Research Article Zangarini, Rajan, Danilenko, Berry, Traversa & Veal
centrations in tumor samples obtained from patients enrolled on the study. No published assay currently exists to measure pegcantratinib in tumor tissues, with LC–MS/MS method development for PEGylated drugs facing significant challenges due to the hetero- geneity and relatively high MW of the PEG fraction. The mass spectrums generated show broad continuous peaks due to the size and charge distributions of PEG that are difficult to interpret and generally not usable for quantitative analysis [9]. However, recent papers have demonstrated that PEGylated therapeutic agents undergo collision-induced dissociation in the ioniza- tion source (in-source CID) of the mass spectrometer, generating nonPEGylated precursor ions that can be used for drug quantification [10,11]. We have developed a sensitive and specific HPLC-
MS/MS assay, coupled with in-source CID, for direct quantitative measurement of pegcantratinib in human skin tumors. The method requires a small amount of tumor tissue, simple homogenization and extrac- tion and short analysis time. After HPLC separation, the PEGylated small molecule undergoes in-source fragmentation in the instrument ionization source, generating a small molecule pegcantratinib-specific fragment ion that can be used as surrogate for the quan- titative analysis. We validated the method according to the European Medicines Agency (EMA) guidance on bioanalytical method validation [12] and demonstrated its applicability to determine the drug’s tumor distri- bution in skin tumor punch biopsies obtained from patients with CYLD mutations participating in the current Phase Ib/IIa clinical trial.
Experimental Standards & chemicals Analytical reference standards of pegcantratinib and CT340, used as internal standard (IS), were provided by Creabilis Therapeutics Srl (Colleretto Giacosa,
Italy) and are shown in Figure 1. Analytical grade methanol, acetonitrile, DMSO and formic acid were purchased from Fisher Scientific (Leicester, UK).
Biological specimens Human skin tumor biopsies were collected as routinely excised tumor from patients. These samples were snap frozen and stored at -80°C until analysis. Control tumor samples (no drug treatment) were used to pre- pare the standards and quality control (QC) samples. Biopsies to be analyzed were excised at the end of treat- ment, subdivided into three different layers (top, mid- dle and bottom), snap frozen and stored at -80°C prior to analysis of pegcantratinib levels.
Standard & QC solutions Stock solutions of pegcantratinib for standards and QCs were prepared in DMSO at a concentration of 5 mg/ml. All stock solutions were diluted serially in methanol to generate working solutions with final pegcantratinib concentrations of 25, 50, 100, 500, 1000 and 2500 ng/ml for standards, and 80, 800 and 1600 ng/ml for working solutions of QCs. These work- ing solutions were used to prepare the standard points of the calibration curve and the QC samples in homog- enate control tissue, respectively. A stock solution of the IS (CT340) was prepared at 5 mg/ml in DMSO. The IS working solution was prepared at a concentra- tion of 10 μg/ml by diluting the stock solution with methanol. All stock and working solutions were stored at -20 °C prior to use.
Tissue homogenate, standards & QC samples Control tumor samples were weighed and homogenized using an Ultra Turrax homogenizer in 1% formic acid (1:5 ratio; w/v) and were used for preparation of cali- bration curve and QC samples. Calibration curve stan- dards were prepared by adding 10 μl of the working
Figure 1. Chemical structures. Chemical structures and proposed surrogate ion structures of (A) Pegcantratinib and (B) CT340.
28017 Bioanalysis (2017) 9(3) future science group
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58
