Distribution of clomipramine, citalopram, midazolam, and metabolites in skeletal tissue after chronic dosing in rats

Abstract

In recent years, the use of skeletal tissue as an alternative matrix in forensic toxicology has received new interest. In cases where extreme decomposition has taken place, analysis of skeletal tissue is often the only option left. In this article, a fully validated method is presented and the distribution of clomipramine, citalopram, midazolam, and metabolites after chronically administration is examined within skeletal tissue. Rats were chronically dosed with respectively clomipramine, citalopram, or midazolam. Extracts were quantitatively analyzed using liquid chromatography−electrospray ionization−tandem mass spectrometry (LC−ESI−MS/MS). Clomipramine, citalopram, and metabolites, respectively desmethylclomipramine and desmethylcitalopram are shown to be detectable in all bone types sampled. Midazolam and its metabolite α‐OH‐midazolam could not be detected. The absence of midazolam in extracts gives an indication that drugs with pKa values under physiological pH are badly or not incorporated in bone tissue. Bone and post‐mortem blood concentrations were compared. A range of different bone types was compared and showed that the concentration is strongly dependent on the bone type. In concordance with previous publications, the humerus shows the highest drug levels. Skeletal tissue concentrations found ranged from 1.1 to 587.8 ng/g. Comparison of the same bone type between the different rats showed high variances. However, the drugs−metabolite ratio proved to have lower variances (<20%). Moreover, the drugs−metabolite ratio in the sampled bones is in close concordance to the ratios seen in blood within a rat. From this, we can assume that the drugs−metabolite ratio in skeletal tissue may prove to be more useful than absolute found concentration.

Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications

“…During the last decade, the interest in the usage of skeletal tissue as an alternative specimen in forensic toxicology has seen a revival [12][13][14]. It is shown that the bone tissue acts as a depot for certain drugs [12].…”

In‐Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry

“…higher than the quantitation limits of the corresponding published HPLC-ESI-MS/MS methods (0.5 ng mL À1 for clomipramine, [36] 0.1 ng mL À1 for chlorphenamine, [37] and 5.1 ng mL À1 for atenolol [38] )h owever these methods used QQQ MS instruments,w hich offer better quantitative performance and higher sensitivity in the multiple reaction mode (MRM) than the quadrupole Ion Tr ap mass analyzer used here, [39,40] and chlorphenamine method utilized aLLE step in which the extract was evaporated to dryness and reconstituted in as maller volume. [37] Ab etter sensitivity was enabled for pindolol by the in-syringe EkE approach (LOQ of 6.1 ng mL À1 in human serum) compared to the HPLC-ESI-MS/MS method (LOQ of 10 ng mL À1 in human serum [41] ) where the latter used protein precipitation.…”

In‐Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry

“…Chemie higher than the quantitation limits of the corresponding published HPLC-ESI-MS/MS methods (0.5 ng mL À1 for clomipramine, [36] 0.1 ng mL À1 for chlorphenamine, [37] and 5.1 ng mL À1 for atenolol [38] )h owever these methods used QQQ MS instruments,w hich offer better quantitative performance and higher sensitivity in the multiple reaction mode (MRM) than the quadrupole Ion Tr ap mass analyzer used here, [39,40] and chlorphenamine method utilized aLLE step in which the extract was evaporated to dryness and reconstituted in as maller volume. [37] Ab etter sensitivity was enabled for pindolol by the in-syringe EkE approach (LOQ of 6.1 ng mL À1 in human serum) compared to the HPLC-ESI-MS/MS method (LOQ of 10 ng mL À1 in human serum [41] ) where the latter used protein precipitation.…”