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Solid-phase microextraction (SPME) is an alternative method to dialysis and ultrafiltration for the determination of plasma protein binding (PPB) of drugs. It is particularly advantageous for complicated analytes where standard methods are not applicable. Di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a lead compound of novel thiosemicarbazone anti-cancer drugs, which entered clinical trials in 2016. However, this agent exhibited non-specific binding on filtration membranes and had intrinsic chelation activity, which precluded standard PPB methods. In this study, using a simple and fast procedure, we prepared novel SPME fibers for extraction of DpC based on a metal-free, silicon string support, covered with C18 sorbent. Reproducibility of the preparation process was demonstrated by the percent relative standard deviation (RSD) of ≤ 9.2% of the amount of DpC extracted from PBS by several independently prepared fibers. The SPME procedure was optimized by evaluating extraction and desorption time profiles. Suitability of the optimized protocol was verified by examining reproducibility, linearity, and recovery of DpC extracted from PBS or plasma. All samples extracted by SPME were analyzed using an optimized and validated UHPLC-MS/MS method. The developed procedure was applied to the in vitro determination of PPB of DpC at two clinically relevant concentrations (500 and 1000 ng/mL). These studies showed that DpC is highly bound to plasma proteins (PPB ≥ 88%) and this did not differ significantly between both concentrations tested. This investigation provides novel data in the applicability of SPME for the determination of PPB of chelators, as well as useful information for the clinical development of DpC. Graphical abstract.

Novel thiosemicarbazone metal chelators are extensively studied anti-cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC-MS/MS method for the simultaneous quantification of 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1-E and M1-Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C18 column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid-phase extraction on 96-well plates. This method was validated over the concentration range of 0.18-2.80 μM for Bp4eT, 0.02-0.37 μM for both M1-E and M1-Z, and 0.10-1.60 μM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration-time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti-cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class. Copyright © 2013 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher's web site.

The aim of this study was to develop and validate HPLC methods for the determination in plasma of two novel thiosemicarbazone anti-tumour drugs developed in our laboratories (Dp44mT and N4mT). The appropriate separations were achieved using a HS F5 HPLC column with the mobile phase composed of a mixture of either acetate buffer/EDTA or EDTA and acetonitrile (62:38 and 50:50, v/v, respectively). The plasma samples were pretreated with SPE (phenyl and C18, respectively). Furthermore, these methods were successfully applied to in vitro plasma stability experiments. The investigation has clearly shown that both thiosemicarbazones are markedly more stable in plasma than their aroylhydrazone forerunners.

Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a promising analogue of the dipyridyl thiosemicarbazone class currently under development as a potential anti-cancer drug. In fact, this class of agents shows markedly greater anti-tumor activity and selectivity than the clinically investigated thiosemicarbazone, Triapine®. However, further development of DpC requires detailed data concerning its metabolism. Therefore, we focused on the identification of principal phase I and II metabolites of DpC in vitro. DpC was incubated with human liver microsomes/S9 fractions and the samples were analyzed using ultra-performance liquid chromatography (UPLC(TM)) with electrospray ionization quadrupole-time-of-flight (Q-TOF) mass spectrometry. An Acquity UPLC BEH C(18) column was implemented with 2 mM ammonium acetate and acetonitrile in gradient mode as the mobile phase. The chemical structures of metabolites were proposed based on the accurate mass measurement of the protonated molecules as well as their main product ions. Ten phase I and two phase II metabolites were detected and structurally described. The metabolism of DpC occurred via oxidation of the thiocarbonyl group, hydroxylation and N-demethylation, as well as the combination of these reactions. Conjugates of DpC and the metabolite, M10, with glucuronic acid were also observed as phase II metabolites. Neither sulfate nor glutathione conjugates were detected. This study provides the first information about the chemical structure of the principal metabolites of DpC, which supports the development of this promising anti-cancer drug and provides vital data for further pharmacokinetic and in vivo metabolism studies.

Dexrazoxane (DEX) is the only clinically used drug effective against anthracycline-induced cardiotoxicity and extravasation injury. However, the mechanism of its cardioprotective action still remains elusive. This paucity of comprehensive data is at least partially caused by the analytical difficulties associated with selective and sensitive simultaneous determination of the parent drug and its putative active metabolite ADR-925 in the relevant biological material. The aim of this study was to develop and validate the first LC–MS/MS method for simultaneous determination of DEX and ADR-925 in the isolated rat neonatal ventricular cardiomyocytes (NVCMs) and the cell culture medium. The analysis was performed on a Synergi Polar-RP column using the gradient profile of the mobile phase composed of 2 mM ammonium formate and methanol. Electrospray ionization and ion trap mass analyzer were used as ionization and detection techniques, respectively. NVCMs were precipitated with methanol and the cell culture medium samples were diluted with the same solvent prior the LC–MS/MS analysis. The method was validated within the range of 4–80 pmol/10 6 NVCMs and 7–70 pmol/10 6 NVCMs for DEX and ADR-925, respectively, and at the concentrations of 8–100 μM for both compounds in the culture cell medium. The practical applicability of this method was confirmed by the pilot analysis of NVCMs and the corresponding cell medium samples from relevant in vitro experiment. Hence, the LC–MS/MS method developed in this study represents a modern analytical tool suitable for investigation of DEX bioactivation inside the cardiomyocytes. In addition, the basic utility of the method for the analysis of DEX and ADR-925 in plasma samples was proved in a pilot experiment.

The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.

This study was focused on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method development for quantification of a novel potential anticancer agent, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), in aqueous media. Solid Bp4eT was found to consist predominantly of the Z isomer, while in aqueous media, both isomers coexist. Sufficient separation of both isomers was achieved on a Synergi 4u Polar RP column with a mobile phase composed of 2 mM ammonium formate, acetonitrile, and methanol (30:63:7; v/v/v). The photo diode array analysis of both isomers demonstrated different absorption spectra which hindered UV-based quantification. However, an equal and reproducible response was found for both isomers using an MS detector, which enables the determination of the total content of Bp4eT (i.e., both E- and Z- isomeric forms) by summation of the peak areas of both isomers. 2-Hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT) was selected as the internal standard. Quantification was performed in selective reaction monitoring using the main fragments of [M+H](+) (240 m/z for Bp4eT and 229 m/z for N4mT). The method was validated over 20-600 ng/ml. This procedure was applied to a preformulation study to determine the proper vehicle for parenteral administration. It was found that Bp4eT was poorly soluble in aqueous media. However, the solubility can be effectively improved using pharmaceutical cosolvents. In fact, a 1:1 mixture of PEG 300/0.14 M saline markedly increased solubility and may be a useful drug formulation for intravenous administration. This investigation further accelerates development of novel anticancer thiosemicarbazones. The described methods will be useful for analogs currently under development and suffering the same analytical issue.