4.4. Statistical Analysis

Validation of the HPTLC-IS methodology was performed with regard to following parameters: linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). For this purpose, native α-LA was used as the standard compound and treated like the modification except for the addition of BITC for excluding any effect of treatment and reconditioning. Subsequently, a calibration series was prepared from the treated non-modified α-LA stock solution. The standard solutions were separated on silica gel and then detected by immunological reaction with antibodies.

Following the documentation of the plates, densitometric evaluation was performed at 650 nm using a TLC Scanner 4 (CAMAG AG, Muttenz, Switzerland). Densitograms recorded with the TLC scanner can be used for quantitative analysis. Linearity was validated using a 15-point calibration curve (n = 3) and was verified by means of Mandel’s fitting test. Homogeneity of variance was assigned regarding all values obtained [83,84].

Limit of detection (LOD) and limit of quantification (LOQ) were determined using the calibration method DIN 32645:2008-11 (n = 3) via the calibration curve (concentrations between 0.2–3 µg). Accuracy and precision were determined by analyzing the closeness of individual densitometric measurements of the band (same concentration of α-LA, c = 1.8 µg, n = 12).