and female rats were found (P > 0.05) for TBHQ and M2, at the doses of 350 and 700 mg/kg. The exploratory assessment of dose proportionality for TBHQ and its metabolites by the power model indicated that the TBHQ exposure increased in a more-than-dose-proportional manner, but the exposures of the metabolites M1-M4 increased in a less-than-dose-proportional manner, across the dose of 7-700 mg/kg.

Using liquid chromatography–ion trap mass spectrometry to determine pharmaceutical residues in Taiwanese rivers and wastewaters

To establish their environmental concentrations and to support surface water protection programs, we have undertaken a preliminary study of the concentrations of selected acidic and neutral pharmaceutical residues (clofibric acid, ketoprofen, ibuprofen, diclofenac and carbamazepine) in Taiwanese river and wastewater samples. These pharmaceutical residues were extracted from the water samples through the Oasis HLB solid-phase extraction (SPE). The analytes were then identified and quantified using liquid chromatography–ion trap mass spectrometry with dual-polarity electrospray ionization in the product ion scan mode. The limits of quantification (LOQs) ranged between 0.5 and 20 ng l −1 for 250 ml samples of water. We investigated the intra- and interbatch precision and accuracy at two levels of concentration. The selected analytes were detected at concentrations ranging from <0.5 to 960 ng l −1 in wastewater treatment plant effluents and river water samples.

Isolation and quantification by high-performance liquid chromatography–ion-trap mass spectrometry of androgen sulfoconjugates in human urine

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography–ion-trap mass spectrometry (LC–MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C 18 column (150 mm × 4.6 mm I.D., 5 μm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89–107%), repeatability (3.4–22%) and intermediate precision (5.8–22%) over the range of 2–200 ng/mL (corresponding to 1.5–147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Cleavage of normal and pathological forms of α-synuclein by neurosin in vitro

Neurosin is one of the serine proteases predominantly expressed in the central nervous system. Neurosin is presumed to play an important role in the degradation of alpha-synuclein (alpha-syn), since a previous study showed that neurosin degrades alpha-syn, inhibits polymerization of alpha-syn in vitro, and exists in Lewy bodies. However, the details of alpha-syn degradation by neurosin are little known. We investigated neurosin-mediated cleavage of alpha-syn by immunoblotting and liquid chromatography-ion trap mass spectrometry (LC/MS/MS). We also compared alpha-syn degradation by neurosin between phosphorylated and non-phosphorylated forms of alpha-syn, and between mutant and wild-type alpha-syn. Neurosin cleaved alpha-syn at specific sites. The major cleavage site was localized between Lys80 and Thr81 within the NAC region (E61 to V95), which is important for alpha-syn aggregation, and accordingly may preclude alpha-syn polymerization. Meanwhile, alternative, minor forms of processing also occur. They conserve the NAC region with truncation of the C-terminal region, and accordingly may contribute to alpha-syn polymerization. Phosphorylated alpha-syn was more resistant to degradation by neurosin than non-phosphorylated alpha-syn. The A30P mutant was more resistant to degradation than the wild-type and other alpha-syn mutants. This resistance to neurosin-mediated degradation of phosphorylated alpha-syn and the A30P mutant, which are, respectively, posttranslational and genetic factors related to the development of Parkinson's disease (PD), provides supporting evidence that neurosin is involved in the pathogenesis of PD.

Determination of Tertiary‐butylhydroquinone and Its Metabolites in Rat Serum by Liquid Chromatography–Ion Trap Mass Spectrometry

A new method applying sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) for analyzing tertiary-butylhydroquinone (TBHQ) and its metabolites in rat serum was validated. Using an extracted ion chromatogram (EIC) of m/z 149, free TBHQ was observed in rat serum after dosing TBHQ at 350 mg/kg to male and female Sprague-Dawley (SD) rats. Four major metabolites of TBHQ were identified-a TBHQ-sulfate, two TBHQ-sulfate-derived substances and a TBHQ-glucuronide through MS(n) spectra. Besides its simplicity, the sample treatment allows one to obtain a very good recovery of analysts, namely around 95%. This result suggests that the method described here is useful for the analysis of TBHQ and its metabolites in rat serum. Moreover, the metabolism of TBHQ was investigated using the method. After oral administration, TBHQ appear to be more completely absorbed and bio-transformed by males than females, which may result in higher acute oral toxicity of TBHQ for males than females.

