Quantification of olanzapine and its three metabolites by liquid chromatography–tandem mass spectrometry in human body fluids obtained from four deceased, and confirmation of the reduction from olanzapine N-oxide to olanzapine in whole blood in vitro

Abstract

Purpose Quantification of olanzapine (OLZ) and its metabolites such as N-desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N-oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. Methods OLZ and its three metabolites were extracted from 40 μL each of body fluids by two-step liquid–liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. Results The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 ℃ in whole blood in vitro. Conclusions To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC–MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

A Review of Advances in Bioanalytical Methods for the Detection and Quantification of Olanzapine and Its Metabolites in Complex Biological Matrices

“…With a decrease in the grain size of the bed, the degree of their packing increases, and thus, the grain surface area per volume unit increases, which results in an increase in the efficiency of the column. Thanks to this, better resolution and shorter time of olanzapine analysis can be achieved [ 19 , 26 , 29 , 40 , 51 , 77 , 112 , 114 ]. The first developed methods of chromatographic separation of olanzapine were achieved by isocratic elution, i.e., with a constant composition of the mobile phase throughout the analysis [ 32 , 34 , 35 , 36 , 37 , 39 , 40 , 42 , 43 , 44 , 46 , 47 , 50 , 52 , 53 , 54 , 56 , 57 , 62 , 63 , 65 , 66 , 67 , 68 , 69 , 70 , 73 , 74 , 75 , 76 , 90 , 91 , 93 , 94 , 102 , 105 ].…”

“…However, it fails when analyzing mixtures containing many chemical compounds with little variation in polarity or molecular weight, as in the case of methods involving many other antipsychotics and their metabolites in addition to olanzapine. Therefore, to separate complex mixtures, gradient elution is used, consisting of a continuous or abrupt change in the composition of the mobile phase during the analysis [ 14 , 19 , 25 , 26 , 27 , 29 , 33 , 38 , 41 , 45 , 48 , 49 , 51 , 58 , 59 , 64 , 71 , 72 , 77 , 78 , 79 , 80 , 81 , 82 , 92 , 95 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 ].…”

“…Quantification for tandem mass spectrometers was performed using Multiple Reaction Monitoring (MRM) mode [ 14 , 15 , 19 , 25 , 26 , 29 , 32 , 33 , 34 , 39 , 40 , 41 , 45 , 48 , 49 , 51 , 52 , 54 , 58 , 64 , 66 , 67 , 70 , 71 , 73 , 78 , 79 , 80 , 81 , 82 , 89 , 94 , 95 , 108 , 109 , 110 , 111 , 112 , 114 ] or Single Reaction Monitoring (SRM) mode [ 27 , 50 , 56 , 59 , 72 , 93 ], in which the selected ion passes through the Q1 quadrupole and then undergoes fragmentation in the collision chamber. The ion at m / z 313.2 is a protonated precursor [M+H]+ in Q1 full-scan spectra.…”

“…The team of H. Nozawa (2023) pointed out that the addition of ascorbic acid during sample collection carries the risk of reducing the concentration of olanzapine N-oxide as a result of its conversion to the parent compound, i.e., olanzapine itself. Moreover, N-oxides are relatively easily reduced, not only by compounds such as ascorbate but also under alkaline conditions during storage or extraction, and olanzapine N-oxide may decompose to the parent compound [ 114 ]. In some of the published studies, olanzapine was unstable in biological material, and the addition of ascorbic acid prevented the concentration from decreasing during storage.…”

“…Optimization of olanzapine measurement conditions using the LC-MS technique includes optimization of the ion source and interface parameters depending on the conditions of chromatographic analysis, such as flow and composition of the mobile phase, and on the properties of the compound itself. The most common ionization methods used in olanzapine bioanalysis are chemical ionization (APCI) [45,50,51,64,65,72] and electroscattering (ESI) [14,19,[25][26][27]29,[32][33][34][35][39][40][41]43,48,49,[52][53][54][56][57][58][59]66,67,70,71,73,[78][79][80][81][82]89,94,95,[108][109][110]113,114]. Regardless of the ionization me...…”