24 and 5.24, respectively; while the resolution factor was 6.88. The asymmetry of the peak for m-cresol and PTH were found to be 1.29 and 5.29, respectively; while the tailing factor parameter for m-cresol and PTH was found to be 1.29 and 1.14, respectively. For replicate injections of m-cresol standard; the % RSD of the main peak selleck chemical Palbociclib area was found to be below 0.7%, and there was no significant variation in the retention time (<0.1 min). The PTH and m-cresol peaks were thus found to be well resolved, and the tailing factor was within the limits. Available PTH formulation in the market contains 100 ��g/mL of m-cresol and the range was selected as 75�C120 ��g/mL. Detection limit (LOD) and quantification limit (LOQ) are not required for the study.
Different brands of reverse phase C18 columns were used (Jupiter column and Grace Vydac column) and compared in terms of percentage variation of principal peak area of m-cresol standard. Experiments were conducted using a system suitability standard. Percentage variation between principal peak of m-cresol was not more than 5% in all samples when compare to the area of principal peak of m-cresol from specificity samples. Retention time of the principal peak of m-cresol was found to be around 5.3 min and 3.9 min whereas the principal peak of PTH was found to be around 10.9 and 4.7 min on Jupiter and Grace Vydac columns, respectively. Relative retention rime with Grace Vydac and Jupiter columns of PTH with respect to m-cresol was found to be 1.2 and 2.1, respectively. The principal peak in both samples was separated by base-to-base while overlapping their chromatograms.
On the basis of relative retention time, a Jupiter column was selected for method validation. Method validation Specificity Specificity of the method for m-cresol in the presence of PTH, and excipients such as mannitol, sodium acetate, and glacial acetic acid was studied in terms of resolution of peaks observed and peak area of m-cresol. To verify this, PTH, formulation buffer, mobile phase, and m-cresol standard were injected onto HPLC separately. Three different concentrations of m-cresol (75, 100, and 120 ��g/mL) were prepared in the mobile phase as well as in the formulation buffer and were injected in triplicate onto HPLC. From Figures 2(a and b) it is evident that there is no interference and the method developed is specific for m-cresol.
Linearity and range The ICH guidelines for an assay method recommend that 80�C120% of stated value can be considered as a range of a Entinostat method. We decided to study linearity in the concentration range from 75 to 120 ��g/mL as the concentration of m-cresol in the drug product is 100 ��g/mL. m-Cresol standard (3 mg/mL) was used for preparation of different concentrations ranging from 75 to 120 ��g/mL, by considering 100 ��g/mL as 100%. Five different concentrations were considered with three replicates of each concentration (n = 15).