Establishment and comparison of three novel methods for the determination of the photodynamic therapy agent 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) in human serum

• The first clinical pharmacokinetic study of HPPH in Chinese was reported.
• Three novel methods were proposed and validated for the determination of HPPH.
• Comparison of fluorescence and MS platform for HPPH quantification was achieved.
• Data consistency was found among proposed three methods.
• Microplate reader method was recommended because of very rapid determination.

2-[1-Hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) is a second-generation photosensitizer that has been applied in clinical studies of photodynamic therapy for a variety of malignant lesions. Based on the differences in selectivity and labour intensity, three novel methods – fluorescence detection coupled with high performance liquid chromatography (LC–FLD), LC-tandem mass spectrometry (LC–MS/MS) and fluorescence-based microplate reader methods – were developed for the determination of HPPH in human serum, which allowed comparison of fluorescence and MS platform for HPPH quantification. All three methods have been validated and successfully applied to support the clinical pharmacokinetic study of HPPH. The concentrations measured by LC–FLD matched those by LC–MS/MS with a correlation coefficient (r = 0.994) and coefficient of determination (r2 = 0.989). Data consistency was also found between the measurements of microplate reader and LC–MS/MS with a correlation coefficient (r = 0.999) and coefficient of determination (r2 = 0.998), indicating that fluorescence assay, the low cost alternative with a relatively poorer selectivity, is clearly suitable for the quantification of HPPH. Calibration curves in the methods of LC–FLD and microplate reader were linear (r ˃ 0.998) over the concentration range from 50 to 5000 ng/mL, and linearity was obtained over the concentration range from 5 to 1000 ng/mL in the LC–MS/MS method. Compared with the other two methods, the fluorescence-based microplate reader method with proven high selectivity should be strongly recommended because of obvious advantages such as the lowest labour intensity, the lowest instrument cost, a better sensitivity than LC–FLD and the very rapid determination of large number of samples (24 samples/40 s).

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