UPLC-MS/MS Conditions

Instrumental Parameters

An Acquity UPLC system comprising an autosampler and a binary pump (Waters, Milford, M A) equipped with a lOpL loop was interfaced with a hybrid linear ion trap triple-quadrupole mass spectrometer (API 4000 QTRAP™ MS/MS system; AB Sciex, Concord, ON, Canada) equipped with electrospray (Turbo V) ion source. A Waters Acquity ВЕН C18 (2.1 mmx50mm, 1.7 pm) column at 30°C was used for separation of the components. Mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), and the components were eluted at the rate of 0.3 mL/min using a gradient program—3% to 30% В from 0 to 3.5 min. 30% to 60% В from 3.5 to 5 min, 60% to 92% В from 5 to 7min, from 7 to 8 min, followed by a return to the initial condition from 8 to 10.5 min. Sample injection volume was 5 pL. The mass spectrometer conditions in negative ionization mode were ion spray voltage (IS) 4200, turbo spray temperature (ТЕМ) 550°C, nebulizer gas (GS 1), heater gas (GS 2) and curtain gas (CUR) at 20 psi, and in positive ionization mode were ion spray voltage (IS) 5500, turbo spray temperature (ТЕМ) 550°C,nebulizer gas (GS 1) 50 psi, heater gas (GS 2) at 50 psi and curtain gas (CUR) at 20 psi. The interface heater was on, nitrogen was used as the CAD and the scan type used was MRM.

Compound-dependent Parameters

The mass spectrometric conditions were optimized by infusing 50ng/mL solutions of the analytes dissolved in methanol at a flow rate of lOpL/min using a Harvard “22” syringe pump (Harvard Apparatus, South Natick, MA, USA). For the MRM quantitation, the highest abundance of precursor-to-product ions for each compound was chosen. The dwell times for both the parent and the internal standard were set at 200 ms. For full-scan ESI-MS analysis, the mass range scanned was from m/z. 100 to 1000. Analyst 1.5.1 software package (AB Sciex) was used for instrument control and data acquisition.

The contents of thirteen bioactive compounds in fruits and leaves of ten Piper species were used for PCA using the software STATISTICA 7.0. When the contents of investigated compounds were below' the quantitation limit or not detected in the samples, the values were considered zero.

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