1220 infinity 2 lc

MYG (500 mg tablets contains 17.5 mg of guggulsterones) were purchased from AVN Ayurveda Formulations Pvt Ltd. Madurai, India. Midazolam (purity 98.0%), E & Z—guggulsterone (purity 98.0%) were also purchased from SIGMA-ALDRICH Company (St. Louis, MO). HPLC grade acetonitrile and Methanol were from Merck Company (Darmstadt, Germany). VIVID CYP450 Screening Kit and VIVID Substrates were purchased from Invitrogen Drug Discovery Solutions, USA. VIVID CYP3A4 Red (Cat. no. P2856). Ketoconazole was obtained as a gift sample from M/s Micro Labs Pvt. Ltd, Hosur, Tamil Nadu, India. All other chemicals were of analytical grade and used without further purification. All analysis was performed with liquid chromatographic system, (AGILENT technologies 1220 Infinity II LC using PDA detector). LC solution software is used to record the data. C18- 250 mm × 4.6 mm & 5 µm). other apparatus included Camag Linomat V (Switzerland) sample applicator, CAMAG automatic TLC sampler III, Remi compact laboratory high speed centrifuge, Electronic balance and PCI Stainless steel analytical ultra Sonicator bath.

Recombinant E. coli cell growth was monitored by measuring optical density at 600 nm (OD₆₀₀) using microplate reader (SpectraMax iD3, Molecular Devices, USA). GABA and glutamate concentrations in the culture broth were determined using a high-performance liquid chromatograph system (1220 Infinity II LC, Agilent Technologies Inc., USA) equipped with a UV detector and ZORBAX SB-C18 column (250 × 4.6 mm) (Agilent Technologies Inc.) as previously reported [19 (link)]. Acetate and glucose concentrations were measured by HPLC equipped with an Aminex HPX-76H column (300 × 7.8 mm) (Bio-Rad, USA) and RI detector as previously reported [22 (link)].

In brief, the method for preparing ECE and DK was as follows: EC was collected in April on Jeju Island, South Korea. First, EC was washed with running water to remove salt, sand, and epiphytes attached to the surface. Then, it was lyophilized and ground into a dry powder, which was extracted with 80% ethanol at room temperature for 24 h. The isolation of DK was performed according to a previously published method [20 (link)]. The BUCHI pure chromatography system (BUCHI, Pure C-850 FlashPrep, Flawil, Switzerland) was used for DK separation. Chromatography was performed on Agilent Technologies 1220 Infinity II LC with a column (poroshell 120 C18, 4.6*100 mm, 4µm). The mobile phase consisted of A; DW (+0.1% Formic acid), B; MeOH (+0.1% Formic acid) as followed: (0 min A; 63% B; 37%, 0–10 min A; 45% B; 55%, 10–12 min A; 63% B; 37%, 12–20 min A; 63% B; 37%). The gradient elution was performed as follows: the flow rate was 0.4 mL/min, and the injection volume was 1 mL. Detection was performed at UV length 230 nm. (Supplementary Materials, Figure S1 illustrates the HPLC chromatography analysis data for the isolated DK).

In this chromatographic system, PDA detector is used (AGILENT technologies 1220 Infinity II LC). LC solution software is used to record the data. C18-250 mm × 4.6 mm & 5 µm, using a mobile phase containing Acetonitrile: water (ACN) (70:30% v/v). A flow of 1 ml/min, injection volume of 10 µl, detection wavelength of 220 with the run time of 5mins and instrument was functioned at ambient temperature. The peak purity was checked with the photodiode array detector. Quantification of compound is determined by peak-area method. The quantification of midazolam & alpha hydroxy midazolam was performed using HPLC–MS techniques by the peak-area method. SIM mode is performed to determine the target ions (m/z 326 for midazolam, m/z342.50 for alpha hydroxy midazolam and m/z 237 for internal standards). The quantification was estimated with a signal-to-noise ratio of > 10, a precision of RSD < 20% and RE < 15% verified with five consecutive replicates. Limit of Detection (LOD) were 0.17 μg/ml⁻¹ for Midazolam and 0.12 μg/ml⁻¹ for alpha hydroxy midazolam, Limit of Quantification (LOQ) was 0.85 μg/ml⁻¹ for Midazolam and 0.6 μg/ml⁻¹ for alpha hydroxy midazolam. The linear range used was 5.0–1000 ng/mL in rat plasma for all analytes. The outcome of chromatographic validation showed that analytical methods were suitable for this analysis.