Liquid chromatography-Orbitrap High Resolution Research Articles

Analysis of chemical variations between raw and wine-processed Ligustri Lucidi Fructus by ultra-high-performance liquid chromatography-Q-Exactive Orbitrap/MS combined with multivariate statistical analysis approach.

Ligustri Lucidi Fructus (LLF) is the dried and mature fruit of Ligubtrum lucidum Ait., which has the effect of nourishing the liver and kidney, brightening the eyes and promoting the growth of black hair. Wine-processed LLF is commonly used in traditional Chinese medicine; however, the processing mechanisms are still unclear. Herein, a system data acquisition and mining strategy was designed to investigate the chemical profile differences between the raw and wine-processed LLF, based on high-performance liquid chromatography-Orbitrap high resolution mass spectrometry coupled with multivariate statistical analysis including principal component analysis and partial least square analysis. Afterwars, a total of 55 components were found to be the main contributors to the significant difference between raw and wine-processed LLF by comparison with chromatographic behaviors, intact precursor ions, and characteristic MS fragmentation patterns. In addition, 10 main constituents of raw and wine-processed LLF were simultaneously determined by UHPLC-MS/MS for analyzing the content variations. Some structural transformation mechanisms during wine processing were deduced from the results. The results may provide a scientific foundation for deeply elucidating the wine-processing mechanism of LLF.

Fluorine and nitrogen functionalized magnetic graphene as a novel adsorbent for extraction of perfluoroalkyl and polyfluoroalkyl substances from water and functional beverages followed by HPLC-Orbitrap HRMS determination

Most of the reported magnetic adsorbents are difficult to absorb multi-class of per- and polyfluoroalkyl substances (PFASs), especially the short-chain PFASs. In this work, a novel fluorine and nitrogen functionalized magnetic graphene (G-NH-FBC/Fe2O3) was first synthesized and characterized by scanning electron microscope (SEM), Fourier Transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM). The as-prepared G-NH-FBC/Fe2O3 was utilized as adsorbents for the magnetic solid-phase extraction (MSPE) of 19 PFASs from water and functional beverages and showed excellent adsorption capacity probably due to the hydrophobic interaction. Under the optimal pretreatment and instrumental conditions, a selective and sensitive high performance liquid chromatography Orbitrap high resolution mass spectrometry (HPLC-Orbitrap HRMS) method was developed for the determination of PFASs. Results indicated that the proposed method had favorable linearity (R2 ≥ 0.994) within a wide range of concentrations. Limit of detection (LOD) and limit of quantification (LOQ) for the developed method ranged from 3 ng/L to 15 ng/L and 10 ng/L to 49 ng/L, respectively. Finally, the method was successfully applied to determine PFASs in drinking water, river water, tap water, factory drainage and functional beverages with recoveries ranging from 71.9% to 117.6% and relative standard deviation of <10%. The prepared G-NH-FBC/Fe2O3 was easy to recycle and could be reused for five times without significant decrease in extraction recoveries of PFASs. These results demonstrated that this novel magnetic G-NH-FBC/Fe2O3 could efficiently enrich PFASs and the proposed method is reliable and robust for the determination of PFASs in water and beverage samples.

In vivo toxicity assessment of Clinopodium vulgare L. water extract characterized by UHPLC-HRMS

Clinopodium vulgare L. (Lamiaceae) was used in the traditional Bulgarian medicine for treatment of wounds, diabetes and gastric ulcers. In this study we aimed at safety assessment of C. vulgare lyophilized water extract (CVE) characterized by ultra high-performance liquid chromatography–Orbitrap high resolution mass spectrometry (UHPLC-HRMS). The acute and sub-acute toxicity of CVE was determined in two rodent species (mice and rats), and two routes of administration – intraperitoneal (i.p.) and oral (p.o.). LD50 (i.p.), were found to be 675 mg/kg (mice) and 500 mg/kg (rats). An acute i. p. administration resulted in central nervous system toxic effects. LD50 (p.o.) was higher than 2000 mg/kg for both species. In sub-acute oral administration, CVE did not exert any toxic effect on hematology, blood and urine biochemistry, and histomorphology in pancreas, liver, spleen and kidney. In addition, based on accurate masses, MS/MS and comparison with standards, a variety of flavonoids, caffeic acid oligomers and saponins were tentatively elucidated in CVE. Rosmarinic acid was the major compound. In conclusion, CVE did not cause hematological, biochemical and histopathological changes after oral administration and it is safe for internal use. The obtained UHPLC-HRMS profile revealed CVE as a new rich source of water soluble caffeic acid oligomers.

