Automated micro-solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry analysis of pesticides in foods extracted with ethyl acetate.

Generic extraction methods for the multi-compound pesticide analysis of food have found their solid place in laboratories. Ethyl acetate and acetonitrile extraction methods have been developed as fast and easy to handle standard multi-compound methods, both feature benefits and limitations. The direct injection to gas chromatography can be impaired by a high burden of coextracted matrix, resulting in deterioration of the chromatographic system and matrix effects, requiring frequent maintenance. Therefore, common clean-up methods, such as dispersive solid-phase extraction, freeze-out of fats, or gel permeation chromatography, have been applied in clean-up. Automated clean-up using micro-solid-phase extraction (µSPE) is a recent development with several demonstrated advantages when employed in the analysis of pesticides and other contaminants in foods extracted with acetonitrile, but it has not yet been evaluated in this application using ethyl acetate for extraction. In this study, an automated procedure using µSPE cartridges was developed and established on an x,y,z robotic sampler for the raw extract clean-up and preparation of diluted samples for injection on a GC-MS/MS system. Validation experiments for 212 pesticides, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in lettuce, avocado, raspberry, paprika, egg, and liver extracts were performed using µSPE with MgSO4, PSA, C18, and CarbonX. The performance in routine operation is briefly discussed.

Benchmarking of Two Peptide Clean-Up Protocols: SP2 and Ethyl Acetate Extraction for Sodium Dodecyl Sulfate or Polyethylene Glycol Removal from Plant Samples before LC-MS/MS.

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.

Multiresidue analysis of pesticides in three Indian soils: method development and validation using gas chromatography tandem mass spectrometry.

The paper reports a multiresidue method that was validated on 220 multi-class pesticides in three major Indian soils, namely, (i) new alluvial soil (NAS); (ii) red lateritic soil (RS) and (iii) black soil (BS) from three different regions. An ethyl acetate-based extraction method with a freezing-out cleanup step was employed for sample preparation, followed by gas chromatography-tandem mass spectrometric analysis. The method that was initially optimized on BS worked satisfactorily for the other two soil matrices. At the spiking level of 10 µg/kg (LOQ), the recoveries were satisfactory (within 70-120%) with precision-RSDs, ≤20% (n = 6) for 85, 88.6, and 89% of compounds in BS, RS, and NAS respectively. At 20 µg/kg, the method performance was satisfactory in each soil for all pesticides. When this validated method was applied to analyse 25 field samples, 6 pesticides were detected in them. In each case, precision (RSD) was <20%. The method sensitivity, accuracy and precision complied with the SANTE/2020/12830 guidelines. The method can be applied for environmental monitoring and risk assessment purposes, thus aiding in regulating pesticide usage in agricultural fields. The limitations and future scope of the study are also discussed.HighlightsA multiresidue method is reported for simultaneous analysis of multi-class pesticides in diverse soilsThe method was validated on 220 pesticides in new alluvial, red lateritic and black soilsSample preparation involved extraction with ethyl acetate and cleanup by a freezing stepThe residues were estimated by gas chromatography tandem mass spectrometry (GC-MS/MS)The method accuracy and precision complied with the EU's SANTE guidelines.

Preliminary verification of the anti-hypoxia mechanism of Gentiana straminea maxim based on UPLC-triple TOF MS/MS and network pharmacology.

BACKGROUND: Anoxia is characterized by changes in the morphology, metabolism, and function of tissues and organs due to insufficient oxygen supply or oxygen dysfunction. Gentiana straminea Maxim (G.s Maxim) is a traditional Tibetan medicine. Our previous work found that G.s Maxim mediates resistance to hypoxia, and we found that the ethyl acetate extract had the best effect. Nevertheless, the primary anti-hypoxia components and mechanisms of action remain unclear. METHODS: Compounds from the ethyl acetate extraction of G.s Maxim were identified using UPLC-Triple TOF MS/MS. Then Traditional Chinese Medicine Systematic Pharmacology Database was used to filtrate them. Network pharmacology was used to forecast the mechanisms of these compounds. Male specific pathogen-free Sprague Dawley rats were randomly divided into six groups: (1) Control; (2) Model; (3) 228 mg/kg body weight Rhodiola capsules; (4) 6.66 g/kg body weight the G.s Maxim's ethyl acetate extraction; (5) 3.33 g/kg body weight the G.s Maxim's ethyl acetate extraction; (6) 1.67 g/kg body weight the G.s Maxim's ethyl acetate extraction. After administering intragastric ally for 15 consecutive days, an anoxia model was established using a hypobaric oxygen chamber (7000 m, 24 h). Then Histology, enzyme-linked immunosorbent assays, and western blots were performed to determine these compounds' anti-hypoxic effects and mechanisms. Finally, we performed a molecular docking test to test these compounds using Auto Dock. RESULTS: Eight drug-like compounds in G.s Maxim were confirmed using UPLC-Triple TOF MS/MS and Lipinski's rule. The tumor necrosis factor (TNF) signaling pathway, the hypoxia-inducible factor 1 (HIF-1) signaling pathway, and the nuclear factor kappa-B (NF-κB) signaling pathway was signaling pathways that G.s Maxim mediated anti-anoxia effects. The critical targets were TNF, Jun proto-oncogene (JUN), tumor protein p53 (TP53), and threonine kinase 1 (AKT1). Animal experiments showed that the ethyl acetate extraction of G.s Maxim ameliorated the hypoxia-induced damage of hippocampal nerve cells in the CA1 region and reversed elevated serum expression of TNF-α, IL-6, and NF-κ B in hypoxic rats. The compound also reduced the expression of HIF-1α and p65 and increased the Bcl-2/Bax ratio in brain tissue. These findings suggest that G.s Maxim significantly protects against brain tissue damage in hypoxic rats by suppressing hypoxia-induced apoptosis and inflammation. Ccorosolic acid, oleanolic acid, and ursolic acid had a strong affinity with core targets. CONCLUSIONS: The ethyl acetate extraction of G.s Maxim mediates anti-hypoxic effects, possibly related to inhibiting apoptosis and inflammatory responses through the HIF-1/NF-κB pathway. The primary active components might be corosolic, oleanolic, and ursolic acids.

