Highly sensitive UHPLC—MS/MS method for quantification of ethylenediamine-N,N′-di-2-(3-cyclohexyl) propanoic acid derivatives in mouse serum

Abstract

Summary. A series of new (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate esters has shown cytotoxic activity towards human leukemic cell lines. The aim of this study was to develop and validate a bioanalytical method for quantification of (S,S)-O, O-diethyl-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate dihydrochlorides (DE-EDCP) and its metabolite, substituted propanoic acid (EDCP), in mouse serum by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Structural analog, derivative of 1,3-propanediamine, was used as an internal standard (IS). Sample preparation employed protein precipitation by acetonitrile and subsequent centrifugation. Optimal UHPLC separation conditions were set to achieve simultaneous determination of both compounds in a short run time of 6 min. Additionally, the selected reaction monitoring (SRM) mode developed in this method allowed a highly sensitive, accurate, and precise identification of compounds of interest. The lower limit of quantitation (LOQ) was 1.3 ng mL −1 for DE-EDCP and 0.3 μg mL −1 for EDCP. The calibration curves were linear over the concentration range of 1.3-26.7 ng mL −1 and 0.3-6.7 μg mL −1 for DE-EDCP and EDCP, respectively. Precision (%CV) and accuracy (%RE) for DE-EDCP and EDCP ranged from 3.5% to 16.0% and from 1.8% to 14.4%, respectively.The validation process was performed in accordance with the regulatory guidance/guideline, and all of the obtained results met the established acceptance criteria. The newly developed and validated UHPLC-MS/MS method is rapid, sensitive, and selective, and it can be successfully applied to drug monitoring in nonclinical studies.

Estimation of passive gastrointestinal absorption and membrane retention using PAMPA test, quantitative structure-permeability and quantitative structure-retention relationship analyses of ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoic acid and 1,3-propanediamine-N,N'-di-2-(3-cyclohexyl)propanoic acid derivatives

“…The system was incubated 16 h at room temperature. After the incubation, concentrations of each investigated substance in starting solution -CD(0), donor solution after incubation -CD(t) and acceptor solution after incubation -CA(t) were measured in triplicate by UHPLC/MS-MS method [14]. Mobile phase A was composed of ammonium acetate (5 mM) -trifluoroacetic acid (99.9:0.1, v/v), whereas mobile phase B was composed of methanol -trifluoroacetic acid (99.9:0.1, v/v).…”

Prediction of in vivo Bioavailibility by in vitro Characterization of Ethylenediamine Dipropanoic Acid Derivatives with Cytotoxic Activity

“…Preparation of all standard solutions is described in a previously published study. 9 Sample preparation is also described previously. 9 All these solutions were prepared by two different analysts in sending and receiving laboratories.…”

“…DE-EDCP, EDCP and IS were eluted by using a mobile phase as previously described. 9 Quantitation was achieved by the MS-MS detection in the positive ionization mode for the DE-EDCP, EDCP and IS. The MS operating conditions were optimized as following: the capillary voltage was 4500 V, the gas temperature was set to 340 °C and gas flow was 10 l/min and the nebulizer pressure was 35 psi.…”

“…For non-clinical study of cytotoxic activity of the DE-EDCP and its potential metabolite (S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoic acid dihydrochloride (EDCP), there has been previously developed and validated the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) bio-analytical method. 9 Bioanalytical methods should be robust. 10 Evaluation of reproducibility (transferability) of bio-analytical method is becoming increasingly important 11-13 since the bio-analytical methods are often used in different laboratories during non-clinical and clinical studies.…”

Inter-Laboratory Reproducibility of Bioanalytical Uhplc-MS/MS Method

“…Confidence intervals of 95% of intercept and slope explain if their value differ from value zero (intercept) and value one (slope) only by chance, allowing conclusion of method agreement and correction action if necessary. [18] Selectivity The selectivity of the method was evaluated by analysing six independent drug-free mouse serum samples with the corresponding spiked plasma at LLOQ.…”