(T1230-06-33) Development and Validation of LC-MS/MS Assay Methods to Determine Ubrogepant and Atogepant in Human Breast Milk

Purpose: Ubrogepant is a calcitonin-gene related peptide (CGRP) receptor antagonist for the acute treatment of migraine and atogepant is a potent, selective, competitive oral CGRP receptor antagonist for the preventive treatment of episodic migraine in adults. Studies in pregnant women have shown that migraine improves or disappears during pregnancy but reappears within one to four weeks post-partum, making migraine a concern in lactating women. The pharmacokinetics of ubrogepant and atogepant in lactating women and their excretion into breast milk have not been evaluated clinically. Consequently, a lack of evidence regarding infant exposure to ubrogepant or atogepant through breast feeding may prevent patients usage of these drugs. The objective of this study was to develop a novel LC-MS/MS bioanalytical method to support the evaluation of the pharmacokinetic attributes associated with excretion of ubrogepant and atogepant in breast milk.

Methods: Calibration curves in human breast milk were prepared for ubrogepant and atogepant (1.00 - 1000 ng/mL) and samples extracted by protein precipitation with stable-labelled internal standards via addition of acetonitrile. Chromatographic separation was performed in reversed-phase mode with a Waters XBridge C18 (2.1 x 30 mm, 3.5 µm) analytical column set at 50°C with a mobile phase of acetonitrile and 0.02% acetic acid (aq). Following isocratic elution, phospholipids were flushed from the column at high flow rate with 95% acetonitrile. Parent ions were formed by positive ion electrospray on a SCIEX API 5000 with MRM transitions m/z 550.2 > 264.1 and 604.2 > 264.1 for ubrogepant and atogepant, respectively.

Results: A novel LC-MS/MS method was developed and validated for the determination of ubrogepant and atogepant in human breast milk. This high throughput chromatography demonstrated the elution of atogepant and ubrogepant at 1.26 and 1.54 minutes respectively. The lysophosphatidylcholines and phosphatidylcholines which are notorious to suppress MS signal, were demonstrated to be well separated from the analytes and were flushed out of the column at each injection to avoid late elution interference and allow method reproducibility and robustness. The extraction recovery was quantitative for each of ubrogepant and atogepant. Calibration curves from 1.00 - 1000 ng/mL were generated using a weighted (1/x²) linear least-squares regression for each analyte. Precision and accuracy (%CV ≤9.9%, %bias between -9% and 8%), specificity, matrix effect and matrix factor met acceptance criteria (ICH M10 Guideline) based upon controls from six individual breast milk donors. Ubrogepant and atogepant were stable in breast milk for at least 25 h at 4°C, 240 days for ubrogepant and 83 days for atogepant at -80°C and four freeze-thaw cycles; post-extraction stability was established for 168 h at 4°C.

Conclusion: The method developed for the quantitation of ubrogepant and atogepant in human breast milk was successfully validated and demonstrated to be specific, precise and accurate, and therefore suitable for clinical sample analysis


Precision and Accuracy for Ubrogepant and Atogepant in Human Breast Milk.


Matrix Effect for Ubrogepant and Atogepant in Human Breast Milk.


Chromatograms of an Extracted LLOQ (1.00 ng/mL) and Blank Samples of Ubrogepant and Atogepant in Human Breast Milk.