1200 series liquid chromatograph

Bruker Esquire HCT mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) and controlled by ChemStation software (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany) was used.
Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C18 column at 30°C. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: 0–4.0 min (10-80% B), 4.0–8.0 min (80-80% B), 8.0–9.0 min (80-10% B), and 9.0–13.0 min (10-10% B). The flow rate was 0.4 mL/min.
The quantification was performed by the peak area method. The determination of target ions was performed in selective ion monitoring mode (m/z 240 for bupropion, m/z 268 for metoprolol, m/z 326 for midazolam, m/z 180 for phenacetin, m/z 198 for omeprazole, m/z 271 for tolbutamide, and m/z 237 for IS) and positive ion electrospray ionization interface. Drying gas flow was set to 7 L/min and temperature to 350°C. Nebuliser pressure and capillary voltage of the system were adjusted to 25 psi and 3,500V, respectively.

¹H NMR and ¹³C NMR analyses were performed at 25 °C on a Bruker AC 300 spectrometer at 300 MHz and 75 MHz, respectively, using chloroform-d as the solvent and tetramethylsilane as the internal standard. HPLC analyses were conducted by an Agilent Technologies 1200 series liquid chromatograph provided with a G1379B degasser, a G1312A pump, and a G1329A autosampler. Separations were attained by means of a Discovery C-18 column (3 μm particle size; 150 mm length; 4.6 mm i.d., Merck KGaA, Darmstadt, Germany). MS/MS spectra were recorded by using an API 4000 Q-Trap (AB Sciex, Framingham, MA, USA) mass spectrometer. Detailed instrumental conditions have been previously reported [24 (link)].

Carotenoids were analysed with a RP-HPLC technique according to the method described by Czaplicki et al. [23 ]. Briefly, the analysis was carried out using a 1200 series liquid chromatograph manufactured by Agilent Technologies (Palo Alto, CA, USA), equipped with a diode array detector (DAD) from the same manufacturer. Separation was performed at 30 °C on a YMC-C30 150 × 4.6 mm, 3 µm column (YMC-Europe GmbH, Dinslaken, Germany). The binary mobile phase consisted of methanol (solvent A) and methyl tert-butyl ether (MTBE) (solvent B). The solvent gradient was as follows: 0–5 min, 95% A, 1 mL/min; 25 min, 72% A, 1.25 mL/min; 33 min, 5% A, 1.25 mL/min; 40–60 min, 95% A, 1 mL/min.
The absorbance was measured at the wavelength of 450 nm. Compounds were identified based on retention times of commercially available standards (Sigma-Aldrich, Poznań, Poland).

Adrenal glands were placed in a cold Ca²⁺- and Mg²⁺-free Hank’s solution (Sigma–Aldrich, Spain) and transferred to 500 μL of 0.1 N perchloric acid before being sonicated (Sonics material vibracell, Dambury, CT, USA) and, subsequently, centrifuged at 3000 rpm for 10 min at 4 °C (tabletop centrifuge, Hettich, Germany). The supernatant was collected, centrifuged at 3000 rpm for 2 min at 4 °C (Eppendorf, Germany), and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A system consisting of a 1200 Series Liquid Chromatograph coupled to a 6410B Triple Quadrupole Mass Spectrometer (LC-MS; Agilent Technologies, Madrid, Spain) was used [57 (link)], and CAs were determined using a positive mode electrospray ionization; likewise, the pellet was analyzed for protein content.

Tocopherols content was carried out with HPLC according to the method described by Dimić et al. [4 (link)]. The sample in n-hexane solution (1%, m/v) was injected into the chromatographic system. The analysis was performed using a 1200 series liquid chromatograph manufactured by Agilent Technologies (Palo Alto, CA, USA), equipped with a fluorescence detector. The separation was done on a LiChrospher Si 60 (250 mm × 4 mm, 5 μm) column (Merck, Darmstadt, Germany). The fluorescence detector was set at 290 nm excitation wavelength and 330 nm emission wavelength. Peaks were identified separately based on retention times determined for α-, β-, and γ-tocopherol standards (Merck, Darmstadt, Germany). The amounts of tocopherols in the extracts were calculated as mg tocopherols in a 100 g oil sample.

The LC-MS system was composed by an Agilent 1200 series liquid chromatograph which consisted on a binary pump, a degasser, an autosampler, a column heater coupled to a single quadrupole mass spectrometer from Agilent 6140 (Agilent Technology, Santa Clara, CA, USA). Positive electrospray mode employed for all analytes and detection was performed with selected ion monitoring. The ChemStation software also from Agilent Technology controlled the LC-MS system and processed the data. The operating parameters were drying gas temperature (350 °C), drying gas flow (12 L/min), nebulizing gas pressure (35 psi) and capillary voltage 2000 V. Molecular masses of the precursor ions of all detection antibiotics was shown in Section 3.4. The separation of the antibacterial substances was performed on a Kintex octadecyl C₁₈ (100 × 2.6 mm, 5 μm) column protected by a RP₁₈ guard column (4.0 × 3.0 mm, 5 μm), both from Phenomenex, operated at 25 °C. The mobile phase consisted of 0.1% formic acid in Milli-Q water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient used was 0–1 min, 5% B; 1–15 min, 15% B; 15–26 min, 36% B; 26–29 min, 100% B; 29–30 min, 100% B; before returning to 5% B in 1 min, with a final hold at 5% B until 36 min. The flow rate was 0.4 mL/min and the injection volume was 15 μL.

Analysis of content of tocopherols was carried out with HPLC-FLD technique, according to the method described by Dąbrowski et al. [4 ]. The oil in n-hexane solution (1%, m/v) was injected into the chromatographic system. The analysis was performed using a 1200 series liquid chromatograph manufactured by Agilent Technologies (Palo Alto, CA, USA), equipped with a fluorescence detector. The separation was done on a LiChrospher Si 60 (250 mm × 4 mm, 5 µm) column (Merck, Darmstadt, Germany). A 0.7% isopropanol solution in n-hexane at a 1 mL/min flow rate was used as a mobile phase. The fluorescence detector was set at 296 nm for excitation and 330 nm for emission. Peaks were identified on the basis of retention times determined for α, β, γ and δ tocopherol standards (Merck, Darmstadt, Germany) separately, and their content was calculated using external calibration curves.