Determination of unbound prednisolone, prednisone and cortisol in human serum and saliva by on‐line solid‐phase extraction liquid chromatography tandem mass spectrometry and potential implications for drug monitoring of prednisolone and prednisone in saliva

Abstract

Prednisolone (PLN) and prednisone (PN) are widely used glucocorticoids. Drug monitoring of PLN and PN is not routinely done owing to the need for multiple blood sampling and challenging measurement of unbound PLN and PN in blood. Here we present a robust method for quantification of cortisol, PLN and PN in serum, ultrafiltrate and saliva by on-line solid-phase extraction LC-MS/MS. The method is linear for the three analytes over the range of 6-1400 nmol/L for serum and 2-450 nmol/L for ultrafiltrate and saliva. Within-run precision of all three analytes was <10% and total precision was <15%. This method was applied to create time-concentration profiles of cortisol, PLN and PN after an oral dose of prednisolone in a healthy volunteer. Salivary levels of PLN correlated well with ultrafiltrate levels (p < 0.01), while this correlation was only marginal for PN (p = 0.052). The PN/PLN ratio was significantly higher in saliva than in ultrafiltrate and serum (p < 0.01). Addition sums of both metabolites in saliva showed excellent correlation with those of ultrafiltrate (p < 0.01). These findings have not been presented before and may have important implications for future studies concerning drug monitoring of PLN and PN in saliva.

High‐throughput determination of cortisol, cortisone, and melatonin in oral fluid by on‐line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry

“…Based on previous reports, a specificity test was performed to evaluate interference by prednisolone (molecular formula C 21 H 28 O 5 ) and prednisone (molecular formula C 21 H 26 O 5 ) on levels of CORTol (molecular formula C 21 H 30 O 5 ) and CORTone (molecular formula C 21 H 28 O 5 ) . Potential interference derives from prednisolone and CORTone being isobars, prednisolone and prednisone having naturally occurring M + 2 isotopes with the same molecular weight as CORTol and CORTone, respectively, and, given the chemical similarity of all these molecules, chromatographic coelution is possible . In the specificity test, an aqueous solution of prednisolone and prednisone at 400 nmol/L and 250 nmol/L, respectively, was used to simulate the maximum levels expected in human oral fluid following administration of 80 mg prednisolone (the maximum therapeutic dose) …”

“…Potential interference derives from prednisolone and CORTone being isobars, prednisolone and prednisone having naturally occurring M + 2 isotopes with the same molecular weight as CORTol and CORTone, respectively, and, given the chemical similarity of all these molecules, chromatographic coelution is possible . In the specificity test, an aqueous solution of prednisolone and prednisone at 400 nmol/L and 250 nmol/L, respectively, was used to simulate the maximum levels expected in human oral fluid following administration of 80 mg prednisolone (the maximum therapeutic dose) …”

Alternative Matrices for Therapeutic Drug Monitoring of Immunosuppressive Agents Using LC–MS/MS

“…In addition, the free fraction is dose-dependent and exhibits circadian variability (approximately 22% higher in the morning) [74]. The PLN plasma unbound fraction demonstrates a high correlation with the salivary level and a lower correlation with the concentration of the prodrug prednisone (PN) [75,76]. Total and free concentrations of PLN+PN in the OF and plasma display an excellent association [76] (Table 2).…”

Monitoring Prednisolone and Prednisone in Saliva

“…16 In brief, we obtained ultrafiltrate for the separation of unbound analytes using Centrifree YM-30 centrifugal filtration units (30,000 MW cutoff; Millipore Ireland BV, Country Cork, Ireland). 16 In brief, we obtained ultrafiltrate for the separation of unbound analytes using Centrifree YM-30 centrifugal filtration units (30,000 MW cutoff; Millipore Ireland BV, Country Cork, Ireland).…”

“…In analogy to cortisol, the unbound or free fraction of PLN is biologically active; only unbound molecules can readily diffuse into target tissues and cells and are therefore considered clinically most relevant. 16 In the present study, the relationship between salivary and (free) serum predniso(lo)ne levels is further explored in a population of healthy volunteers after administration of a single oral dose of PLN. One way to overcome this problem might be the measurement of PLN levels in saliva, as concentrations in saliva may represent free PLN levels in blood.…”