Comparison of Packed-Column Supercritical Fluid Chromatography−Tandem Mass Spectrometry with Liquid Chromatography−Tandem Mass Spectrometry for Bioanalytical Determination of (R)- and (S)-Ketoprofen in Human Plasma Following Automated 96-Well Solid-Phase Extraction

Abstract

The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)-and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma.After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC.(R,S)-2-(3-Benzoylphenyl)propionic acid (ketoprofen, kt; Figure 1), is a potent nonsteroidal, anti-inflammatory drug that is commonly formulated as a tablet for oral administration or, in some countries, as a topical gel or cream. In both cases, the active is formulated as a racemic mixture. It is well known that the (S) form of kt contains the intrinsic pharmacologic activity, 1 and for this reason, it is desirable to determine the plasma concentrations of the individual enantiomers. The estimated bioavailability of orally dosed ketoprofen is g92%, 2 and the maximum plasma concentrations reach low-microgram per milliliter levels after a typical 25-mg dose. 2,3 With peak concentrations in that range, LC with UV detection has been established to provide adequate sensitivity for obtaining full pharmacokinetic (PK) curves. 4-10 Typical chiral LC-UV methods reported in the literature have analysis times of approximately 10-25 min and lower limits of quantitation (LLOQ) of 25 ng/mL. Other approaches for bioanalytical determination of ketoprofen are GC/MS based with reported 10-20-min analysis times. 11-15 One of these methods 14

Chiral separations in sub‐ and supercritical fluid chromatography

“…[57], an LC-MS/MS method was compared with an SFC-MS/MS method for the determination of ketoprofen enantiomers in human plasma. A Chirex 3005 column with id of 2 mm in LC and 4 mm in SFC, provided the near baseline R s of ketoprofen enantiomers after optimization of the separation conditions.…”

Clinical and pharmaceutical applications of packed‐column supercritical fluid chromatography

“…For example, ketoprofen [25] and phenylbutazone [26] are prescription drugs that have been measured in human plasma and serum samples using pSFC techniques. Additionally, the metabolite of phenylbutazone, oxyphebutazone, was determined simultaneously with its parent compound [26].…”

Recent applications of stereoselective chromatography

“…This technique has been applied in the bioanalysis of ketoprofen enantiomers in the study of their pharmacokinetics in humans. 104 …”