Quantification of Four Isoflavones in Forages with UPLC®-MS/MS,Using the Box–Behnken Experimental Design to Optimize Sample Preparation

A performant method for the simultaneous quantification of daidzein, genistein, formononetin, and biochanin A in forages using an UPLC^®-MS/MS was developed and fully validated. The ultrasound-assisted extraction and enzymatic hydrolysis used in the sample preparation step were optimized using the Box–Behnken experimental design. The optimal extraction conditions used for a representative mix of forage plants were 80 °C, 10 min, and 55 % methanol, and for hydrolysis, they were 20 °C, 18 h, and pH = 6. The chromatographic separation was achieved using an Acquity UPLC^® HSS T3 column, with a water/methanol linear gradient containing 0.01 % of formic acid at a 0.55 mL min⁻¹ flow rate. The four isoflavones were detected by ESI mass spectrometry in positive ion MRM mode. The method allows high throughput analyses of samples and showed an adequate linear regression model for all isoflavones over a range from 5 to 125 ng mL⁻¹. There were good intra- and inter-day precisions (≤8.2 and ≤7.6 %) and accuracy (≤11.4 and ≤7.1 %). The recovery rates were in an acceptable range of 70–120 %, except for biochanin A, where the rate was about 50 %. Good method repeatability was also observed, and there was no matrix effect or carryover problem. The sample extracts were stable for at least 6 days of storage at -21 and 6 °C. The method proved to be sensitive, precise, and accurate for discriminating a wide variety of forages likely to be grazed by ruminants according to their isoflavone contents and to observe the impact of storage process on isoflavone content in forages.

     

Fast gas chromatography characterisation of purified semiochemicals from essential oils of Matricaria chamomilla L. (Asteraceae) and Nepeta cataria L. (Lamiaceae)

The chemical composition of Matricaria chamomilla L. and Nepeta cataria L. essential oils was determined by GC-MS on an apolar stationary phase by comparison of the characteristic fragmentation patterns with those of the Wiley 275L database. The GC-MS chromatograms were compared with those obtained by fast GC equipped with a direct resistively heated column (Ultra Fast Module 5% phenyl, 5 mx 0.1 mm, 0.1 microm film thickness). Analytical conditions were optimised to reach a good peak resolution (split ratio=1:100), with analysis time lower than 5 min versus 35-45 min required by conventional GC-MS. The fast chromatographic method was completely validated for the analysis of mono- and sesquiterpene compounds. Essential oils were then fractionated by column chromatography packed with silica gel. Three main fractions with high degree of purity in E-beta-farnesene were isolated from the oil of M. chamomilla. One fraction enriched in (Z,E)-nepetalactone and one enriched in beta-caryophyllene were obtained from the oil of N. cataria. These semiochemical compounds could act as attractants of aphid's predators and parasitoids.