Spectral peak purity evaluations are often used to control inadequate selectivity issues appearing in liquid chromatography separations coupled with UV-Vis photodiode detection, despite known limitations related to (a) the lack of characteristic absorption bands in UV-Vis for target analytes and overlapping related impurities; (b) increased similarity between the spectral characteristics of co-eluting compounds; (c) large concentration differences between overlapping compounds; (d) perfect co-elution cases. We propose an alternative protocol to evaluate differences between spectra collected over a peak’s elution interval. After normalizing the acquired spectra, linear regression was applied between each pair of spectra, resulting in slope, intercepts, and correlation coefficient sets, from each comparison. The means and standard deviations of each variable are thus computed. An ellipsoid in the 3D Cartesian space illustrates these computations, having the mean coordinates as center and 2 × standard deviations of the variables as the axes. The volume of the ellipsoid should be related to the similarity between the compared spectra; a smaller volume indicates a higher spectral similarity. This approach was evaluated with respect to the (a) concentration of the analytes; (b) spectral acquisition parameters (spectral resolution, spectral frequency acquisition); (c) spectral similarity between overlapping peaks; (d) perfect co-elution situation; (e) influence of spectral processing (derivation, wavelength rationing), using test solutions of carbamazepine (a,b), acetyl cysteine and enalapril maleate (c), or nitrazepam and diazepam (d,e).

Clozapine and its metabolites require close therapeutic monitoring (TDM) in patients due to poor correlation between the administrated doses and resulting plasma concentrations, the narrow therapeutic interval, high inter-individual variability, and the risk of serious side effects once toxic levels are exceeded. The aim of the study was to develop a simple (relatively cheap) LC-UV method for the quantification of clozapine and its metabolites in plasma and urine samples. For sample preparation, liquid-liquid extraction (LLE) in n-octanol was more efficient and less limiting in injection volumes compared to the in-situ formation of SUPRAS. When analyzing urine, an alkalinization step before extraction was required. The proposed method produced linear concentration responses with/without internal standard (IS) for the target analytes, with LLOQs within the targeted range of 50 ppb and %RSD within the acceptable 15% range. Furthermore, sample stability studies proved that pre-extracted samples were stable for the short term at room temperature and long-term when frozen.