Pitfall in the high-throughput quantification of whole blood cyclosporin A using liquid chromatography-tandem mass spectrometry

Abstract

In a growing number of laboratories the technique of liquid chromatography-tandem mass spectrometry is used for the quantification of cyclosporin A in whole blood, employing cyclosporin D as the internal standard. Cyclosporin A is extensively metabolized in vivo; in liquid chromatography-tandem mass spectrometry respective metabolites can give rise to both parent and product ions that are isobaric with ions commonly used for the detection of cyclosporin A and cyclosporin D, respectively. In this article it is demonstrated that limited chromatography with co-elution of such metabolites together with cyclosporin A and cyclosporin D can lead to incorrect results.Keywords: cyclosporin A; cyclosporin D; liquid chromatography-tandem mass spectrometry; specificity.In tandem-mass spectrometry the detection of target analytes is based on their physical decomposition behavior; the molecular mass-specific selection of an intact precursor ion of an analyte and of the respective product ion results in very high analytical specificity. This high degree of specificity can limit the requirements of sample preparation and chromatography and may enable ''high-throughput'' routine methods. However, the specificity of tandem-mass spectrometry should not be assumed to be absolute and unlimited. Given the complexity of human body fluids as a matrix and the extensive metabolization of certain target analytes, the specificity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be a critical issue. This is demonstrated by an observation reported here.With the intention of switching the quantification of whole blood cyclosporin A (CsA) from immunoassay technology to LC-MS/MS, we decided to modify the LC-MS/MS method that we currently use for the quan-

Development and validation of UHPLC method for the determination of cyclosporine A in biological samples

“…When we focus on CsA determination in other, different types of biological tissues, many more methods have been published which employ modern and complex MS techniques (Brignol et al ., ; Zhou et al ., ; Streit et al ., ; Salm et al ., ; Vogeser and Spoehrer, ; Zaater et al ., ; Ansermot et al ., ; Bogusz et al ., ; Li et al ., ; Mora et al ., ; Brozmanova et al ., ; Kanduru et al ., ; Onoune et al ., ; Zhi‐gang et al ., ; Di Tommaso et al ., ; Karapirli et al ., ; Rodriguez‐Aller et al ., ; Guada et al ., ; Tszyrsznic et al ., ). The use of LC/MS and LC/MS/MS techniques enabled much shorter analysis times to be achieved, with lower LOD and LOQ, and other validation parameters at acceptable levels.…”

Pitfalls Associated with the Use of Liquid Chromatography–Tandem Mass Spectrometry in the Clinical Laboratory

“…This breakdown results in a fragment that is isobaric to cy- closporine D, which is a widely used internal standard for cyclosporine A measurement (6 ). This case demonstrates that in therapeutic drug monitoring that employs LC-MS/MS, potential interfering compounds that are related to isobaric metabolites can be identified only in postdose patient samples that contain relevant endogenously formed drug metabolites (Fig.…”

Pitfalls in measuring the endocannabinoid 2-arachidonoyl glycerol in biological samples

“…Similar interferences can arise from in-source fragmentation of conjugate metabolites (12). Potential isomer and metabolite interference has already been reported for LC-MS/MS measurement of methylmalonic acid (13), 25-hydroxyvitamin D 3 (14), and cyclosporin A (15). Consequently, for specific detection of some target analytes, rather long chromatographic run times with appropriate separation efficacy may be mandatory, even for LC-MS/MS.…”