Materials and methods
The following beta-blockers used in this study were pharmaceutical formulations purchased in a local pharmacy: ATE (50 mg ATE, CAS 29122-68-7, 1A Pharma GmbH, Vienna, Austria), BIS (1.25 mg BISfumarate, CAS 104344-23-2, Sandoz GmbH, Kundl, Austria), MET (47.5 mg MET succinate, CAS 98418-47-4, Hexal Pharma GmbH, Vienna, Austria), and PRO (10 mg PRO-hydrochloride, CAS 13071-11-9, AstraZeneca GmbH, Vienna, Austria). Their physico-chemical properties can be seen in Table 1. Concentrations of the beta-blockers given within this manuscript refer to the substance, not to the salt. Diclofenac sodium salt (CAS 15307-79-6) used as internal standard (ISTD) was purchased from Sigma-Aldrich (Steinheim, Germany). The cress seeds “smooth cress, broad-leaf” (Kiepenkerl, Everswinkel, Germany) were purchased in a local garden shop.
Ultrapure water (18 MΩ cm) was obtained by a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany). Acetonitrile (ACN, CAS 75-05-08, HPLC grade) and methanol (MeOH, CAS 67-56-1, HPLC grade) were supplied by VWR Chemicals (Vienna, Austria). Formic acid (FA, CAS 64-18-6, 96%) was from Sigma-Aldrich (Steinheim, Germany) and hydrochloric acid (HCl, CAS 7647-01-0, 37%) from Fisher Chemical (Vienna, Austria).
A total of 1000 mg L−1 stock solutions were prepared individually in MeOH. Each solution was further diluted for irrigation in tap water to a final concentration of 1 mg L−1. The concentration of 1 mg L−1 was selected as it was low enough to avoid interferences with plant growth on the one side and high enough to facilitate accurate quantitative analysis on the other side.
A 1260 Infinity II modular HPLC system (Agilent Technologies, Santa Clara, USA) was used with an Agilent InfinityLab Poroshell 120 Bonus-RP column (3.0 × 100 mm, 2.7 μm) combined with a C18 Guard column (4 × 3.0 mm, Phenomenex).
For quantification, an Agilent Technologies 6460 Triple Quadrupole mass spectrometer (QQQ-MS) equipped with a Dual Agilent Jet Stream Electrospray Ionization (Dual AJS-ESI) source was used as a detector. The Dual AJS-ESI was operated in positive mode. Nitrogen was used as drying gas and sheath gas. The temperature of drying gas and sheath gas was 275 °C, both with a flow rate of 11 L min−1. The nebulizer gas pressure was 45 psi, the capillary voltage was 4000 V, and the nozzle voltage was 1000 V. The fragmentor voltage was set for each compound individually as listed in Table 2 together with the precursor, quantifying, and qualifying ions as well as their respective collision energies.
For tentative identification of possible metabolites, an Agilent Technologies 6560 DTIM QTOF-MS equipped with a Dual AJS-ESI source and a gas kit (Alternate Gas Kit, Agilent Technologies) was used as a detector, which was operated in positive mode. Nitrogen was employed as drying gas and sheath gas (both at a flow rate of 10 L min−1); the gas temperatures were maintained at 275 °C. The fragmentor voltage was 400 V, the capillary voltage 3500 V, the nozzle voltage 1000 V, and the nebulizer gas pressure set to 50 psi.
The mobile phase consisted of ultrapure water with 0.1% formic acid (v/v) (A) and acetonitrile with 0.1% formic acid (v/v) (B). The gradient elution was employed as follows: 0% B from 0 until 0.2 min, rising to 90% B within 5 min, hold 90% B for 2 min, and re-conditioning with 0% B for 5 min.
For the quantitative analysis, a matrix-matched calibration with the addition of an internal standard (ISTD) was done automatically by the autosampler of the HPLC system. Therefore, the following autosampler procedure was employed: needle wash (2 s); draw 10 μL of matrix (untreated cress root extract or untreated cress leaf extract); needle wash (2 s); draw 1, 5, or 10 μL of sum standard (0.1, 1, 10 mg L−1); needle wash (2 s); draw 2 μL of ISTD (1 mg DCF L−1); mix in the air (5 times); and inject.
For the quantitative analysis of the plant extracts used for the time study, the ISTD was added automatically by the autosampler of the HPLC system. The following autosampler procedure was employed: needle wash (2 s), draw 10 μL of plant extract (treated cress root extract or treated cress leaf extract), needle wash (2 s), draw 2 μL of ISTD (1 mg DCF L−1), mix in the air (5 times), and inject.
The matrix-matched calibration was indispensable since a significant difference in signal intensities was observed for the two different plant matrices (root/leaf). Thereby, a reduced slope was obtained for the calibration curve related to the leaf matrix.
Germination and growing of cress
The cress seeds were germinated for 7 days in tap water on a propagator tray. Then, the watering solution was exchanged with drug-containing water. The cress plants were grown for another 8 days whereby water was added on a daily basis to compensate for evaporation. Cress plants were always grown as triplicates for each beta-blocker.
Harvesting, extraction, and analysis of cress
The propagator tray was divided into 4 sections and the plant parts were harvested after day 1, day 2, day 4, and day 8. In the process of harvesting, the roots and leafs of the cress plants were separated. The plant parts were washed twice with ultrapure water, blotted dry, and about 1.5 g roots or leafs (wet weight) were weighed in. An example for this procedure can be seen in Fig. 1.
A total of 3 mL 0.1 M HCl was added to the plant material and the mixture was homogenized with an UltraTurrax (Type TP18/10, Janke&Kunkel IKA-Labortechnik, Staufen, Germany). The slurry was centrifuged at 4000 rpm for 8 min and the supernatant was filtered with 0.45 μm Rotilabo® nylon syringe filters (Roth, Karlsruhe, Germany) into 1.5-mL HPLC glass vials (VWR, Vienna, Austria) and the samples were stored at − 80 °C until analysis.
Data recording and evaluation
For data, recordings of Agilent MassHunter Workstation LC/MS Data Acquisition for 6400 Series Triple Quadrupole (Version 10.0 SR1) and Agilent MassHunter Optimizer (Version 10.0 SR1) were used. Quantitative analysis was done using Agilent MassHunter Quantitative Analysis for QQQ (Version 10.1).
For qualitative data evaluation, Agilent MassHunter Qualitative Analysis B.07.00 was used.