Ec10 2 nucleoshell c18 2.7 m column

The previously obtained crude extracts (see Section 2.3) were diluted in methanol to a final concentration of 1 mg/mL. Mass spectra were recorded on a micrOTOF-Q mass spectrometer (Bruker, Billerica, MA, USA) with ESI-source coupled with a HPLC Dionex Ultimate 3000 (Thermo Scientific, Darmstadt, Germany) using an EC10/2 Nucleoshell C18 2.7 µm column (Macherey-Nagel, Düren, Germany). The column temperature was 25 °C. MS data were acquired over a range from 100 to 3000 m/z in positive mode. Auto MS/MS fragmentation was achieved with rising collision energy (35–50 keV over a gradient from 500 to 2000 m/z) with a frequency of 4 Hz for all of the ions over a threshold of 100. HPLC begins with 90% H₂O containing 0.1% acetic acid. The gradient starts after 1 min to 100% acetonitrile (0.1% acetic acid) in 20 min. 5 µL of a 1 mg/mL sample solution was injected; and, flow rate was set to 0.3 mL/min.

The preservation alcohol of all eleven P. persiae specimens from Lavan Island, Iran was combined and subsequently analyzed for chemical composition (Group 1 or G1). The preservation alcohol of the single specimen of P. persiae from Bandar Lengeh, Iran was analyzed separately (G2), as was the one specimen of P. verruculata from Bangka Island, Indonesia (G3). The EtOH of each group was evaporated under vacuum conditions, the residue was re-dissolved in 100 µL methanol and analyzed by a micrOTOF-QIII mass spectrometer (Bruker) with ESI-source coupled with an HPLC Dionex Ultimate 3000 (Thermo Scientific) using an EC10/2 Nucleoshell C18 2.7 µm column (Macherey–Nagel). The column temperature was 25 °C. MS data were acquired over a range from 100–3,000 m/z in positive mode. Auto MS/MS fragmentation was achieved with rising collision energy (35–50 keV over a gradient from 500–2000 m/z) with a frequency of 4 Hz for all ions over a threshold of 100. HPLC begins with 90% H₂O containing 0.1% acetic acid. The gradient starts after 1 min to 100% acetonitrile (0.1% acetic acid) in 20 min. 5 µl of a 1 mg/ml sample solution (MeOH) was injected into a flow of 0.3 ml/min^{66 (link)}.

Mass spectra were recorded on a micrOTOF-QII mass spectrometer (Bruker) with ESI-source coupled with a HPLC Dionex Ultimate 3000 (Thermo Scientific) using an EC10/2 Nucleoshell C₁₈ 2.7 µm column (Macherey-Nagel) at 25 °C. MS data were acquired over a range from 100 to 3000 m/z in positive mode. Auto MS/MS fragmentation was achieved with rising collision energy (35–50 keV over a gradient from 500 to 2000 m/z) with a frequency of 4 Hz for all ions over a threshold of 100. HPLC (flow: 0.3 mL/min) conditions start with 90% A (H₂O + 0.1% AcOH) and 10% B (acetonitrile + 0.1% AcOH). A gradient over 20 min to 100% B starts after 1 min. 5 µl of a 1 mg/mL sample solution (MeOH) were injected.