Determination of sugammadex in human plasma, urine, and dialysate using a high-performance liquid chromatography/tandem mass spectrometry assay.

Sugammadex (Bridion®, Merck Sharp & Dohme Corp., Oss, The Netherlands) is a modified γ-cyclodextrin which has the ability to reverse the neuromuscular blockade induced by the steroidal neuromuscular blocking agents rocuronium and vecuronium. The objective of the current study is to describe the bioanalytical methods that have been developed and validated according to US Food and Drug Administration guidelines on bioanalytical method validation, and subsequently applied to determine total sugammadex (i.e., free sugammadex plus sugammadex bound to the neuromuscular blocking agent) in human heparinized plasma, urine and dialysate. Sugammadex was extracted from human plasma and urine using solid phase extraction with Isolute HAX 96-well extraction plates; no extraction was performed on dialysate samples. Samples from plasma, urine, and dialysate were analyzed on a Polaris® C18-A PEEK (polyaryletheretherketone) analytical column (50 mm × 4.6 mm internal diameter, 5 µm) with a linear mobile phase gradient of 0.1% (v/v) formic acid in water:methanol from 70:30 to 20:80. The flow rate was 1 mL/min with a total run time for each injection of 6 min. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ion mode with a turbo ion-spray interface to quantify the concentration of sugammadex. Inter- and intra-assay precision and accuracy were within pre-defined acceptance limits. The presence of rocuronium did not interfere with the assay in plasma, urine or dialysate; similarly, vecuronium did not interfere with the plasma assay (not tested for interference in urine or dialysate). Sugammadex was found to be stable in plasma, urine and dialysate in the short-term at room temperature, in the long-term at -20°C, and after several freeze/thaw cycles. The validated bioanalytical methods developed here have been successfully applied in a series of clinical studies for the determination of total sugammadex in plasma, urine and dialysate.

Two sensitive and rapid chromogenic assays of fondaparinux sodium (Arixtra) in human plasma and other biological matrices.

Fondaparinux is a synthetic selective inhibitor of factor Xa recently approved for thromboprophylaxis after major orthopedic surgery. Determination of its concentration gives valuable insight into specific pharmacokinetics or safety studies. The aim of the study was to develop direct, sensitive, precise and accurate assays of fondaparinux sodium in different biological matrices. Consistency with the recommended chromogenic assay for low molecular weight heparin required a similar method. However, recent data indicated some variability in the determination of anti-Xa level between commercial chromogenic assays. Consequently, we developed and validated two chromogenic methods (A and B) for assaying fondaparinux in plasma and other biological matrices. The assays are calibrated with fondaparinux, a pure chemical entity, and the result is expressed as amount (microg) of the fondaparinux calibrator. Results showed that precision was lower than 5.2% in plasma or plasma water and 13% in placental medium. The accuracy was lower than 7.6% in plasma or plasma water and 10.2% in placental medium. The lower limit of quantification in plasma was 0.042 microg/mL with automated Method A and 0.019 microg/mL with Method B. The assay was not affected by the source of the samples, the presence of blood cells, EDTA, citrate or repeated cycles of freezing and thawing. The two chromogenic assays calibrated with fondaparinux sodium reach the equivalence criteria for plasma samples and provide reliable and reproducible results.