We were asked to provide measurement of rivaroxaban in breast milk in support of a research study by OB-GYN at our institution. We developed an assay as a modification of our existing LC-MS/MS assay for plasma rivaroxaban. We report results of validation of this assay for research-use-only (RUO). LC-MS/MS was performed using Shimadzu UFLC coupled to ABSciex 3200MD MS/MS. Rivaroxaban and rivaroxaban-d4 (internal standard, IS) were purchased from Santa Cruz Biotechnology (Dallas, TX). Chromatography was performed using a Phenomenex C8 guard column (4x3 mm) and a Phenomenex Synergi 4 µm Fusion-RP 80 Å LC column (50 x 4.6 mm) at 34 °C. Mobile phases were H2O/0.1% formic acid (A) and MeOH/0.1% formic acid (B). Time profile relative to sample injection was (minutes, %B): (-1,50;1,50;2.5,100;4.7,100;5,50). Retention time of Rivaroxaban was 4.7 min. Q1/Q3 positive-ion pairs were 436/145 for rivaroxaban and 440/145 for rivaroxaban-d4. Cow’s milk was used as a surrogate for human breast milk in this evaluation. Stock standards of rivaroxaban in milk (500 ng/mL) were prepared by addition of 5 µL methanolic rivaroxaban stock (1 mg/mL) to 10 mL milk (consumer grade, pasteurized, homogenized, vitamin D-supplemented bovine milk). These stock standards were aliquoted by 500 µL and kept frozen (-20 °C) until use. From this stock, new standards (100, 67, 33, 0 ng/mL) were prepared using milk as diluent on each day of method validation. Samples were prepared for LC-MS/MS as follows: 100 µL sample was combined with 200 µL IS in MeOH and vortexed for 10 s; samples were incubated for 5 min at room temperature and centrifuged for 10 min at 3000 g; 100 µL of supernatant was combined with 100 µL of a 50:50 combination of A:B mobile phases; after vortexing for 10 s, this solution was centrifuged for 10 min at 3000 g; finally, 100 µL of supernatant was placed in the LC autosampler for measurement of rivaroxaban by LC-MS/MS. Sample-to-sample interval was 6.5 min. Recovery of rivaroxaban from dilutions in milk compared to dilutions in MeOH was 55%. The standard curve was linear across all calibrators (0-500 ng/mL; r>0.99). Interassay coefficients of variation (CVs) for controls were 4.6% (50.8 ± 2.4 ng/mL; n=20) and 3.1% (26.0 ± 0.8 ng/mL; n=19). Lower limit of quantitation (LLOQ) was set at 5 ng/mL, having a signal to noise ratio (S/N) >100 and CV <10%. By mixing studies, no analytical interferences were observed for a large array of common therapeutic drugs and drugs of abuse. Conclusions: The LC-MS/MS assay for rivaroxaban in milk was based on our existing plasma assay but required development of new procedures for preparation of calibrators and processing of samples. The analytical performance characteristics of the LC-MS/MS assay for rivaroxaban in milk were deemed to be acceptable for use as an RUO assay.
