The LOD and the LLOQ were determined as the lowest concentration steroid spiked into Schirmer strip calibrators, giving a minimum signal-to-noise ratio (S/N) >3:1 for LOD and >8:1 for LLOQ in replicate analyses (n = 10). The method linearity was evaluated using Schirmer strip calibrators, including two blank Schirmer strip calibrators (with and without addition of internal standard solution) over a period of 5 days. Linearity was defined as deviation from the assigned calibrator values of ≤10%, with the exception of the LLOQ/lowest calibrator for which deviation of ≤15% was accepted. Precision and accuracy for each steroid were evaluated intra- and inter-day using QC material described above. For intra-assay imprecision, a sequence of five replicates of QC in Schirmer strips were analysed in a single batch, and for inter-assay studies, each QC was measured in singleton in batches over a 5-day period . The intra- and inter-assay imprecision were expressed as a CV and deviation from assigned values. The analyte recovery indicates the efficiency of the extraction process and was evaluated following the strategy reported by Matuszewsky et al. . Thus recovery was assessed spanning the analytical range of the assay by analysis of the Schirmer strip QCs and conducted for each steroid. Recovery was calculated as the analyte peak area of the extracted QC in Schirmer strip as a percentage of the peak area of a blank Schirmer strip spiked to the equivalent concentration after the extraction process . Analyses were conducted in triplicate.