High-performance liquid chromatography with fluorescence detection for mycophenolic acid determination in saliva samples

Abstract

Background For therapeutic drug monitoring (TDM) of mycophenolic acid (MPA), which is frequently proposed, saliva might be a suitable and easy-to-obtain biological matrix. The study aimed to validate an HPLC method with fluorescence detection for determining mycophenolic acid in saliva (sMPA) in children with nephrotic syndrome. Methods The mobile phase was composed of methanol and tetrabutylammonium bromide with disodium hydrogen phosphate (pH 8.5) at a 48:52 ratio. To prepare the saliva samples, 100 µL of saliva, 50 µL of calibration standards, and 50 µL of levofloxacin (used as an internal standard) were mixed and evaporated to dryness at 45 °C for 2 h. The resulting dry extract was reconstituted in the mobile phase and injected into the HPLC system after centrifugation. Saliva samples from study participants were collected using Salivette® devices. Results The method was linear within the range of 5–2000 ng/mL, was selective with no carry-over effect and met the acceptance criteria for within-run and between-run accuracy and precision. Saliva samples can be stored for up to 2 h at room temperature, for up to 4 h at 4 °C, and for up to 6 months at − 80 °C. MPA was stable in saliva after three freeze–thaw cycles, in dry extract for 20 h at 4 °C, and for 4 h in the autosampler at room temperature. MPA recovery from Salivette® cotton swabs was within the range of 94–105%. The sMPA concentrations in the two children with nephrotic syndrome who were treated with mycophenolate mofetil were within 5–112 ng/mL. Conclusions The sMPA determination method is specific, selective, and meets the validation requirements for analytic methods. It may be used in children with nephrotic syndrome; however further studies are required to investigate focusing on sMPA and the correlation between sMPA and total MPA and its possible contribution to MPA TDM is required. Graphical abstract

Liquid chromatography–tandem mass spectrometry method for mycophenolic acid and its glucuronide determination in saliva samples from children with nephrotic syndrome

“…The highest LLOQ for MPA was found by Alsmadi et al [ 14 ] and it amounted to 150 ng/mL. As the highest saliva MPA concentration in samples collected from children included in our study was 532 ng/mL, it seems unnecessary to apply the wider concentration range, up to 2000 ng/mL, as we did in our previous study [ 23 ].…”

“…The results of the LC–MS/MS method were compared with those obtained using the HPLC-FLD method, which was previously developed [ 23 ]. Since MPAG concentrations were not determined in the previous study, and only two children (one boy and one girl, both aged 12 years) were included in the MPA analysis, we limited our comparison to these specific cases, encompassing 16 MPA concentrations.…”

“…The correlation of the data was tested using Pearson correlation analysis as the analyzed data were normally distributed. To compare results from the LC–MS/MS method with those obtained using the HPLC-FLD method [ 23 ], we assessed Passing-Bablok regression and Bland–Altman plot. In all analyses, a p-value of 0.05 was considered significant.…”

“…In our previous study [ 23 ], we successfully applied high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for MPA determination in saliva, but were unable to detect MPAG. Consequently, we explored the possibility of determining both MPA and MPAG in saliva using the liquid chromatography tandem mass spectrometry (LC–MS/MS) method.…”