Monique Zangarini (Presenter)
Newcastle University
Authorship: M. Zangarini (1), P. Berry (1), F Raynaud (2), P Jones (3), D. Edwards (3) and G.J. Veal (1)
(1) NICR, Newcastle University, Newcastle upon Tyne, UK; (2) CRUK Cancer Therapeutics Unit, The Institute of Cancer Research, London, UK; (3) Cancer Research UK Centre for Drug Development, London, UK
| Short Abstract A LC-MS/MS method was developed and validated to quantify CCT245737, a small molecule orally active CHK1 inhibitor, in human plasma. The method requires 20µL of plasma and involve acetonitrile deproteinisation after addition of the labelled CCT245737 (IS). Detection was obtained by SRM, following the transitions m/z 379.8→360.2 for CCT245737 and m/z 384.0→324.2 for the IS. It is sensitive, precise and accurate with overall precision ≤8.5%, accuracy in the range 96%–102% and high recovery ≤93.4%. The LLOQ is 20ng/ml. The assay was validated in the range 20-20000 ng/ml. This is the first method validated to measure CCT245737 in human plasma. |
Long Abstract
Background: CCT245737 ((R)-5-((4-((morpholin-2-ylmethyl)amino)-5-(trifluoromethyl) pyridin-2-yl)amino)pyrazine-2-carbonitrile) is an orally active small molecule inhibitor of Checkpoint kinase 1 (CHK1) discovered at the Cancer Therapeutics Unit, Institute of Cancer Research, and developed for use in an oncology setting. CHK1 is activated in response to DNA damage, which may be a consequence of cancer itself, or may be associated with the use of chemotherapy or radiotherapy. Interfering with CHK1 function can produce substantial increases in the sensitivity of tumour cells to a variety of chemotherapeutic agents. We have developed and validated a bioanalytical method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of CCT245737 in human plasma, supported by previous work in this area carried out by the Cancer Therapeutics Unit of The Institute of Cancer Research in London.
Methods: A sensitive and specific HPLC-MS/MS (API 4000) assay was developed and validated according to EMA guidelines for quantitative measurement of the CHK1 inhibitor, CCT245737 in human plasma. The sample preparation procedure required a small volume of plasma (20 µl) and involved protein precipitation with acetonitrile after addition of a stable labelled CCT245737 ((13C15N)-CCT245737) as internal standard (IS). Reversed-phase chromatography under gradient conditions (mobile phase A: 10 mM ammonium acetate + 0.5 % ammonia; mobile phase B: acetonitrile) was applied with separation on a Kinetex 2.6µm C18 column (100 Å, 50 x 4.6 mm) coupled with a guard column of the same material, at a flow rate of 0.5 ml/min. Detection was obtained by selected reaction monitoring (SRM), following the transitions m/z 379.872 → 360.200 for CCT245737 and m/z 384.086 → 324.200 for the IS. The method is rapid and selective, allowing good resolution of peaks in 4 min, and has been validated according to EMA guidelines on bioanalytical method validation. The validation parameters assessed were: selectivity, anticoagulant comparison, matrix effect, recovery, limit of quantitation (LOQ), linearity and range, dilution integrity, carryover of analyte and IS, intra/inter-assay precision and accuracy and stability under different conditions.
Results: The bioanalytical method was fully validated and exhibited good sensitivity, precision, and accuracy, with overall precision expressed as CV ≤ 8.5 % and accuracy in the range 96-102%. The method developed was shown to be selective, with an absence of interfering components (<7 % of the LOQ and <1 % for the IS in six batches of plasma evaluated). Recovery was high (≥ 93.4 %) and consistent, with CV ≤ 8.3 %. No matrix effect was observed in six independent matrix batches including haemolysed plasma (CV 3.6%). The study also assessed potential anticoagulant effects, using plasma obtained with sodium citrate, K2EDTA and lithium heparin. No effect of different anticoagulants was observed; the CV calculated was ≤ 4.8 % and accuracy within the range 97-100 %. The LOQ was determined to be 20 ng/ml, with precision and accuracy of 5 % and 97 %, respectively. The assay developed was linear in the range 20-20,000 ng/ml, as demonstrated by a coefficient of determination (r2 value) of ≥ 0.998, with precision ≤ 4.3 % and accuracy within the range 97-102 %. The carry-over effect was excluded by injecting mobile phase samples and extracted blank plasma samples after the upper limit of quantification (ULOQ). This guaranteed a peak response no higher than 5% of LOQ. Intra-day precision and accuracy were ≤ 5.6 % and within the range 97-100 %, respectively, while the inter-day precision and accuracy were ≤ 3.4 % and within the range 99-102 %, respectively. CCT245737 is stable in human plasma for at least 4 hours at room temperature, for 7 days at 4°C, before and after extraction, and over 3 freeze-thaw cycles.
Conclusion: We have successfully developed and validated a LC-MS/MS method to measure the anti-tumour agent CCT245737 in human plasma samples.
References & Acknowledgements:
Acknowledgements: Work supported by Cancer Research UK and the ICR is acknowledged for providing the starting method.
| Description | Y/N | Source |
| Grants | yes | Cancer Research UK |
| Salary | yes | Cancer Research UK |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |