Ltq xl linear ion trap instrument

The only source of AKH, the corpora cardiaca, were dissected from the heads of adult specimens of each of the 34 lepidopteran species under investigation with the aid of a stereomicroscope at 20 to 40-fold magnification. The neuroendocrine glands were placed into a microcentrifuge tube containing 80% v/v methanol, extracted as described previously (Gäde et al., 1984 (link)), and dried in a vacuum-centrifuge. Thereafter, extracts were dissolved in either distilled water for use in biological assays, or in 50 μl of aqueous formic acid for liquid chromatography tandem positive ion electrospray mass spectrometry (LC-ESI) on an LTQ XL linear ion trap instrument (Thermo Fisher Scientific, San Jose, CA, United States), as previously outlined in detail (Kodrík et al., 2010 (link)).
Exact mass and elemental composition were acquired by LC-ESI high-resolution mass spectrometry (HRMS) using the same Jupiter RP Proteo column and gradient elution, but a HRMS Orbitrap Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a HESI-II ion source. Positive ESI mass spectra were scanned every 2.1 s and were acquired at resolution R = 70,000 with an internal lock mass m/z 622.02896 of hexakis(2,2-difluoroethoxy)phosphazene using the mass range of 450–1250 Da.

LC–MS analysis was performed using a Finnigan Surveyor plus system (Thermo Scientific, San Jose, CA, USA), equipped with a quaternary MS pump with an integrated degasser and an autosampler with an integrated column oven. The chromatographic separation was performed on a Kinetex C18 column (5 µm, 50 mm × 4.6 mm ID) preceded by a C18 security guard cartridge (4.0 mm × 3.0 mm ID), both from Phenomenex (Torrance, CA, USA). Separation was attained using an isocratic elution with the flow rate of 0.5 ml/min. The column and tray temperature was set at 25°C and 10°C, respectively. The mobile phase consisted of 0.5% formic acid in water and methanol (45:55 v/v). The run time of the analysis was 3 min.
Mass spectrometry was performed on an LTQ XL linear ion trap instrument (Thermo Scientific, San Jose, CA, USA), coupled with heated electrospray ionization (HESI-II) probe operated in the positive ion mode. After optimization, the parameters in the source were set as follows: source voltage of 4.5 kV, source heater temperature of 250°C, sheath gas flow of 30 arb, and auxiliary gas flow of 10 arb. The mass spectrometer was operated in the selected reaction monitoring (SRM) channels. Fragment ion m/z 180 → 163 was used for memantine quantification, while m/z 152 → 135 was used for amantadine quantification. Thermo Fisher Xcalibur software was used for the analysis.