Following the criteria described above limit of detection (LOD) and lower limit of quantification (LLOQ) were established to be: 0.30 and 0.50 ng/mL for CORT; 0.1 and 0.29 ng/mL for CCONE; 0.05 and 0.10 ng/mL for 11-DECOL; 0.05 and 0.08 ng/mL for ADIONE; 0.02 and 0.08 ng/mL for TESTO; 0.05 and 0.08 ng/mL for 17-OHP. The only exception was PROG, for which LOD and LLOQ coincided with the lowest concentration Schirmer strip calibrator (0.05 ng/mL). The correlation between steroid concentration and signal intensities, including two blank Schirmer strip calibrators, was linear: from 0.50 to 20.80 ng/mL for CORT; from 0.10 to 1.00 ng/mL for 11-DECOL; from 0.08 to 2.91 ng/mL for ADIONE; from 0.08 to 1.00 ng/mL for TESTO; from 0.08 to 4.20 ng/mL for 17-OHP; and from 0.05 to 4.20 ng/mL for PROG. For CCONE the linearity was observed from 0.29 to 4.03 ng/mL, but the method resulted to be not linear for this steroid. CCONE has the same nominal molecular mass as 11-DECOL, and even if the chromathographic conditions were able to chromatographically resolve CCONE and 11-DECOL (Figure 1), the signal for CCONE after LC-MS/MS analysis of Schirmer strip samples was found to be low, reflecting the typical low endogenous concentration of this steroid. Table 1 shows the investigated linear ranges, the calculated calibration functions, and the corresponding correlation coefficients (r2). The r2 for all analytes was >0.993 over their concentration ranges, and all calculated concentrations for the calibrators were within ±10% of the assigned values. The intra- and inter-assay coefficient of variation (CV) and deviation from assigned values was ≤15.2% and ≤13.9% for all analytes, respectively (Table 2) . The mean analytical recovery from the extraction at each Quality Control (QC) level for all steroids, without internal standard correction, was also summarized in Table 2. Recovery using analyte peak areas exclusive of internal standard normalization gives an indication of the efficiency of analyte extraction resulting from the chosen method of sample preparation. According to the principle of isotope dilution internal standardization, the addition of isotopically labelled internal standards for each analyte prior to the extraction should compensate for any variation in ionisation efficiency due to residual matrix effects and for losses in recovery of the extraction process .