Determination of steroid hormones and their metabolite in several types of meat samples by ultra high performance liquid chromatography—Orbitrap high resolution mass spectrometry

A new analytical method based on ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap high resolution mass spectrometry (Orbitrap-HRMS) has been developed for the determination of steroid hormones (hydrocortisone, cortisone, progesterone, prednisone, prednisolone, testosterone, melengesterol acetate, hydrocortisone-21-acetate, cortisone-21-acetate, testosterone propionate, 17α-methyltestosterone, 6α-methylprednisolone and medroxyprogesterone) and their metabolite (17α-hydroxyprogesterone) in three meat samples (chicken, pork and beef). Two different extraction approaches were tested (QuEChERS “quick, easy, cheap, effective, rugged and safe” and “dilute and shoot”), observing that the QuEChERS method provided the best results in terms of recovery. A clean-up step was applied comparing several sorbents, obtaining the best results when florisil and aluminum oxide were used. The optimized method was validated, obtaining suitable results for all validation parameters in the three meat matrices evaluated. Recovery values ranged from 70% to 103% (except for prednisone in beef samples), meanwhile repeatability and reproducibility were obtained at values lower than 18% and 21%, respectively. The limit of quantification (LOQ) was established for most of the compounds at 1.0 μg/kg, except for testosterone in chicken and hydrocortisone-21-acetate and cortisone-21-acetate in pork at 2.0 μg/kg. Decision limit (CCα) and detection capability (CCβ) values ranged from 1.0–2.7 μg/kg and 1.9–5.5 μg/kg, respectively, in the three matrices. Finally, thirty one meat samples were analyzed and two hormones, progesterone and hydrocortisone, were detected in a beef and pork sample at 1.7 and 2.8 μg/kg respectively.

Multiresidue Method for the Rapid Determination of Pesticide Residues in Tea Using Ultra Performance Liquid Chromatography Orbitrap High Resolution Mass Spectrometry and In-Syringe Dispersive Solid Phase Extraction.

A method based on in-syringe dispersive solid phase extraction (IS-D-SPE) and ultra performance liquid chromatography Orbitrap high resolution mass spectrometry for the multiresidue analysis of 117 pesticides in tea was developed. Full scan mode was acquired over an m/z range of 100–800 with Orbitrap resolution at 70000, followed by full scan/dd-MS2 mode for confirmation. The identification criteria of retention time and mass accuracy tolerance was ±0.20 min and ±5.0 ppm, respectively. MS/MS fragment ions obtained dd-MS2 were necessary to identify the pesticides with the same molecular mass weight. The IS-D-SPE technique involved a mixture of 200 mg PSA, 100 mg C18, and 15 mg multiwalled carbon nanotubes for the cleanup of tea matrix. Good linearity (R2 > 0.99) for 117 pesticides was obtained. Satisfactory recoveries in the range of 70–120% were obtained for 105 pesticides, while intraday and interday precisions were below 20%. Limits of quantification were generally 10 μg kg–1. Finally, this method was employed to analyze 117 pesticides in 70 tea samples.

Target analysis of primary aromatic amines combined with a comprehensive screening of migrating substances in kitchen utensils by liquid chromatography–high resolution mass spectrometry

An analytical strategy including both the quantitative target analysis of 8 regulated primary aromatic amines (PAAs), as well as a comprehensive post-run target screening of 77 migrating substances, was developed for nylon utensils, using liquid chromatography–orbitrap-high resolution mass spectrometry (LC–HRMS) operating in full scan mode. The accurate mass data were acquired with a resolving power of 50,000 FWHM (scan speed, 2Hz), and by alternating two acquisition events, ESI+ with and without fragmentation. The target method was validated after statistical optimization of the main ionization and fragmentation parameters. The quantitative method presented appropriate performance to be used in official monitoring with recoveries ranging from 78% to 112%, precision in terms of Relative Standard Deviation (RSD) was less than 15%, and the limits of quantification were between 2 and 2.5µgkg−1. For post-target screening, a customized theoretical database was built for food contact material migrants, including bisphenols, phthalates, and other amines. For identification purposes, accurate exact mass (<5ppm) and some diagnostic ions including fragments were used. The strategy was applied to 10 real samples collected from different retailers in the Valencian Region (Spain) during 2014. Six out of eight target PAAs were detected in at least one sample in the target analysis. The most frequently detected compounds were 4,4′-methylenedianiline and aniline, with concentrations ranging from 2.4 to 19,715µgkg−1 and 2.5 to 283µgkg−1, respectively. Two phthalates were identified and confirmed in the post-run target screening analysis.

Comprehensive analytical strategy for biomonitoring of pesticides in urine by liquid chromatography–orbitrap high resolution mass spectrometry

In this study we propose an analytical strategy that combines a target approach for the quantitative analysis of contemporary pesticide metabolites with a comprehensive post-target screening for the identification of biomarkers of exposure to environmental contaminants in urine using liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS). The quantitative method for the target analysis of 29 urinary metabolites of organophosphate (OP) insecticides, synthetic pyrethroids, herbicides and fungicides was validated after a previous statistical optimization of the main factors governing the ion source ionization and a fragmentation study using the high energy collision dissociation (HCD) cell. The full scan accurate mass data were acquired with a resolving power of 50,000 FWHM (scan speed, 2Hz), in both ESI+ and ESI− modes, and with and without HCD-fragmentation. The method – LOQ was lower than 3.2μgL−1 for the majority of the analytes. For post-target screening a customized theoretical database was built, for the identification of 60 metabolites including pesticides, PAHs, phenols, and other metabolites of environmental pollutants. For identification purposes, accurate exact mass with less than 5ppm, and diagnostic ions including isotopes and/or fragments were used. The analytical strategy was applied to 20 urine sample collected from children living in Valencia Region. Eleven target metabolites were detected with concentrations ranging from 1.18 to 131μgL−1. Likewise, several compounds were tentatively identified in the post-target analysis belonging to the families of phthalates, phenols and parabenes. The proposed strategy is suitable for the determination of target pesticide biomarkers in urine in the framework of biomonitoring studies, and appropriate for the identification of other non-target metabolites.

