The mobile phase consisted of a water/ACN/99 % acetic acid mixture (64/35/1, v/v/v).

Chromatographic separation was done at a 0.7 mL/min flow rate, with detection conducted at a 288 nm wavelength. The injected volume was 20 μL. The analysis took 10 min and etoposide retention time was 6.4 min (Fig. 2). Chromatographic analysis makes it possible to identify one or more compounds characterized by a chromatographic peak and its retention time. The area under the peak represents the concentration of each compound. Thus, component concentration in solution was monitored by comparing peak areas against a calibration plot. Fig. 2 Chromatograms of a 400-mg/L etoposide solution and a NaCl mTOR inhibitor 0.9 % blank 2.2.2 Validation of the Analytical Method Validation is essential to demonstrate that the method is adapted to its use. Validation was conducted by evaluating common parameters defined by the International Conference on Harmonization (ICH) [7] such as specificity,

response function, linearity, accuracy, precision (repeatability and intermediate precision) and limits of detection (LOD) and quantification (LOQ). The parameters were determined by Compound C the statistical analysis of six calibration plots. 2.2.2.1 Specificity Specificity was investigated by comparing the chromatogram of a blank sample with the chromatogram of the solution under study. HPLC is a selective method that separates different components on a column. The specificity of the method was assessed by analysing an etoposide solution. Figure 2 shows the chromatogram resulting from the injection of a 400 mg/L etoposide solution and of a blank sample of NaCl 0.9 %. 2.2.2.2 Linearity The calibration range was constructed based on 11 calibration standards (25, 50, 100, 150, 200, 250, 500, 750, 1,000, 1,250 and 1,500 mg/L). Linearity was investigated for six calibration plots recorded on six different days (one plot a day). The average equation parameters for the six linear regressions were: $$ y = 3,787, 945 x + 29,207 \, (r^ 2 = 0.999). $$ A statistic comparison

of the calibration curves DOK2 was conducted through normalised analysis of variance. Variances were found homogenous by a Bartlett–Selleckchem CRT0066101 Levine test (p < 0.00001). 2.2.2.3 Accuracy Accuracy expresses the closeness of agreement between the values accepted as conventionally true (referred to as the standard) and an estimated value (called the medium) obtained by applying the analysis technique a number of times. As shown in Table 1, accuracy values expressed by the theoretical value were below 5 % except for the lowest quality control established at 6.7 %. Table 1 Fidelity and accuracy data of the analytical method Quality controls (mg/L) 35 180 220 900 1,100 1,350 Repeatability              CVr (%) 2.8 0.5 4.8 4.9 1.2 0.9  Bias (%) 6.7 4.5 6 4.4 0.5 0.2 Intermediate fidelity              CVi (%) 2.2 0.3 1.6 2.6 1.7 1.6  Bias (%) 2.3 2.1 0.1 0.6 −2 −2.1 2.2.2.