Article pubs.acs.org/ac
Rapid Quantification of Digitoxin and Its Metabolites Using Differential Ion Mobility Spectrometry-Tandem Mass Spectrometry Caroline Bylda,†,‡ Roland Thiele,† Uwe Kobold,† Alexander Bujotzek,† and Dietrich A. Volmer*,‡ †
Roche Diagnostics GmbH, Penzberg, Germany Institute of Bioanalytical Chemistry, Saarland University, Saarbrücken, Germany
‡
S Supporting Information *
ABSTRACT: This study focuses on the quantitative analysis of the cardiac glycoside drug digitoxin and its three main metabolites digitoxigenin-bisdigitoxose, digitoxigeninmonodigitoxose, and digitoxigenin using electrospray ionization-differential ion mobility spectrometry-tandem mass spectrometry (ESI-DMS-MS/MS). Despite large molecular weight differences, gas-phase separation of the four compounds in the DMS drift cell was not possible, even by utilizing additional volatile chemical modifiers. Baseline separation was achieved after adduct formation with alkali metal ions, however, and efficiency was shown to improve with increasing size of the alkali ion, reaching optimum conditions for the largest cesium ion. Subsequently, an assay was developed for quantification of digitoxin and its metabolites from human serum samples and its analytical performance assessed in a series of proof-of-concept experiments. The method was applied to spiked human serum pools with concentration levels between 2 and 80 ng/mL. After a short reversed-phase chromatographic step for desalting the sample, rapid DMS separation of the analytes was carried out, resulting in a total run time of less than 1.5 min. The instrumental method showed good repeatability; the calculated coefficients of variation ranged from 2% to 13%.
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similar to the selected ion monitoring (SIM) mode of the quadrupole MS. Significant improvements of DMS separation efficiency are obtained by adding polar chemical modifiers to the buffer gas.5−9 These modifiers alter the drift behavior of ions as a result of dynamic clustering/declustering processes that increase the difference between high and low field drift velocities. During the low field, ions form clusters with modifier molecules, resulting in larger CCS than “naked” ions and resulting in slower movement. During the high field, clusters are dissociated and mobilities increase.10,11 Schneider et al. systematically demonstrated enhancement effects for a wide range of chemical modifiers (e.g., isopropanol, ethyl acetate, acetonitrile), thus reducing considerably the number of modifiers that have to be tested for a specific application.5,11 Beneficial effects of different modifiers have been shown for small molecules,12 pharmacologically active substances,13 and chiral molecules, such as amphetamine-type compounds.14 Further improvements have been achieved by implementing multicomponent modifiers, which increased peak capacity and sensitivity for detection of low molecular weight drugs and allowed fine-tuning separations in specific applications.15 Drift times were also strongly influenced in ESI-IMS-MS after adding nitrobenzene as a modifier, which has the ability to form large clusters with small target ions.16 We have recently shown that
on mobility spectrometry (IMS) separates ionized analytes based on their mobilities in an electric field in the gas phase.1 The mobility depends on its mass, charge, and shape.2 In classical IMS, ions are separated in a drift cell filled with inert buffer gas at atmospheric pressure in a constant low electric field. Ions passing through the buffer gas are subjected to a number of collisions and these processes will be influenced by the collision cross sections (CCS) of the analyte ions. Ions of different size and shape are thus separated in the drift tube. Differential ion mobility spectrometry (DMS) and field asymmetric ion mobility spectrometry (FAIMS) separate ions based on changes of ion mobility in alternating electric fields.3 An asymmetric electric waveform (separation voltage, SV), comprising a short high voltage (10−30 kV/cm) and a longer low voltage (
