Spd m20a pda

The HPLC system (Shimadzu Company, Japan) was used to assess drug concentration in the prepared sample, which included a thermostated CTO-20AC column oven and SIL20AC autosampler, as well as an SPD-M 20A PDA detector. Shimadzu Corporation’s Lab solution programme (version 5.53 SP3C) was used for data collection and processing. An Agilent C18 column (4.6 mm 250 mm, 5 m; Thermo ScientificTM Hypersil GOLDTM, MA, USA) was used for chromatographic separation. The injection volume was set at 20 μL. The 90:10 (v/v) methanol and water mobile phase for the chromatographic separation was filtered before being utilised isocratically at room temperature at a flow rate of 1 mL/min, and SB was detected at 288 nm [36 ].

The fractionation of venom components was performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using an LC-20A Prominence HPLC system (Shimadzu Co., Kyoto, Japan) with a C18 column (Luna C18(2), 250 × 4.6 mm, 5 μm, 100 Å). Separation was achieved by using a linear gradient (0–100% of B) for 30 min, after 5 min isocratic elution (0% B), at a 1 mL/min constant flow. The employed buffers were A (0.1% acetic acid) and B (0.1% acetic acid in 90% acetonitrile). Eluates were monitored by a Shimadzu detector SPD-M20A PDA; the wavelengths 280 nm—used to detect tryptophan and tyrosine in the sample—and 214 nm, which is absorbed by the peptide bond, were used so that peptides lacking trp and/or tyr were not missed. Fractions were manually collected every 5 min and then lyophilized for later use. Optimized gradients were developed for further separation of active fractions: for F3, a gradient of 5–40% of B in 40 min was used, and for F3.7 and F3.8, a 5–60% gradient of B in 35 min was used.

The aqueous extract of PV was quantitatively analyzed by HPLC. The HPLC system (Shimadzu, Kyoto, Japan) with a CBM-20A controller, LC-20AD pump, SPD-M20A PDA detector, and a SIL-20A autosampler was used for the analysis of PV. Data collection was performed using Shimadzu Lab Solution. All chromatographic separations were performed on a CAPCELL PAK C₁₈ UG120 column (250 mm × 4.6 mm, 5 μm, Shiseido, Tokyo, Japan) with 0.1% acetic acid/acetonitrile gradient system at ambient temperature with detection at 254 nm. The amount of rosmarinic acid and caffeic acid in the water extract of PV were determined.

Samples were run on an Agilent Eclipse XDB 5 µm C₁₈ (250 × 4·6 mm) analytical column at a flow rate of 1 ml/min for 20 min at 23·4°C with an autosampler (Shimadzu SIL) on an HPLC containing a LC20AB pump (Shimadzu), and a Shimadzu SPD-M20A PDA. Mobile phase consisted of 47:47:6 methanol, acetonitrile and chloroform. Samples were analysed against an external standard curve prepared using retinyl acetate (US Pharmacopeia); standards were prepared in duplicate daily from stock solutions after analysis on a spectrophotometer at 325 nm to quantify absorbance. Concentration was calculated using a molar extinction coefficient of 0·155 for retinyl acetate in ethanol⁽³¹ (link)⁾.

Accurate LC-MS data of cyanobacterial extracts were recorded with a Waters Acquity I-Class UPLC system and a Waters Synapt G2 HDMS mass spectrometer. High-resolution electrospray ionization-mass spectrometry (HRESI-MS) data for synthetic compounds and cyanobacterial extracts were obtained by direct infusion of methanolic solutions on a Waters Synapt HDMS QTOF mass spectrometer (Waters Corporation, Milford, MA). HPLC analyses for synthetic intermediates were performed using a Shimadzu LC-20-AT Series separations module equipped with Shimadzu SPD-M20A PDA (photo diode array) multiple wavelength detectors (180 nm-800 nm). For indole-isonitrile compounds, UV detector was set at 310 nm with a 5 nm slit-width. The overall system, CBM-20 was controlled using LC Solutions software. Raw data was plotted using Origin® software program after exporting absorbance data as an ASCII-formatted file. Analytical separations of stereoisomers (of cis and trans) mixtures were carried out on Daicel® (normal phase) AS chiral column. A 10% isopropanol/ 90% hexanes mixture was used as elution medium with a flow rate of 1 mL/min in an isocratic mode. Individual retention times for indole-isonitriles are reported along with analytical data for each isomer.