Marked regression of abdominal fat amyloid in patients with familial amyloid polyneuropathy during long-term follow-up after liver transplantation

To elucidate whether the amount of tissue-deposited amyloid in familial amyloid polyneuropathy (FAP) patients decreases or increases over the long-term course after liver transplantation (LT), we examined changes in histopathological and biochemical characteristics of abdominal fat amyloid in the transplanted patients with a postoperative history of more than 10 years. Using a series of aspirated abdominal fat tissues from 6 FAP patients with transthyretin (TTR) Val30Met variant, the severity of amyloid deposits was examined and the composition ratio of wild type-to-variant TTR in fat amyloid was assayed by liquid chromatography-ion trap mass spectrometry (LC-MS/MS). Histopathological examination of abdominal fat tissues demonstrated a significant decrease or disappearance of amyloid deposits in all 6 patients. On LC-MS/MS analysis, the contribution of wild-type TTR to the composition ratio in amyloid fibrils was markedly increased in all patients after LT. This is the first report showing pathological evidence that deposited amyloid in FAP patients with long posttransplantation courses can gradually regress or disappear.

Metabonomics in cancer diagnosis: mass spectrometry-based profiling of urinary nucleosides from breast cancer patients

Modified nucleosides are formed post-transcriptionally in RNA. In cancer disease, the cell turnover and thus RNA metabolism is increased, yielding higher concentrations of excreted modified nucleosides. In the presented study, urinary ribonucleosides were used to differentiate between breast cancer patients and healthy volunteers. The nucleosides were extracted from urine samples using affinity chromatography and subsequently analyzed via liquid chromatography ion trap mass spectrometry (LC-IT-MS). The peak areas were related to the internal standard isoguanosine and to the urinary creatinine level. For bioinformatic pattern recognition we used the support vector machine. We examined 113 urine samples from breast cancer patients (stage Tis-T4) and 99 control samples from healthy volunteers. We achieved a sensitivity of 87.67% and a specificity of 89.90% when including 31 nucleosides. The medical metabonomics concept based on the urinary nucleoside profile reveals a significantly improved classification compared with currently applied breast cancer biomarkers such as CA15-3.

Characterization of glutathione conjugates of chloroacetanilide pesticides using ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Glutathione S-transferases (GSTs) isolated from maize were used to catalyze the conjugation of glutathione (GSH) with chloroacetanilide herbicides, producing stable conjugates that were structurally characterized using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QqToF-MS) and liquid chromatography/ion trap mass spectrometry (LC/IT-MS). Enzyme-mediated dechlorination of alachlor, metolachlor, and propachlor resulted during GSH conjugation as revealed by the mass spectra of the conjugates, which was confirmed by the loss of the chlorine isotopic signature and from high accurate mass measurements. Several fragmentation patterns in the mass spectra of the chloroacetanilide-GSH conjugates can be used to verify the identities of the enzyme reaction products, such as characteristic ions corresponding to the neutral loss of glutamic acid residue (129 Da) and water (18 Da) observed in the product ion spectrum. For the first time, data are presented showing detection of chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates.

Comparison of four extraction methods for the analysis of 24 pesticides in soil samples with gas chromatography–mass spectrometry and liquid chromatography–ion trap–mass spectrometry

With a view to analyse multiresidues of pesticides in soil samples, a new ultrasonic solvent extraction (USE) was compared to the European Norm DIN 12393 for foodstuff (extraction with acetone, partitioning with ethylacetate/cyclohexane and clean-up with gel permeation chromatography (GPC)), the QuECheRS method and a pressurised liquid extraction (PLE) method. Pesticides were analysed with both GC-MS and HPLC-MS/MS. The reference materials were the EUROSOIL 7 and its subsoil SO26 as well as a sea sand. All the substances were observed to be linear in the range of 4–800 ng g −1 for the European Norm DIN 12393, 7–1400 ng g −1 for the USE method and 20–4000 ng g −1 for the QueCheRS and the PLE methods. Limits of detection (LOD) and limits of quantification (LOQ) were with HPLC between 0.006 and 0.23 ng g −1 and between 0.022 and 0.77 ng g −1, respectively, with the exception of diuron (LOD up to 11.8 ng g −1; LOQ up to 39.2 ng g −1). With GC they ranged from 3.0 to 87.5 ng g −1 and from 10 to 292 ng g −1, respectively. All substances could be recovered with USE as well as with the QuECheRS method; the European Norm DIN 12393 could not recover carbendazim and metamitron; the PLE carbendazim, metamitron and monolinuron. For the remaining substances, recoveries at a 500 ng g −1 fortification level ranged from 10.9 to 96.3% with the USE. In comparison, the QuECheRS method was the most efficient extraction method with recoveries from 27.3 to 120.9%. It was followed by the European Norm DIN 12393 with recoveries between 6.8 and 108.1% and the PLE with recoveries from 12.2 to 153.2%. Recoveries were higher from the EUROSOIL 7 than from the SO 26. The repeatability expressed in term of standard deviation was below 20% for all substances and all materials.