(W0930-09-50) Development and Validation of a Blood Micro Sampling Methodology to Facilitate Conduct of Pharmacokinetic Studies in Pregnant and Postpartum Patients

Purpose: Pregnancy is associated with several physiological changes altering the pharmacokinetics (PK) of certain drugs. Since limited pregnancy data is available, dosing recommendations for drugs are extrapolated from studies carried out in non-pregnant patients. Application of micro blood sampling as a tool for remote blood sampling is a convenient way for performing studies in this population. A novel micro blood sampling device called Volumetric Absorptive Micro Sampling (VAMS) developed in 2014 allows consistent blood volume collection following fingerpick. We hypothesize that a micro sampling tool such as the VAMS device will optimize conduct of pharmacokinetic studies in pregnant women and provide substantial data to predict alterations in the clearance and exposure of buprenorphine during and after pregnancy. In this study, we have developed and validated an LC-MS/MS assay to measure buprenorphine and attempted to quantify its metabolites simultaneously in 20 µL blood samples using VAMS.

Methods: Two stages of sample extraction-desorption from the polymeric VAMS tip as well as analyte extraction has to be considered. To maximize recovery, we tested multiple solvents in different ratios and vortexing/sonication/shaking conditions. Use of a methanol-water based solvent along with an acidic additive formic acid, in addition to physical agitation using a lateral shaker was the desorption step. For analyte extraction, protein precipitation, LLE and solid-phase extraction were tested. Protein precipitation using acetonitrile was concluded to give best recovery in a clinical laboratory setting. The acetonitrile step also contains the Internal Standard: 500 pg/mL (buprenorphine-D_4, norbuprenorphine-D_3,),1 ng/mL (buprenorphine-D₄-3-β-D-glucuronide, norbuprenorphine glucuronide-D₃) was added to the samples. The Eppendorf tubes were then vortexed, followed by centrifugation at 15,000 rpm for 12 min at 8 °C. Samples were dried under stream of air, and reconstituted using mobile phase.

Concentrations of buprenorphine, norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide in VAMS and blood samples were determined using a Waters Acquity H class model with a C18 1.7 μm, 2.1 x 100 mm column. A sample volume of 5 μL was injected on column. The calibration curves were generated by plotting the response ratio of buprenorphine to buprenorphine-D4, norbuprenorphine to norbuprenorphine-D3, buprenorphine-3-β-D-glucuronide to buprenorphine-D4-3-β-D-glucuronide, norbuprenorphine glucuronide to norbuprenorphine glucuronide-D3 against nominal concentration of the corresponding analytes in blood samples. Accuracy and Precision were evaluated using intra- and inter-day coefficient of variation. Three samples for each QC were analyzed on a single day for intra-day, and three samples were measured on three consecutive days for inter-day CV. The extraction recovery of buprenorphine and three of its metabolites was performed by comparing the responses obtained from neat standard QC samples with the responses obtained from extracted blank human blood spiked with analytes including internal standard. The effect of matrix was analyzed by comparing response from extracted blank blood samples spiked with drug post extraction with the absolute response of neat solvent spiked with drug. Finally, stability of the VAMS samples was tested at RT for 24 hrs, 3 days and 4 days as well as at -80⁰ C for a period of 4 days.

Results: The method displayed linearity over a range of concentrations for buprenorphine (1-20 ng/ml), buprenorphine glucuronide and norbuprenorphine glucuronide (2-40 ng/mL) with acceptable intra and inter day precision and accuracy for Buprenorphine. The recoveries for all analytes in VAMS samples were above 100% over the concentration range tested, and BUP showed acceptable recovery. However, the recoveries of NBUP, BUPG and NBUPG were not within acceptable limits indicating significant overestimation using the VAMS method. For stability, no significant degradation of samples at any of the conditions tested compared to the duplicates analyzed immediately after sampling was observed, however the variability between duplicates was large.

Conclusion: To our knowledge, this is the first study conducted using VAMS to quantify buprenorphine and its metabolites. We have developed and validated a rapid, sensitive, and robust UPLC-MS/MS assay quantify the concentration of buprenorphine in 20 µL VAMS blood samples. The advantages of this method include simple sample processing, ultra-low volume requirement, acceptable BUP recovery and short run time. Further work is needed to accurately assay the metabolites.


Blood desorption in VAMS using aqueous extraction solvent


Standard Curves for (A) Buprenorphine (B) Norbuprenorphine (C) Buprenorphine-3-glucouronide (D) Norbuprenorphine-3-glucouronide


Results from top-left to right: Precision, Accuracy, Recovery, Matrix effect and Stability for all four analytes using VAMS