Single Instrument AMS and IRMS for Microdose Mass Balance Studies

Introduction

Complete tracer recovery in mass balance studies remains difficult for large dose exposures containing high radioactivity. Low or micro-dose studies use high sensitivity AMS for precisely quantifying ¹⁴C/C in aliquots of excreta and accessible fluids with much lower isotope tracer concentrations over longer periods of time. AMS provides sample-size independent ¹⁴C concentrations from representative mg-sized fractions of a collection. A carbon concentration of the sample converts the isotope concentration to the drug concentration of the collection. We perform precise IRMS with the same instrument to obtain total carbon content of samples using a ¹³C spike resulting in accurate drug masses in all pools, including the highly variable pool excreted in urine.

Method

Collection aliquots from ¹⁴C-microdose studies are spiked with ¹³C-glycine. Total collection volume or mass is recorded, but the specific sample fraction of total is not needed. The sample and spike volumes are obtained from precise positive displacement pipettes for isotope dilution calculation. Samples are prepared to reduced fullerene solids and ionized to negative carbon ions in a Cs sputter ion source for AMS analysis. AMS measures the ¹³C/¹²C ratio to ±0.5% along with the ¹⁴C/¹²C ratio to ±1%. Isotope dilution of ¹³C determines the carbon content of the sample while the ¹⁴C concentration determines the labeled drug content. The sample concentration is applied to the total collection volume or mass for an accurate account of pool drug concentrations.

Data

Urinary excretion was followed from humans for up to 3 weeks after 200 nCi, ≤100 µg doses of ¹⁴C-labeled drugs and vitamins. AMS measured the label concentration per gram carbon in the urine. Carbon content of 200 urine samples ranged from 1.50 mg/ml to 12 mg/ml, with a coefficient of variation (CV) of 0.5% across the entire range as measured by the ¹³C spike using the IRMS capability of the instrument. ¹⁴C concentrations in urine collections ranged between 34.5 to 0.1 pM over the excretion period with precisions (CV) of 0.4 - 4.5% over that range. This precision is inclusive of both quantification of the ¹⁴C by AMS and the measurement of carbon content by ¹³C IRMS made on the same samples in a single instrument. Typical sensitivities for mass balance over long periods from microdoses will be shown.

Figure 1: Linearity in the response from 0.1 to 4% carbon using a sucrose based surrogate matrix.
The method showed greater than 90% accuracy over the range of 0.2 to 4% carbon.

Novel Aspect

Combination AMS and IRMS provides high accuracy tracer excretion data over long time periods to obtain high total recovery information. This approach solves the urine carbon content problem in a single measurement that does not propagate errors associated with off-line Carbon Analysis.

• "Triple Collector High Energy" IR-AMS provide ¹⁴C (drug) and total C (matrix) in a single measurement
• Quantitative results without quantitative extraction
• Can be applied to any matrix (skin, tears, tumor biopsies) — eliminating a second total C analysis by C/N analyzer
• Cost efficiencies to Mass Balance studies
• New clinical paradigms