Robustness of an analytical chromatographic method for separation of cystatin c has been veri fi ed. Changes in many parameters were carried out, such as, wavelength, column oven, mobile phase composition, chromatographic column.

Imperative changes have altered the ef fi ciency of the chromatographic separation; such changes include pH alteration of the mobile phase as well as alkyl sulfonate molarity changing.

All robustness conditions showed no major effect on the chromatographic separation of the analyte except with the changes related to TFA and alkyl sulfonate ion pair reagents. Peak area RSD, asymmetry and No. of theoretical plates were < 0.7%, < 1.2 and > 10,000, respectively. Results obtained using mobile phase after 6 months of storage have proven its stability and possibility of use. Gradient elution mode was utilized to elute cystatin c with a UV detection of 224 nm. Ace and Waters C8 (150 x 4.6 mm i.d., 5 μ m) as chromatographic columns were used.