Identification of phase II pharmaceutical metabolites in reclaimed water using high resolution benchtop Orbitrap mass spectrometry

This study described an analytical method for the identification of common phase II pharmaceutical metabolites in reclaimed water using liquid chromatography high resolution Orbitrap mass spectrometry after solid phase extraction (SPE). Orbitrap mass spectrometer was operated at resolution of 70000 in MS mode and 35000 in data-dependent MS/MS mode, without using lock mass. Firstly, parent drugs and their metabolites were tentatively identified based on accurate mass using a mass tolerance of 5ppm. A detailed examination of the extracted ion chromatograms (XICs) for all potential metabolites revealed the presence of two phase II metabolites of sulfamethoxazole, acetylsulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. Secondly, the high resolution data-dependent MS/MS spectra of each compound were further investigated using metabolic profiling software. After comparing characteristic ions obtained in MS/MS mode with those predicted by the software and reported in previous studies, the two phase II metabolites were positively identified in reclaimed water. Lastly, the two metabolites were detected and quantified in the reclaimed water samples collected during a period of one month. As a result, averaged concentrations of sulfamethoxazole, acetylsulfamethoxazole and sulfamethoxazole glucuronide were calculated at 2848±1367ngL−1, 1980±1410ngL−1, and 2859±1526ngL−1, respectively. The two metabolites represented 54% of the source of sulfamethoxazole in reclaimed water suggesting the importance of measuring pharmaceutical metabolites in the environment. To our knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples.

Multi-residue method for the detection of veterinary drugs in distillers grains by liquid chromatography–Orbitrap high resolution mass spectrometry

Distillers Grain (DG) is an important by-product of ethanol production. The ethanol production process uses only the starch portion of the plant and all the remaining nutrients, protein, fat, minerals, and vitamins remain in DGs, a valuable feed material for livestock. The use of antimicrobial drugs is helpful to limit harmful bacterial growth during the early part of the fermentation process. This can lead to the possible presence of contaminants in the by-products that are used in the food and feed industries, resulting in a major concern for the development of bacterial resistance in both humans and animals. To facilitate the detection of antimicrobial and other commonly used veterinary drugs in DGs, a liquid chromatography–high resolution mass spectrometry (LC–HRMS) method was developed targeting a wide range of 12 chemical classes of anti-bacterial substances and drugs, such as ionophore and non-ionophore authorized coccidiostats, banned coccidiostats, macrolides, tetracyclines, nitroimidazoles, amphenicols, quinolones, sulphonamides, tranquilizers, non-steroidal anti-inflammatory drugs and benzimidazoles. Following a simple and fast extraction step with a mixture of organic solvents, the extract was directly injected into the LC coupled to an Orbitrap mass analyzer. The identification of residues is based on accurate mass measurement. The high mass resolution of 50,000 full width at half maximum (FWHM) and corresponding narrow mass windows permitted a very selective and sensitive detection of the analytes in such a complex matrix. A single-laboratory validation procedure was carried out evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance for precision and trueness, and allowed the determination of the compounds at low concentration. The proposed multi-method demonstrated that liquid chromatography coupled to an Orbitrap mass spectrometer is a promising analytical technique, suitable for official residue control of a variety of veterinary drugs in DGs supporting feed safety policies.

Comprehensive qualitative and quantitative determination of pesticides and veterinary drugs in honey using liquid chromatography–Orbitrap high resolution mass spectrometry

A database has been created for the simultaneous analysis of more than 350 pesticides and veterinary drugs (including antibiotics) using ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry (UHPLC–Orbitrap-MS). This is a comprehensive exact mass database built using the Exactive-Orbitrap analyzer. The developed database includes exact masses of the target ions and retention time data, and allows the automatic search of the included compounds. Generic chromatographic and MS conditions have been applied. The presented database is suitable for qualitative analysis, and it was also evaluated for quantitative purposes in routine analysis, after the optimization and validation of a generic extraction method in honey samples. Adequate recovery and precision values for most of the studied analytes were obtained and the limits of detection (LOD) ranged from 1 to 50μgkg−1. For pesticides, LODs were always lower than the MRLs established by European Union in honey, except for a few compounds. This method was applied to the analysis of 26 real honey samples and some pesticides (azoxystrobin, coumaphos, dimethoate and thiacloprid) were detected in 4 samples. Azoxystrobin and coumaphos were determined in two different samples (organic honey) at 1.5μgkg−1 and 5.1μgkg−1. Veterinary drugs were not detected in the analyzed samples.