Development and validation of a liquid chromatographic tandem mass spectrometric method for the analysis of patulin in apple and apple juice.

This study reports a robust and sensitive method for rapid testing of patulin in apple and apple juice. The method involved extraction of homogenised samples (10 g) with ethyl acetate (10 mL) and clean up by dispersive-solid phase extraction using primary secondary amine (25 mg/mL). Prior to the LC-MS/MS analysis, the cleaned extract was reconstituted in methanol/water (2:8). The optimised LC-MS condition provided a symmetric peak of patulin within a short LC-runtime of 5 min. The recoveries at the limit of quantification (0.005 mg/kg) and higher levels were satisfactory (> 80%), with the precision-RSDr (< 11%). In an inter-laboratory comparison study involving 13 accredited laboratories, the reproducibility-RSDR and HorRat values ranged between 4.80 and 6.08% and between 0.18 and 0.23 respectively, indicating a satisfactory method-precision. The z-scores of the participating laboratories were within ± 2. When the method was applied to incurred samples, the contamination range was 0.008-0.225 mg/kg and 0.018-0.034 mg/kg for apple and juice respectively, demonstrating a satisfactory performance in terms of precision. Based on the solvent standard, matrix-matched standard and standard-addition approaches, the calibration graphs provided similar quantitative performances. Because of its reliability, robustness and time-effectiveness, the method can be recommended for regulatory testing purposes.

Evaluation of Biological Activity of Mastic Extracts Based on Chemotherapeutic Indices.

BACKGROUND: Most previous mastic investigators have not considered its potent cytotoxicity that may significantly affect the interpretation of obtained data. In the present study, we re-evaluated several biological activities of mastic extracts, based on chemotherapeutic indexes. MATERIALS AND METHODS: Pulverized mastic gum was extracted with n-hexane and then with ethyl acetate or independently with methanol or n-butanol. Tumor specificity (TS) of the extracts was determined by their cytotoxicity against human malignant and non-malignant cells. Antibacterial activity was determined by their cytotoxicity against bacteria and normal oral cells. Antiviral activity was determined by their protection of viral infection and cytotoxic activity. Cytochrome P-450 (CYP) 3A4 activity was measured by ß-hydroxylation of testosterone. RESULTS: Ethyl acetate extract showed slightly higher tumor specificity (TS=2.6) and one order higher antibacterial activity (selectivity index (SI)=0.813) than other extracts (TS=1.4-2.5; SI=0.030-0.063). All extracts showed no anti-human immunodeficiency virus (HIV) activity, but some anti-herpes simplex virus (HSV) activity, which was masked by potent cytotoxicity. They showed strong inhibitory activity against CYP3A4. CONCLUSION: Ethyl acetate extraction following the removal of cytotoxic and CYP3A4 inhibitory substances by n-hexane can enhance antitumor and antibacterial activity of mastic.

Study on chemical constituents and in vitro anti-inflammatory activity of ethyl acetate extraction part from ethanol extract of the roots of Ardisia virens / 中国药房

