Determination of sirolimus blood concentration using high-performance liquid chromatography with ultraviolet...

Different HPLC methods have been developed and used to determined sirolimus blood concentrations. These methods show different performance characteristics, mostly related to peak interference, recovery, assay sensitivity, and turnaround times.

We adapted, improved, and validated an HPLC method with UV detection for measurement of sirolimus in whole blood clinical samples.

The standards, quality controls, or patient samples (0.25 or 0.5 mL) and internal standard (desmethoxysirolimus) were extracted with 1-chlorobutane. After evaporation, the extract was reconstituted in a 70% acetonitrile/water mixture and analyzed onto a reverse-phase C18 column at 50 degrees C under a flow rate of 1.0 mL/min in the HPLC system. Ultraviolet detection was performed at 278 nm, with sensitivity setting of 0.010 AUFS. Identification of peaks of interest was by retention time; quantification of sirolimus was based on a peak area ratio.

Analytic recovery ranging from 96 to 120% (CV = 3.7 to 16.8%; bias = -4.2 to 16.7%) was observed throughout the assay's linear range (2.5-150.0 ng/mL). The lower limit of quantification for both sample volumes (0.25 or 0.5 mL) was 2.5 ng/mL (CV = 12 and 15%, bias = -1.2 and 4%, respectively). The intra- and interassay imprecision ranged from 6.2 to 14.4% and from 9.1 to 18.6%, with bias ranging from 1.3 to 12.9% and -1.8% to 7.1, for quality control levels of 3, 10, and 20 ng/mL. Whole blood and extracted samples are stable at room temperature and at 4 and -20 degrees C for 1 week and 3 days, respectively. Chromatograms showed good separation free of interfering peaks. A set of 45 samples can be extracted in 2 h, allowing results within 24 h.

This HPLC-UV method shows good and reproducible performance, satisfying all requirements of an assay designated to be applied in therapeutic drug monitoring strategies after organ transplantation.