Development and validation of an analytical method for quantitation of Monobutylphthalate, a metabolite of Di-n-Butylphthalate, in rat plasma, amniotic fluid, fetuses, and pups by UPLC-MS/MS

Abstract

Phthalates have been used for decades as softening agents in the production of plastics, but in recent years have been extensively investigated for their potential hazards to human health and the environment. Di-n-butyl phthalate (DBP), with widespread exposure occurring through a variety of consumer products such as cosmetics and pesticides, is a suspected carcinogen and an endocrine system disruptor in both humans and laboratory animals. Its predominant metabolite is the monoester, monobutyl phthalate (MBP), which can serve as a marker of exposure. To support toxicological studies of DBP in pregnant and lactating rats and their offspring, a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for quantitation of MBP in rat plasma, amniotic fluid, fetuses and whole pup samples. Plasma samples were extracted using a simple protein precipitation with acetonitrile. Extraction and delipidation of pup homogenate was performed using acetonitrile and then submerging the vials in liquid nitrogen. Extracts were analyzed by UPLC-MS/MS in the negative ion mode. The method was successfully validated over the concentration ranges 25-5,000 ng/mL in female Sprague Dawley (SD) rat plasma and 50-5,000 ng/g in SD pup homogenate. Matrix calibration curves were linear (r ≥ 0.99), and the percent relative error (%RE) values were ≤ ±15% for standards at all levels. Absolute recoveries were > 92% in both matrices. The limits of detection (LODs) were 6.9 ng/mL in plasma and 9.4 ng/g in pup homogenate. Acceptable intra- and interday accuracy and precision were demonstrated by mean %RE ≤ ±7.5 and relative standard deviation (%RSD) ≤ 10.1%. Extract stability was demonstrated for ~6 days at various temperatures and freeze-thaw stability was demonstrated after 3 cycles over 3 days. Secondary matrix evaluation was performed for MBP in amniotic fluid and pooled fetus homogenate (mean %RE ≤ ±11.5 and %RSD ≤ 13.7). These data demonstrate that this simple method is suitable for determination of MBP in plasma, amniotic fluid, fetus and pup samples from toxicological studies of DBP.