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the retention times and peak places . A recovery study was performed to validate the accuracy in the developed method. Hence, root samples were spiked with normal stock solutions in the analytes in triplicate at two distinct concentrations. The spiked root samples were extracted with 100 mL ethanol following the procedure Dub inhibitor for sample preparation as described in section 2.3. Finally, the spiked samples were analyzed utilizing the identical experimental and instrumental circumstances as previously described for Dub inhibitor the analysis in the un spiked roots. The recovery was determined by comparing the amount of analyte added towards the root sample as well as the amount of analyte detected in the course of HPLC analysis . 3. Final results and discussion 3.1. HPLC Optimization A number of preliminary studies were conducted to develop an HPLC method for the separation of compounds 1 6 in the C.
alata root extract. The LC separation circumstances in the analytes were optimized by systematically adjusting the methanol and acetonitrile content in the mobile phase using the addition of distinct buffers, such Dasatinib as trifluoroacetic acid, formic acid, and ammonium acetate to obtain superior resolution in the phenolic compounds. The retention times in the analytes decreased with an increase in the amount of methanol in the eluent. This observation was in agreement with a prior report by Ding et al An increase in the amount of acetonitrile in the eluent also resulted inside a decrease in retention time of compounds 1 6. The addition of 10 mM NH4Ac buffer towards the mobile phase resulted in the finest peak resolution of compounds 1 6.
Addition of NH4Ac buffer towards the mobile phase not only improved the resolution, but also resulted in total deprotonation of compounds 1 6 ?. The pH in the mobile phase was also optimized to obtain superior resolution of compounds NSCLC 1 6. Separation at pH 4.8 utilizing NH4Ac buffer resulted in co elution of rhein and kaempferol . Thus, resolution of only compounds 3 6 could be obtained. At pH 8.8 compounds 1 3 co eluted. Full separation of compounds 1 6 were only achieved at pH 6.8 utilizing NH4Ac buffer. The flow rate in the eluent was also optimized at 0.4 mL min for finest resolution and MS detection. The use of flow rates greater than 0.4 mL min resulted in overloading in the mass spectrometer detector. Optimal separation in the analytes was obtained within 45 minutes for normal mixtures too as the C.
alata root extract by use of an isocratic mobile phase of ACN MeOH NH4Ac buffer at pH 6.8 . We optimized the retention times in the analytes utilizing a gradient elution system containing ACN MeOH NH4Ac buffer at pH 6.8 for solvent A Dasatinib and ACN MeOH NH4Ac buffer at pH 6.8 for solvent B, allowing productive separation of all analytes within 30.0 minutes. Nevertheless, we did not use the gradient elution system for quantification in the analytes because it was not reproducible. The phenolic compounds 1 6 were identified in the C. alata root extract by spiking the extracts using the respective standards. Prior to this procedure, all standards were run separately to determine the retention time of each and every analyte. The chromatographic separation of compounds 1 6 is shown in Figure 2A for the normal mixture at 30 ppm as an example, and in Figure 2B for the root extract .
Along with the analyte peaks obtained in Figure 2B, an unidentified first eluting peak was also observed. We've isolated this unknown utilizing flash column chromatography, followed Deubiquitinase inhibitor by purification utilizing preparative HPLC. Nevertheless, soon after performing spectroscopic studies , we concluded that this unknown peak is an impurity composed of a mixture of compounds. No further analysis of this peak was attempted. 3.2. LC MS analysis Simultaneous separation and identification of phenolic compounds 1 6 in the C. alata root extracts were performed by use of LC APCI MS detection. Identification in the peaks was achieved by comparison in the retention times, UV spectra, too as MS data in the separated compounds Dasatinib using the respective standards.
The total ion chromatograms of analytes 1 6 in the normal mixture and root extract were recorded in the scan mode, and are shown in Figure 3A and B, respectively. As noticed in Figure 3B, Dasatinib the peak intensities for aloe emodin and physcion are very low, as a result of their low concentration in the root extract as determined by HPLC in this study. The mass spectra in the phenolic compounds 1 6 in the root extract are presented in Figure 4. The presence of each and every analyte in the root extract was confirmed by its respective ? m z. Along with the ions at ? of compounds 1 6, the ion at m z 239 was registered in the mass spectrum of rhein and aloe emodin as a result of fragmentation of molecular ions in the analyte resulting in ? and ?, respectively. The ions at m z 253 and 271 were also recorded in the mass spectrum of rhein which are assumed to be a fragment derived from the molecular ion resulting in ? and an adduct formation between the ion at m z 239 and methanol , respectively. The ion at