Reversed phase hplc

A ten-μL above aliquot was injected into a reversed-phase HPLC (Waters, Milford, MA, USA). The column was eluted by 30% ethanol and linearly increased to 100% at a flow rate of one mL/min. The peaks were detected at 360 nm. According to the peak time, each individual identifiable peak was collected via a detector. The collected fraction was dried and resolved in 20 μL of ethanol.

Fresh spores were cultured in PDB medium (1.0 × 10⁷ spores mL⁻¹) containing 0 or 0.25 mg L⁻¹ cinnamon oil for 6, 12, and 18 h at 25 °C under 200 rpm shaking condition. The spores and mycelia were collected by centrifugation, and the carbohydrate content of the supernatant was measured utilizing a portable refractometer (LB20T, Suwei, Taiwan, China). The total carbohydrate content of spores and mycelia was determined by a Total Carbohydrate Content Assay kit (BC2710, Solarbio, Beijing, China). The ATP content was measured by an ATP Content Assay Kit (BC0300, Solarbio, Beijing, China) in strict accordance with the product manual.
Fresh spores were cultured in PDB medium (1.0 × 10⁷ spores mL⁻¹) containing 0 or 0.25 mg L⁻¹ cinnamon oil for 2, 4, and 6 days at 25 °C under static condition. After centrifugation, the supernatant was filtered through a 0.22 μm syringe filter (NO. F513152, Sangon, Shanghai, China). Patulin content was evaluated by a reversed-phase HPLC (high-performance liquid chromatography) with UV detection (Waters, Milford, MA, USA). A Waters XTerra RP18 column was used with mobile phases (95% water and 5% acetonitrile). The operational parameters were descripted as Zhou et al. [33 (link)].

Briefly, the fluorescently labelled peptide substrate was chemically synthesized on Rink amide resin using fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) on an Activotec P11 synthesizer (Activotec, Cambridge, U.K.). The peptide TAMRA-(GRGA)₄ consists of four repeats of the arginine-containing tetrapeptide Gly-Arg-Gly-Ala and was N-terminally coupled to the fluorescent group 5(6)-carboxytetramethylrhodamine (TAMRA). After SPPS, the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) side chain protection group on the arginines was chemically removed in a mixture containing 88.9% (v/v) trifluoroacetic acid (TFA, Biosolve, Valkenswaard, The Netherlands), 4.4% (v/v) thioanisole (Arcos Organics, Geel, Belgium), 2.2% (v/v) 1,2-ethanedithiol (Merck, Darmstadt, Germany), and 66.7 mg/mL crystalline phenol (Merck). The peptide was precipitated in 90% (v/v) cold methyl tert-butyl ether and purified through reversed-phase HPLC (Waters Corporation, Milford, MS), on a 250x8.0-mm PepMap C18 column (VDS Optilab, Berlin, Germany). Elution was performed in an increasing acetonitrile gradient in 0.1% (v/v) TFA. Peptides were detected and their relative molecular mass (Mr) confirmed via electrospray ion trap mass spectrometry (AmaZon SL, Bruker, Bremen, Germany). The purified peptides were lyophilized (Speedvac) overnight and stored at -20°C until use.

For each sample, 100 mg leaf tissue was ground with liquid nitrogen into a powder and soaked in 2 ml 100% methanol. The homogenates were centrifuged at 10,000 × g for 10 min and the supernatant was filtered through a syringe with a 0.2 μm cellulose acetate membrane filter (Pall, USA). Then the filtrate was evaporated to dryness under vacuum and dissolved in 500 μl 50% methanol. The final sample was analyzed by reversed-phase HPLC (Waters, USA) so that the tryptophan and serotonin contents could be quantified. The samples were separated on an XTerra RP C18 column (250 × 4.6 mm, 5 μm, Waters) with an isocratic elution of 50% methanol in water containing 0.3% trifluoroacetic acid at a flow rate of 0.4 ml/min. A UV wavelength of 280 nm was used for detection. The standard samples for tryptophan and serotonin were made by Sigma (USA).

DOTA-PD-L1-i or HEHA-PD-L1-i (1 mg) was dissolved in 50 µL of ethanol, adjusting to a final volume of 1 mL with 1 M acetate buffer, pH 5.0 to 20 µL of the PD-L1-i conjugates, 80 µL of ¹⁷⁷LuCl₃ (370 MBq in 0.01M HCl) or ²²⁵AcCl₃ (370 kBq in 0.01M HCl) were added. Finally, the mixture was incubated at 95 °C for 60 min. After labeling, ²²⁵Ac-HEHA-PD-L1-I was purified using solid-phase extraction (Speak C18 cartridge, Waters). Radioactive solutions were diluted in injectable-grade water (1 mL) for further use. For comparative purposes, Lu-DOTA-PD-L1-i was also prepared under the same conditions using stable LuCl₃ (anhydrous powder; 99.99% trace metal basis; Sigma-Aldrich: Saint Louis, MO, USA) followed by HPLC purification.
The radiochemical purity of the radioconjugates was determined by reversed-phase HPLC (Waters) with radiometric and UV-vis detectors. The analysis was carried out with a Discovery C18 column (particle size: 5 µm, length: 25 cm, diameter: 4.6 mm). A linear gradient (rate flow: 1 mL/min) of CH₃CN-0.1% TFA (B)/H₂O-0.1%TFA (A), from 100% to 10% of A in 30 min, was used. For ²²⁵Ac-HEHA-PD-L1-i, fractions of 1.0 mL were collected, and the activity was evaluated in a NaI(Tl) detector (Auto In-v-tron 4010; NML Inc., Houston, TX, USA).