Ultra fast liquid chromatograph

The concentration of candesartan in plasma was evaluated. Standard curves were established in corresponding biological matrix using olmesartan as an internal standard. The standard curve range was 1–1,000 ng/mL. Plasma samples were added to a microcentrifuge tube along with cold acetonitrile containing internal standard. The samples were vortexed, centrifuged and the supernatant was transferred to new tubes for evaporation under nitrogen gas. Once dried down, the samples were reconstituted, vortexed, centrifuged and transferred to LC vial for analysis. Candesartan and olmesartan were monitored using a Shimadzu Ultra-Fast Liquid Chromatograph with Shimadzu 8040 Triple Quadrupole Mass Spectrometer (UFLC-MS/MS). The separation was achieved by using 10% methanol in water with 0.1% formic acid and acetonitrile with 0.1% formic acid as the mobile phases and a Phenomenex Kinetex C18 column (50 × 3 mm, 2.6 μm particles) with the appropriate guard column. The triple quadrupole source was positive electrospray ionization using multiple reaction monitoring to monitor both candesartan and olmesartan. The quantitation analysis was performed using Shimadzu’s LabSolutions software, with weighting of the calibration curve model being 1/x².

Incubation was done in refrigerated shaker incubator of model Innova 4230, New Brunswick Scientific Co., Inc., (NJ, USA). Sample analysis was done by using a chiral column purchased from Phenomenex (USA) (Lux Cellulose-4; 250 × 4.6 mm; 5 µ particle size) and an Ultra Fast Liquid Chromatograph of Shimadzu (Kyoto, Japan) equipped with binary pump (LC 20AD), UV/Visible detector (LC 20A) and Rheodyne injector port. Lab solutions software was employed for the HPLC analysis.
Racemic Carvedilol was kindly gifted by Symed labs limited, Medak (India). (S)-(−)-Carvedilol and (R)-(+)-Carvedilol were purchased from the Toronto Pharmaceuticals (Canada). Calcium chloride, sodium alginate, sodium chloride and di-potassium hydrogen phosphate were purchased from Hi Media Laboratories Pvt. Ltd., Mumbai (India). Solvents (n-heptane, isopropanol and methanol) of HPLC grade were purchased from Sigma Aldrich Chemicals Pvt. Ltd., Maharashtra (India). Laboratory grade ethyl acetate was purchased from Finar limited, Ahmadabad, Gujarat (India).

To confirm differential abundance of the specific candidate proteins, purified samples (7 μg total protein) from each subject were digested, and the digestion solution (0.4 μg total protein) was eluted using a linear gradient of 5–65% acetonitrile containing 0.1% formic acid at a flow rate of 0.2 mL/min for 15 minutes on an Ultra-Fast Liquid Chromatograph (Shimadzu, Kyoto, Japan) that was equipped with a C18 column (Jupiter C18, 5 μm, 300 Å, 2.0 × 150 mm; Phenomenex, Torrance, CA, USA). The peptides of each subject’s sample were analyzed repeatedly in MRM mode using QTRAP 5500 (AB SCIEX, Foster City, CA, USA). Only peptide peaks shown at the same retention time in a minimum of 3 MRM chromatograms of the same peptide were deemed as target peptide peaks originating from the protein [20 (link)]. Based on the difference in peptide abundance between the 2 groups, candidate proteins whose retention times were the same among multi-transition chromatograms and transitions with peak height greater than 1000 cps were selected. Three transitions for each target peptide of the candidate proteins were selected based on the formation of higher intensity product ions.