DOI: 10.7910/dvn/qkl6op
title: Neonatal Brain Concentrations of Buprenorphine and Gabapentin in Prenatally Exposed Mouse Pups

description: The rise in rates of opioid abuse in recent years in the United States has led to a dramatic increase in the incidence of neonatal abstinence syndrome (NAS). Despite improved understanding of NAS and its acute symptoms, there remains a paucity of information regarding the long-term effects of prenatal exposure to drugs of abuse on neurological development. This dataset contains LC-MS/MS-derived drug concentrations of the opioid buprenorphine and the commonly co-abused drug gabapentin from the brains of neonatal mouse pups that were prenatally exposed to the drugs via oral dosing of the pregnant dam. The presence of drug in the brains of these pups was used to validate downstream analysis of excitatory and inhibitory synaptic connectivity in the prenatally-exposed animals.

All experiments were conducted in accordance with Marshall University’s Institutional Animal Care and Use Committee (IACUC) guidelines (W.C.R. protocols 696 and 697). Adult C57/Bl6 WT pregnant female mice were ordered from Jackson Laboratory (Bar Harbor, ME). On embryonic day 6 (E6), dams were given access to 1 ml of a 1:1 sweetened condensed milk/water solution served in a plastic 35 mm dish. Starting on E7, dams were given free access to a once daily 1 ml solution of 1:1 condensed milk/water containing either pharmaceutical grade buprenorphine hydrochloride (CIII) (5 mg/kg; Spectrum Chemical, Gardena, CA), gabapentin (30 mg/kg; Spectrum), or vehicle control. Drug doses were calculated based on the weight of pregnant females on E7. Daily dosing continued through the birth of pups. All animals were given ad libitum food (standard mouse chow milled on site) and water.

The brains of the newborn pups were isolated and then shipped on dry ice to the Pharmaceutical Sciences Research Institute at the McWhorter School of Pharmacy (Samford University, Birmingham, AL) for drug concentration analysis. Mouse brain samples were homogenized 1:5 with 5 mM ammonium acetate buffer using an Omni THQ homogenizer (Atlanta, GA). Gabapentin calibration standards, blanks and QCs were prepared by spiking 20 µl of blank control brain homogenate with the appropriate amount of gabapentin to achieve concentrations in ranging from 25-10,000 ng/ml. Internal standard (1 ml of 100 ng/ml Gabapentin-d4 in methanol) was added to precipitate the proteins. After centrifugation for 5 minutes at 13,000 rpm, the supernatant was transferred to glass tubes and the solvent was evaporated under nitrogen at 40°C. The samples were then redissolved in 200 µl of 95/5 10 mM ammonium formate/methanol with 0.1% formic acid and transferred to limited volume autosampler vials and analyzed in positive ion mode by liquid chromatography with tandem mass spectrometry (LC/MS/MS).

Buprenorphine calibration standards, blanks, and quality controls (QCs) were prepared by spiking 100 µl of blank control brain homogenate with the appropriate amount of buprenorphine to achieve concentrations ranging from 0.2-200 ng/ml. Standards, blanks, QCs and samples were spiked with internal standard (10 µl of 50 ng/ml Terfenadine in acetonitrile). Acetonitrile (1 ml) was added to precipitate the proteins. After centrifugation for 5 minutes at 13,000 rpm, the supernatant was transferred to glass tubes and the solvent was evaporated under nitrogen at 50°C. The samples were then redissolved in 200 µl of 50/50 5 mM ammonium acetate/acetonitrile and transferred to limited volume autosampler vials and analyzed in positive ion mode by LC/MS/MS.

The LC/MS/MS system consisted of Shimadzu system (Columbia, MD) equipped with LC20-AD dual HLPC pumps, an SIL20-AC HT autosampler, and a DGU-20A2 in-line degasser. Detection was performed using an Applied BioSystems 4000 QTRAP (Applied Biosystems, Foster City, CA) triple quadrupole mass spectrometer operated in the positive ion mode utilizing electrospray ionization. Mass calibration, data acquisition and quantitation were performed using Applied Biosystem Analyst 1.6.2 software (Applied Biosystems). 

Separation of gabapentin and the gabapentin-d4 from the brain homogenate matrix was achieved from a 10 μl injection of the samples using a Phenomenex Polar C18, 100 X 2 mm 5 µm particle column. The mobile phase was delivered at a flow rate of 400 µl/min using a gradient elution profile consisting of 5 mM ammonium acetate (A) and acetonitrile with 0.1% formic acid (B). The analyte and internal standard were detected using multiple reaction monitoring (MRM) for the following transitions: Gabapentin (m/z 172.2 - 137.0), Gabapentin-d4 (m/z 176.2 - 141.0).

Separation of buprenorphine and terfenadine from the brain homogenate matrix was achieved from a 10 μl injection of the samples using a Phenomenex Luna C18, 100 X 2 mm 5 µm particle column. The mobile phase was delivered at a flow rate of 400 µl/min using a gradient elution profile consisting of 5 mM ammonium acetate with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The analyte and internal standard were detected using multiple reaction monitoring (MRM) for the following transitions: Buprenorphine (m/z 468.4 - 396.1), Terfenadine (m/z 472.4 - 436.3).
description: LC-MS/MS results from the drug concentration analysis are summarized in an Excel spreadsheet. Separate worksheets are included for the gabapentin and buprenorphine-exposed animals, with comparisons to the vehicle-treated controls shown on each sheet. Standard accuracy is reported on the "standards and QC" sheet.