On-cartridge derivatization coupled with solid-phase extraction for the ultra-sensitive determination of minodronic acid in human plasma by LC–MS/MS method

• An ultra-sensitive method for MA bioanalysis with an LLOQ of 10.0 pg/mL.
• Pentamethyl derivatives of nitrogen heterocyclic ring-containing BPs was found.
• D4-MA exhibited a slower methylation rate than MA to yield pentamethyl derivatives.
• The proposed method enabled the ultra-sensitive detection of ZA and RA.
• The yield was affected by acidity, exposure time, and anticoagulant.

Minodronic acid (MA) is a third-generation bisphosphonate (BP). Its high potency allows lower doses to be administered in clinical settings compared with other BPs, which results in extremely low systemic exposure. Therefore, it is essential to develop an ultra-sensitive bioassay for pharmacokinetics studies of MA. In this work, we used on-cartridge derivatization of MA with trimethylsilyldiazomethane to extract MA from plasma samples and improve its LC–MS/MS behavior. The reaction produced a known derivative, tetramethylated MA, and a novel derivative, pentamethylated MA (PMMA). PMMA exhibited a better signal-to-noise ratio, and was monitored for the quantification of MA. However, the derivatization yield of d4-PMMA was much lower and more variable than that of PMMA, which decreased the effectiveness of its correction function as an internal standard. Therefore, a two-cycle derivatization approach was introduced to increase its yield and improve the reproducibility. The calibration curves of MA showed good linearity over the range of 10.0–1000 pg/mL. A lower limit of quantification of 10.0 pg/mL was achieved with acceptable precision (<10.5%) and accuracy (5.0%). The intra- and inter-batch precision of quality control samples was <9.5%, and the accuracy ranged from −2.8% to 0.6%. The stability results showed that MA was stable in human plasma for 6 h at room temperature (25 °C), for 115 days at −20 °C, during three freeze/thaw cycles (from −20 °C to 25 °C), and in post-preparative samples for 24 h at 4 °C. The method was successfully used to characterize the pharmacokinetic profile of MA following an oral dose of 1.0 mg MA hydrate to healthy volunteers (n = 12). The proposed derivatization procedure was also extended to measure other BPs (risedronic acid and zoledronic acid) in human plasma at low pg/mL.

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