A simultaneous extraction method for metabolome and lipidome and its application in cry1Ac and sck-transgenic rice leaf treated with insecticide based on LC–MS analysis

Abiotic stress caused by insecticide treatment is an interesting and challenging topic in plant research. Here a simultaneous extraction method with methyl tert-butyl ether for metabolomics and lipidomics analysis by using rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. The extraction efficiency of lipidome and metabolome based on the current developed method showed an obvious improvement compared with literatures. About 300 metabolites were identified in rice leaf. Method validation was performed and analytical properties including the linearity (R², 0.991–1.000), repeatability (over 87 % peaks with CV < 20 % accounting for over 92 % of total response) and recovery (74.4–119.6 %) were satisfactory. The method was then applied to investigate time-course changes caused by insecticide treatment in transgenic rice with cry1Ac and sck genes and its wild counterpart. Antioxidants including ferulic acid and sinapic acid were down-regulated at 24 h after insecticide treatment. Signaling metabolites including salicylic acid and nicotinamide adenine dinucleotide were significantly up-regulated at 12–24 h after treatment in transgenic rice. More flavonoids were significantly changed in transgenic plants. Results indicated that gene transformation affected the metabolic response of rice to insecticide stress.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.     

A reappraisal of the Pleurotus eryngii complex – New species and taxonomic combinations based on the application of a polyphasic approach,and an identification key to Pleurotus taxa associated with Apiaceae plants

The Pleurotus eryngii species-complex comprises choice edible mushrooms growing on roots and lower stem residues of Apiaceae (umbellifers) plants. Material deriving from extensive sampling was studied by mating compatibility, morphological and ecological criteria, and through analysis of ITS1-5.8S-ITS2 and IGS1 rRNA sequences. Results revealed that P. eryngii sensu stricto forms a diverse and widely distributed aggregate composed of varieties elaeoselini, eryngii, ferulae, thapsiae, and tingitanus. Pleurotuseryngii subsp. tuoliensis comb. nov. is a phylogenetically sister group to the former growing only on various Ferula species in Asia. The existence of Pleurotusnebrodensis outside of Sicily (i.e., in Greece) is reported for the first time on the basis of molecular data, while P. nebrodensis subsp. fossulatus comb. nov. is a related Asiatic taxon associated with the same plant (Prangos ferulacea). Last, Pleurotusferulaginis sp. nov. grows on Ferulago campestris in northeast Italy, Slovenia and Hungary; it occupies a distinct phylogenetic position accompanied with significant differences in spore size and mating incompatibility versus other Pleurotus populations. Coevolution with umbellifers and host/substrate specificity seem to play key roles in speciation processes within this fungal group. An identification key to the nine Pleurotus taxa growing in association with Apiaceae plants is provided.     

High-performance liquid chromatographic determination of diadenosine 5′,5‴-p1,p4-tetraphosphate with precolumn fluorescence derivatization and its application to metabolism study in whole blood

Diadenosine 5′,5‴-p¹,p⁴-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N⁶-ethenoadenosine derivative within 60 min at 80°C. It was separated by reversed-phase HPLC and detected fluorimetrically (excitation and emission wavelengths of 275 and 410 nm, respectively). The detection limit of Ap4A was ca. 0.2 μg/ml in plasma when 10 μl of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 μg/ml) was examined using this method. Half-lives (means ± S.E., n = 3) were 0.88 ± 0.30 min (in rat blood), 13.7 ± 3.6 min (in dog blood and 17.2 ± 1.4 min (in human blood). A marked species difference in the degradation rate of Ap4A in blood was observed.     

Mechanistic Approach to Stability Studies as a Tool for the Optimization and Development of New Products Based on L. rhamnosus Lcr35? in Compliance with Current Regulations

Probiotics are of great current interest in the pharmaceutical industry because of their multiple effects on human health. To beneficially affect the host, an adequate dosage of the probiotic bacteria in the product must be guaranteed from the time of manufacturing to expiration date. Stability test guidelines as laid down by the ICH-Q1A stipulate a minimum testing period of 12 months. The challenge for producers is to reduce this time. In this paper, a mechanistic approach using the Arrhenius model is proposed to predict stability. Applied for the first time to laboratory and industrial probiotic powders, the model was able to provide a reliable mathematical representation of the effects of temperature on bacterial death (R²>0.9). The destruction rate (k) was determined according to the manufacturing process, strain and storage conditions. The marketed product demonstrated a better stability (k = 0.08 months⁻¹) than the laboratory sample (k = 0.80 months⁻¹). With industrial batches, k obtained at 6 months of studies was comparable to that obtained at 12 months, evidence of the model’s robustness. In addition, predicted values at 12 months were greatly similar (±30%) to those obtained by real-time assessing the model’s reliability. This method could be an interesting approach to predict the probiotic stability and could reduce to 6 months the length of stability studies as against 12 (ICH guideline) or 24 months (expiration date).