Quantification of eight benzodiazepines in human breastmilk and plasma by liquid-liquid extraction and liquid-chromatography tandem mass spectrometry: Application to evaluation of alprazolam transfer into breastmilk

Abstract

Breastfeeding is strongly encouraged for infant and maternal health. Benzodiazepines (BZDs) are widely prescribed drugs for symptoms, such as anxiety and insomnia, which many women could experience during the postpartum period. However, limited information is currently available to evaluate the transfer of different BZDs into breastmilk. In order to assess the proprieties of this medication during breastfeeding, robust and sensitive analytical methods to quantify BZDs are required. For this purpose, we developed a method for quantification of BZDs, including alprazolam, bromazepam, clonazepam, clotiazepam, etizolam, flunitrazepam, lorazepam, and CM7116 (a metabolite of ethyl loflazepate), in human breastmilk and plasma using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Sample preparation was performed by a simple liquid-liquid extraction (LLE) with ethyl acetate. For sample preparation of CM7116, the pretreatment process to completely obtain the metabolite was added before the LLE step. The BZDs were separated by a C18 column using a gradient elution of acetonitrile in aqueous ammonium acetate solution, and were detected in the positive ion electrospray mode with multiple reaction monitoring (MRM). Lower limits of quantification (LLOQs) in breastmilk ranged from 0.25 to 0.5 ng/mL, and those in plasma ranged from 0.5 to 1.0 ng/mL. The intra-day and inter-day precision, and accuracy of data were assessed and found to be acceptable. The developed method was successfully applied to measure the concentration of alprazolam in breastmilk and plasma, which were donated by a lactating woman who had been regularly treated with alprazolam. Milk to plasma (M/P) ratios were calculated as 0.52 (before oral administration) and 0.49 (2 h after administration) 3 days after delivery. The M/P ratio 1 month after delivery was calculated as 0.41 (2 h after administration). We estimated that the relative infant dose (RID) values of alprazolam ranged from 3.11 to 4.61%.