OBJECTIVE To isolate and ide ntify the chemical constituents of the root of Ardisia virens and preliminarily evaluate the in vitro anti-inflammatory activity of the compounds. METHODS The ethyl acetate extraction part from 70％ ethanol extract of the root of A. virens were separated and purified by silica gel column chromatography ，ODS column chromatography ， etc. The structures of the compounds were identified according to physical and chemical properties and spectral data. The inflammation model of RAW 264.7 cells was induced by lipopolysaccharide ，and anti-inflammatory activity of the compound was investigated by MTT assay. RESULTS A total of 11 compounds were isolated from the ethyl acetate extraction part ，and were identified as cyclamiretin A （1），α-spinasterol （2），（3S，5R，6S，7E）-3，5，6-trihydroxy-7-megastigmen-9-one （3）， （+）-angelicoidenol（4），octadeca-dienoic acid- 2，3-dihydroxypropyl ester （5），α-linolenic acid （6），glycerol monooleate （7），5， 5′-（4，7-hexadecadlene-1，16-diyl）bisresorcinol（8），1-（3，5-dihydroxyphenyl）heptan-1-one（9），5-heptylresorcinol and （10） 5-n-nonylresorcinol（11）. The in vitro anti-inflammatory results showed that 80，40，20，10，5 μg/mL of compounds 2，8，9 and 10 could reduce the cell survival rate in different degrees. CONCLUSIONS Compounds 1-11 are isolated from this plant for the first time，and compound 8 is a new natural product. Compound 2，8，9 and 10 show certain anti-inflammatory activity in vitro .

Rapid extraction of melamine in powdered milk for direct electrospray ionization tandem mass spectrometry analysis.

A combination of a simple pretreatment for melamine extraction and direct analysis in electrospray ionization tandem mass spectrometry (ESI-MS/MS) is proposed. Three pretreatments were evaluated. The first was based on suppressing interference using acetonitrile. The second used sulphuric acid and trichloroacetic acid to suppress interference and for melamine extraction, respectively. The third used sulphuric acid to suppress milk interference, trichloroacetic acid for melamine precipitation, and ethyl acetate for melamine extraction. However, only the last pretreatment suppressed milk interference in melamine detection and a good linearity (R(2)=0.99) was obtained. The presence of MS/MS 85 on melamine fragmentation spectrum showed the selectivity of this method. The limit of detection and limit of quantification were 0.269 µg L(-1) and 0.897 µg L(-1), respectively. The recoveries and relative standard deviation (RDS) of method were lower than 114% and 7.86%, respectively. Further, the research was extended to elucidate the nature of the melamine in the extract through infrared spectroscopy and microscopy analyses. The precipitate was characterized as melaminium bis(trichloroacetate) dihydrate, which is generated through hydrogen bound formation in an interaction between melamine and trichloroacetic acid. Therefore, a simple, fast, and easy method for melamine extraction and direct ESI-MS/MS analysis was developed.

Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction of herbicides in peanuts.

Liquid-phase extraction coupled with metal-organic frameworks-based dispersive solid phase extraction was developed and applied to the extraction of pesticides in high fatty matrices. The herbicides were ultrasonically extracted from peanut using ethyl acetate as extraction solvent. The separation of the analytes from a large amount of co-extractive fat was achieved by dispersive solid-phase extraction using MIL-101(Cr) as sorbent. In this step, the analytes were adsorbed on MIL-101(Cr) and the fat remained in bulk. The herbicides were separated and determined by high-performance liquid chromatography. The experimental parameters, including type and volume of extraction solvent, ultrasonication time, volume of hexane and eluting solvent, amount of MIL-101(Cr) and dispersive solid phase extraction time, were optimized. The limits of detection for herbicides range from 0.98 to 1.9 µg/kg. The recoveries of the herbicides are in the range of 89.5-102.7% and relative standard deviations are equal or lower than 7.0%. The proposed method is simple, effective and suitable for treatment of the samples containing high content of fat.

Experimental study on the mechanism of anti-anaphylaxis effect of ethyl acetate extraction of Jing-fang powder / 中国药学杂志

OBJECTIVE: To investigat the mechanism of anti-anaphylaxis effect of Jing-fang powder(traditional Chinese medicines) by using initiative systemic anaphylaxis model in mice. METHODS: The model of initiative systemic anaphylaxis in mice was induced by intraperitoneal injection with ovalbumin and DPT vaccine, and the ethyl acetate extraction of Jing-fang powder was intragastricly administrated for 14 d. ELISAs methods were used to measure the levels of total IgE and LTB4 in serum, and the levels of NO, iNOS and LTB4 in lung tissues in model mice. Ultraviolet spectrophotometry was used to measure the PGE2 level in lung tissue, and the index of thymus and spleen were also evaluated. RESULTS: At the dose of 6.67 g·g-1, the ethyl acetate extraction of Jing-fang powder decreased the index of thymus and spleen(P<0.05), as well as suppressed the production of total IgE and LTB4 in serum(P<0.05, and reduced the levels of NO, iNOS, LTB4 and PGE2 in lung tissue in model mice(P<0.05). Meanwhile, the extraction also suppressed the activity of iNOS in lung tissue under the dose of 6.67 and 10.01 g·g-1(P<0.05). CONCLUSION: These results suggest that the ethyl acetate extraction of Jing-fang Powder has potential anti-allergic effect, which the mechanism may be related to reduce the production of IgE, and inhibit the release of metabolic products of arachidonic acid, for example LTB4 and PGE2, and decrease the release of inflammatory allergy mediators NO.