Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography–tandem mass spectrometry

Abstract

A high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 μl whole blood onto FTA cards. A 3 mm disc was cut from the DBS samples and extracted using methanol, followed by liquid–liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C 18 column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0–5000 ng/ml with correlation coefficients ( r 2) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were −0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2–2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from −6.8 to 8.5% and −0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24 h at room temperature and 4 h at 60 °C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤−60 °C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.