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A fast, reliable, inexpensive, and practical method with a low determination limit and high recovery has been developed for the determination of the marijuana metabolite in routine analysis. THC-COOH in urine was validated using liquid chromatography—tandem mass spectrometry (LC—MS/MS). Before an easy single-step extraction with Toxi-Tubes, basic hydrolysis was performed at 60 °C for 30 min. LC—MS/MS analysis takes 2.5 min for each sample, and the retention time of the analyte is 1.75 min. Specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, repeatability, and intermediate precision (inter-day) system suitability parameters were determined in the validation study. The recovery of the extraction method was 88.67 (±5.91). LOD and LOQ values were 1.41 and 5.00 ng mL−1, respectively. The method showed linear response between the values 5.00 and 500.00 ng mL−1. The repeatability was 9.64% (relative standard deviation, RSD%), and the intermediate precisions (RSDR%) were 10.73%, 13.74%, and 8.11% at 10.00, 100.00, and 200.00 ng mL−1 concentration levels, respectively. No statistically significant difference was found in ANOVA analysis, between three consecutive days in intermediate precision study, for 90% confidence level. HorRat values were between 0.34 and 0.61. The method was applied to CEDIA positive samples, obtained from the Trabzon Group Presidency of Turkish Council of Forensic Medicine, successfully.

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Seed germination is a new beginning for the crop life cycle, which is closely related to seed sprouting and subsequent plant growth and development, and ultimately affects grain yield and quality. Salt stress is one of the most important abiotic stress factors that restrict crop production. Therefore, it is highly important to improve crop salt tolerance and sufficient utilization of saline-alkali land. In this study, we identified the phosphorylated proteins involved in salt stress response by combining SEM, 2-DE, Pro-Q Diamond staining and tandem mass spectrometry. The results showed that salt stress significantly inhibited seed germination and starch degradation. In total, 14 phosphorylated protein spots (11 unique proteins) in the embryo and 6 phosphorylated protein spots (4 unique proteins) in the endosperm were identified, which mainly involved in stress/defense, protein metabolism and energy metabolism. The phosphorylation of some proteins such as cold regulated proteins, 27K protein, EF-1β and superoxide dismutase could play important roles in salt stress tolerance.

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Abstract

Palmatine is a compound with good water solubility extracted from Coptis chinensis, Fibraurea recisa Pierre, Cortex Phellodendri Chinensis. Palmatine has good antibacterial activity and mainly used for the treatment of bacterial dysentery, gynecological inflammation, surgical infection, and conjunctivitis. It has anti-diabetic, anti-oxidant, and cognitive-enhancing activities. In this study, we used UPLC-MS/MS to determinate palmatine in rat plasma, and investigated its pharmacokinetics. Coptisine was utilized as an internal standard (IS), and acetonitrile precipitation method was used to process the plasma samples. Chromatographic separation was achieved using a UPLC BEH C18 column using mobile phase of acetonitrile- 0.1% formic acid with gradient elution. Electrospray ionization (ESI) tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive ionization was applied. The results indicated that within the range of 1–500 ng/mL, linearity of palmatine in rat plasma was acceptable (r > 0.995), and the lower limit of quantification (LLOQ) was 1 ng/mL. Intra-day and inter-day precision RSD of palmatine in rat plasma were less than 14%. Accuracy range was between 93.7 and 107.1%, and matrix effect was between 101.6 and 109.4%. The method was successfully applied in the pharmacokinetics of palmatine in rats after oral and intravenous administration. The absolute bioavailability of the palmatine was 15.5% in rats.

Open access

The aim of this study was to assess the impact of hanging position of hunted pheasant carcasses (secured by the head as compared to hanging position secured by the legs) on the biogenic amine concentration in the thigh and breast muscles. The carcasses of feathered game (Phasianus colchicus), left entirely untreated after hunting and placed in a storage space at a pre-set temperature of 7 °C for 21 days were used in the study. Samples of breast and thigh muscles were taken at regular weekly intervals. Measurement of biogenic amines (putrescine, cadaverine, tyramine, tryptamine, histamine, phenylethylamine) was based on high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Higher biogenic amine concentrations were detected in the muscles (both breast and thigh) of pheasants hanging by their legs compared to pheasants hanging by their heads (no statistically significant difference in biogenic amine concentration between monitored groups was, however, established). Higher concentrations of biogenic amines were found in the thigh muscles compared to breast muscles in both monitored groups. The obtained results show, that hanging the carcasses of pheasants during storage by the head is more suitable method in term of biogenic amine concentration than storing carcasses hanging by the legs.

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A comparative proteomic analysis of grain proteins during five grain developmental stages of wheat cultivar Chinese Spring (CS) and its 1Sl/1B substitution line CS-1Sl(1B) was carried out in the current study. A total of 78 differentially expressed protein (DEP) spots with at least 2-fold expression difference were detected by two-dimensional electrophoresis (2-DE). Among these, 73 protein spots representing 55 differentially expressed proteins (DEPs) were successfully identified by matrix-assisted laser desorption/ionization time-offlight tandem mass spectrometry (MALDI-TOF/TOF-MS). Differential protein spots between the two genotypes were analyzed by cluster software, which revealed significant proteome differences. There were 39 common spots (including 33 DEPs) that showed significant difference between the two lines across five grain developmental stages, of which 14 DEP spots (including 11 DEPs) were mainly involved in carbohydrate metabolism that were encoded by the genes on 1B chromosome while 25 DEP spots (including 12 DEPs) were mainly related to stress response and gluten quality that were encoded by 1S1 chromosome. These results indicated that the Sl genome harbors more stress and quality related genes that are potential valuable for improving wheat stress resistance and product quality.

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Eupatilin, mainly derived from Artemisia asiatica (Asteraceae), is an O-methylated flavone with various bioactivities. In the present study, a validated ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was established for the quantification of eupatilin in rat plasma with the internal standard (IS) of tussilagone and the protein precipitation of plasma samples was performed using acetonitrile–methanol (9:1, v/v). The eupatilin and IS were eluted separately on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with the gradient mobile phase consisted of 0.1% formic acid and acetonitrile. The protonated analytes were quantified by multiple reactions monitoring (MRM) mode with an electrospray ionization (ESI) source operated in positive ion mode. The calibration plots were found to be linear over the range from 2 to 1000 ng/mL for eupatilin in rat plasma. Both of the intra-day and inter-day precision variations (RSDs) were ≤13%. The recoveries of eupatilin in rat plasma were between 83.7% and 94.6%, and the accuracy of the method ranged from 95.8% to 107.6%. In addition, the validated method was applied to pharmacokinetic study of eupatilin after an intravenous dose of 2 mg/kg to rats.

Open access
Acta Chromatographica
Authors: Su-su Bao, Jian Wen, Teng-hui Liu, Bo-wen Zhang, Chen-chen Wang, and Guo-xin Hu

Olmutinib (Olita™) is an oral third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) which is used to treat non-small cell lung cancer (NSCLC). A simple, rapid, and sensitive method based on ultra-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) has been developed for the determination of olmutinib. Sample preparation was performed following simple one-step protein precipitation with acetonitrile. Olmutinib and internal standard (dasatinib) were separated on an Eclipse Plus C18 RRHD (2.1 × 50 mm, 1.8 μm) column. The mobile phase consisted of acetonitrile–0.1% formic acid in water with gradient elution. A total run time of 1.7 min was achieved. Detection was performed on a positive-ion electrospray ionization mass spectrometer in multiple reaction monitoring (MRM) mode, using transitions of m/z 487.2 → 402.1 for olmutinib and m/z 488.2 → 401 for dasatinib (IS), respectively. The calibration curve (R 2 = 0.999) was linear over the range of 1–500 ng/mL. The recovery of olmutinib ranged from 85.8% to 95.5%. This method can be applied to pharmacokinetic studies of olmutinib.

Open access

Abstract

Chronic hepatitis B virus (HBV) carriers may develop hepatocellular carcinoma (HCC) by a wide range of mechanisms including angiogenesis. We show that HBV replication induces the expression of angiogenic proteins interleukin 6 (IL6) and cyclooxygenase-2 (Cox2). Interestingly, ibuprofen (a Cox2 inhibitor) is found to attenuate the levels of IL6 and Cox 2 which are induced by HBV replication.

