Enzymatic Hydrolysis  Assess the efficiency of the enzymatic hydrolysis using β-glucuronidase from E. coli  Derivatization  Assess the efficiency of the trimethylsilyl (TMS) derivatization  Limits of Quantification  (LOQ ) The LOQ  shall be determined during method validation as the lowest concentration that can be measured with an uc (%) not greater than (≤) 30% and shall meet the following criteria:  • T, E ≤ 1 ng/mL;  • 5αAdiol, 5β Adiol ≤ 10 ng/mL;  • A, Etio ≤ 500 ng/mL  Measurement Uncertainty , uc (%) Level  A Etio T E Adiols  (5α-, 5β-) T/E The estimated uc (%) shall be not greater than (≤) the uc_Max  (%) value given below  at LOQ  ≤ 30%   at 5 x LOQ ≤ 20%  ≤ 25%   (T, E) > 5 ng/mL   ≤ 15%  (T, E) ≤ 5 ng/mL   ≤ 30%  2.1.2 ITP Analysis Requirements  Sample  The ITP for the quantification of the Markers  of the steroid profile shall be conducted on a single Aliquot .