Pda detector

HRESIMS analysis of the BME was done using a LTQ Orbitrap spectrometer coupled to an HPLC system (PDA detector, PDA autosampler, and pump, ThermoFisher Scientific, Inchinnan, Renfrew PA4 9R, UK). The following conditions were used: capillary voltage of 45 V, capillary temperature of 260 °C, auxiliary gas flow rate of 10−20 arbitrary units, sheath gas flow rate of 40−50 arbitrary units, spray voltage of 4.5 kV, and mass range of 100−2000 amu (maximal resolution of 30,000). For LC-HRESIMS, a Sunfire C₁₈ analytical HPLC column (5 μm, 4.6 mm × 150 mm) was used with a mobile phase of 0 to 100% MeOH over 20 min followed by 100% MeOH over 5 min at a flow rate of 1 mL min⁻¹.

All samples were analyzed on an Accela reversed phase ultra-high performance liquid chromatography (RP-UHPLC) system, equipped with a pump, degasser, autosampler, and photodiode array (PDA) detector (Thermo Scientific, San Jose, CA, United States). Samples (5 μL) were injected onto an Acquity UPLC BEH C18 column (150 × 2.1 mm, particle size 1.7 μm) (Waters, Milford, MA, United States). To ensure the stability of the released compounds, the temperature of the autosampler was kept at 4°C during the analysis. The flow rate was 400 μL/min at 45°C. The binary mobile phases consisted of (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. The elution profile was as follows: Isocratic on 5% B; 0.0–1.5 min, B linearly from 5 to 60%; 1.5–20.0 min, B linearly from 60 to 100% B; 20.0–20.1 min, isocratic on 100% B; 20.1–25.0 min, B linearly from 100 to 5% B; 25.0–26.0 min, isocratic on 5% B; 26.0–31.0 min. UV spectra for methyl p-coumarate and methyl ferulate were recorded at 310 and 320 nm, respectively. No MS data was acquired for the incubation of the model substrates with the FAEs. The decrease in substrate concentration representing activity was determined from a standard curve of the substrates (0.625–50 μg/mL). Data were processed using Xcalibur 2.2 (Thermo Fisher Scientific).

The derivative was isolated from a development batch of insulin lispro substance by repetitive ion exchange chromatography (IEC) using ICS-5000 system with a PDA detector (Dionex, Sunnyvale, USA). 0.1 mg of the insulin lispro containing 0.8% of the derivative was injected on a DNA Pac PA-100, 250 × 4.0 column (Dionex, Sunnyvale, USA). The separation was carried out at 35°C with a linear gradient elution from 0% to 25% eluent B in 30 min at the flow rate 0.5 ml/min. Eluent A was 8 mM phosphate buffer, 67% ethanol pH 7.6 and eluent B was 0.3 M NaCl, 8 mM phosphate buffer, 67% ethanol pH 7.6. The peak of the derivative (relative retention time 0.8; see Fig. 3) was collected. This procedure was repeated several times to obtain sufficient amount of the derivative. All fractions of the derivative were pooled and evaporated to dryness using Concentrator Plus vacuum centrifuge (Eppendorf, Hamburg, Germany) and kept at −20°C until use.

The high-performance liquid chromatography (HPLC) system (Dionex, Thermo Scientific) consisted of an ultimate 3000 model pump (SN 8031808), autosampler (SN 8031972), column oven (SN 8031817), PDA detector (SN 8031310), and Chromeleon software V.6.8 (Dionex, Germany). Weights were measured using Oahu’s balance (RI097) (Switzerland), and pH was detected by using Mettler-Toledo PH meter (TYPE: MP225) (ID: RI015) (Columbus, US). A multichannel stirrer model (MS -52M) (Jeio-Tech, Korea) was used to confirm dissolving, and a stability chamber for accelerated storage conditions at 40 ˚C and 75% RH (RI 091) (Memmert, Germany) was used to evaluate the quality of the product at these harsh conditions. A UV-spectrophotometer (Jasco V 730, Japan) was used to check the suitable wavelength at which SIP showed the maximum absorbance.

HPLC analysis was performed on a Surveyor HPLC system with a PDA detector (Thermo Finnigan, San Jose, CA, USA). The column was Gemini C18 (250 mm × 4.6 mm; Phenomenex, Torrance, CA, USA) operated at 30 °C. Phase A: water/acetonitrile/formic acid (80:10:7 v/v/v); phase B: water/acetonitrile/formic acid (40:50:7 v/v/v). Samples were eluted according to the gradient described by Wang et al. [35 ] and Veberic et al. [36 (link)] with some modifications. Briefly: 0 min, 0% B; 15 min, 30% B; 25 min, 50% B; 35 min, 0% B, with an injection amount of 10 μL and a flow rate of 1 mL·min⁻¹. The anthocyanins were identified by comparing their UV-VIS spectra from 200 to 600 nm to known standards and their retention times; they were detected and quantified at 535 nm.

Qualitative HPLC-PDA/UV-ESI-MS/MS analyses were performed using a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan) equipped with Xcalibur 3.1 software. Analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Fusion-RP column (Phenomenex). The eluent was a mixture of methanol (solvent A) and a 0.1% v/v aqueous solution of formic acid (solvent B). A linear gradient of increasing 55% to 85% A was developed within 45 min. The column was successively washed for 15 min with methanol and equilibrated with 55% A for 10 min. Elution was performed at a flow rate of 0.8 ml/min with a splitting system of 2:8 to MS detector (160 ml/min) and PDA detector (640 ml/min), respectively. The volume of the injected methanol solutions was 20 μl. Analyses were performed with an ESI interface in the positive mode. The ionization conditions were optimized and the parameters used were as follows: capillary temperature, 270 °C; capillary voltage, 29.0 V; tube lens offset, 50.0 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 3.00 arbitrary units; spray voltage, 4.50 kV; scan range of m/z 150–1200. N₂ was used as the sheath and auxiliary gas. PDA data were recorded with 200–600 nm range with preferential channel as the detection wavelength 260 nm.