The mechanism of attenuation of angiogenic proteins by ibuprofen was further investigated. Our results show that HBx is involved in the increase of the expression of Cox2 through the NFκB pathway. However, the expression of Cox2 is decreased when the HBx-expressing cells are incubated with ibuprofen. The contrasting effect of HBx on Cox2 is found to be determined by differential dimer formation among the members of the NFκB family of proteins, including NFκB, RelA, and C-rel. Specifically, HBx alone results in dimer formation between NFκB and RelA, while the combined presence of HBx and ibuprofen leads to the formation of NFκB and C-rel. Additional information on the interaction network involving HBx, ibuprofen, and NFκB pathways is revealed by two-dimensional liquid chromatography-tandem mass spectrometry proteomics analysis. Taken together, our findings provide new insights on the angiogenesis induced by HBV replication.

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The effect of free-range versus cage management system on corticosterone transfer into the eggs was studied in laying hens. Hungarian Yellow laying hens (age: 21 weeks, body weight: 2.0 ± 0.5 kg) were divided into two groups in the spring: Group I, free-range keeping (n = 15 layers, density: ≯ 0.5 bird/m2) in outdoor runs, with continuous access to a commercial layer feed; Group II, hens kept in battery cages (n = 17 layers, density: 2 birds/m2, natural light, continuous access to feed and water). Eggs were collected after a one-week adaptation period on days 2, 7 and 16. Corticosterone (CST) was extracted from homogenised egg samples using an ASE-200 Accelerated Solvent Extractor and then assayed by liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) [Thermo Quest Surveyor high-performance liquid chromatography (HPLC) interfaced via Atmospheric Pressure Chemical Ionisation (APCI) ion source to Finnigan/Thermo Quest LCQ Deca MS/MS] using dexamethasone as internal standard with positive APCI ionisation. CST concentrations of whole eggs laid by free-range hens on days 2, 7 and 16 were 0.370 ± 0.218, 0.259 ± 0.066 and 0.915 ± 0.745 ng·g-1, respectively, while those of eggs laid by caged hens were 0.206 ± 0.157, 0.223 ± 0.165 and 0.184 ± 0.110 ng·g-1 at the above sampling times. It is concluded that in free-range laying hens the sharp changes of environmental weather conditions significantly increased the corticosterone content of eggs, while the environmentally controlled and closed battery cage management technology resulted in relatively uniform corticosterone concentrations in the whole eggs.

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A simple and sensitive liquid chromatography—tandem mass spectrometry method was developed for the quantification of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin, and atorvastatin lactone in rat plasma. Solid-phase extraction was used for preparation of samples. Rosuvastatin was chosen as an internal standard. Chromatographic separation was achieved on ZORBAX Eclipse C18 Analytical, 4.6 × 100 mm (3.5 μm) column with a gradient mobile phase composed of acetonitrile and 0.1% acetic acid, at a flow rate of 400 μL min−1. The column was kept at constant temperature (25 °C), and autosampler tray temperature was set at 4 °C. The following selected reaction monitoring (SRM) transitions were selected: (m/z, Q1 → Q3, collision energy) atorvastatin (559.47 → 440.03, 22 eV), atorvastatin lactone (541.36 → 448.02, 19 eV), ortho-ohydroxyatorvastatin (575.20 → 440.18, 20 eV), para-hydroxyatorvastatin (575.54 → 440.18, 20 eV), and rosuvastatin (482.25 with selected combination of two fragments 257.77, 31 eV, and 299.81, 35 eV) in positive ion mode. The method was validated over a concentration range of 0.5–20 ng mL−1 for ortho-hydroxyatorvastatin and para-hydroxyatorvastatin and 0.1–20 ng mL−1 for atorvastatin and atorvastatin lactone with excellent linearity (r 2 ≥ 0.99). This method demonstrated acceptable precision and accuracy at four quality control concentration levels. The detection limits were 0.1 and 0.13 ng mL−1 for orth-ohydroxyatorvastatin and para-hydroxyatorvastatin, respectively, and 0.05 ng mL−1 for atorvastatin and atorvastatin lactone. All analytes were found to be stable at examined conditions. Validated method was applied for determination of atorvastatin and its metabolites in plasma of experimental animals.

Open access