Symmetry shield rp18

The equipment used for ponazuril assessment consisted of a 2695 separation module and a 2487 ultraviolet detector (Waters Milford, MA, USA). Separation was achieved on a Waters Symmetry Shield RP₁₈ (4.6 mm × 150 mm, 5 µm) column. The mobile phase consisted of 0.1% formic acid in water and acetonitrile (50:50, v/v). The ultraviolet detector was set at 254 nm and the flow rate was 1.1 mL/min.

Standard stock solutions of hydroxysafflor Yellow A (Chengdu Must Bio-Technology CO., Ltd, Chengdu, Sichuan, China) were dissolved in 25% methanol at a concentration of 100.0 μg/mL, allantoin (Chengdu Must Bio-Technology CO.) was dissolved in 100% methanol at a concentration of 200.0 μg/mL. All of them were stored at 4℃. The standard working solutions were prepared by serial dilution of the stock solutions with 25% methanol or 100% methanol.
Chromatographic analysis was performed for simultaneous determination of the PAMs ethanol extracts using an Agilent HPLC 1260 system (Agilent, Palo Alto, CA, USA). All analytes were separated on a Waters Symmetry Shield RP18 (250mm ×4.6mm, 5μm) and maintained at 30℃. As shown in Table 1, the isocratic flow was used for the analysis of each component.

We selected the second instar at day 0 between wild type Dazao and al^c mutant strains for amino acid and catecholamine content analysis. The free amino acids were extracted as follows: the samples were homogenized with 800 µL 0.1 M hydrochloric acid (HCl) in centrifuge tubes, then the homogenate was mixed for 15 min using ultrasonication followed by centrifugation (12,000× g, 4 °C). The supernatant (600 µL) was transferred to a new tube containing 600 µL of 10% sulfosalicylic acid, followed by further centrifugation (12,000× g, 4 °C for 15 min). The supernatants were transferred and filtered with 0.22-µm membranes. A Hitachi (Tokyo, Japan) L-8800 amino acid analyzer physiological fluid system (lithium system) was used for amino acid content analysis. DOPA and dopamine were extracted and quantified according to Koch’s method [21 (link)]. Agilent1260 Infinity HPLC (Santa Clara, CA, USA) and Symmetry Shield RP18 (5 μM, 4.6 × 250 mm, Waters Corp., Milford, MA, USA) columns were used for HPLC analysis. Compared to known standards, amino acids and catecholamine standards were identified based on retention times, as follows: Phe 56.33 min; Tyr, 53.35 min; DOPA 5.858 min; and Dopamine 8.226 min (Supplementary Figure S2A,B).

Quality control comprised radioanalytical high-performance liquid chromatography (HPLC) and instant thin-layer chromatography (ITLC). HPLC analyses were performed on a Waters chromatograph coupled to a 996 photodiode array UV detector (Waters, Vienna, Austria) and a Gabi gamma detector (Raytest RSM Analytische Instrumente GmbH, Straubenhardt, Germany). Data processing and chromatography were controlled with the Empower Software (Waters, Milford, MA, USA). For analyses, a Symmetry Shield RP-18 (5 μm, 4.6 mm × 150 mm) cartridge column (Waters, Eschborn, Germany) was eluted at a 1 mL/min flow rate with a linear gradient system 1 starting from 0% B and advancing to 40% B within 20 min (solvent A = 0.1% aqueous TFA and B = MeCN). ITLC analyses were performed on Whatman 3 mm chromatography paper strips (GE Healthcare, Chicago, IL, USA), developed up to 10 cm from the origin with 5 M ammonium acetate/MeOH 1/1 (v/v) for the detection of reduced hydrolyzed technetium ([^99mTc]TcO₂ × nH₂O), or acetone for the detection of [^99mTc]TcO₄⁻.
All manipulations with beta- and gamma-emitting radionuclides and their solutions were performed by trained and authorized personnel behind suitable shielding in licensed laboratories, in compliance with European radiation safety guidelines and supervised by the Greek Atomic Energy Commission (license #A/435/17092/2019)