Analytical validation of accelerator mass spectrometry for pharmaceutical development

Abstract

The validation parameters for pharmaceutical analyses were examined for the accelerator mass spectrometry measurement of (14)C/C ratio, independent of chemical separation procedures. The isotope ratio measurement was specific (owing to the (14)C label), stable across samples storage conditions for at least 1 year, linear over four orders of magnitude with an analytical range from 0.1 Modern to at least 2000 Modern (instrument specific). Furthermore, accuracy was excellent (between 1 and 3%), while precision expressed as coefficient of variation was between 1 and 6% determined primarily by radiocarbon content and the time spent analyzing a sample. Sensitivity, expressed as LOD and LLOQ was 1 and 10 attomoles of (14)C, respectively (which can be expressed as compound equivalents) and for a typical small molecule labeled at 10% incorporated with (14)C corresponds to 30 fg equivalents. Accelerator mass spectrometry provides a sensitive, accurate and precise method of measuring drug compounds in biological matrices.

Figure 1. Stability of accelerator mass spectrometry measurements of total ¹⁴C in three human serum samples is shown by periodic aliquots obtained from multiple freeze–thaw cycles throughout a year

Coefficient of variations of the measured isotope concentrations were within 3%.

Figure 2. Linearity of accelerator mass spectrometry (AMS) quantitation over five orders of magnitude is demonstrated by a urine dilution series (three or six samples at each concentration), 50 direct comparisons between liquid scintillation counting and AMS measurements of the same materials, and by the well documented range of ¹⁴C dating down to parts per quadrillion concentrations

Figure 3. Total of 52 urine samples having high ¹⁴C concentrations (90–450 Modern) were remeasured 10 days apart

Reproducibility averaged less than 0.01%, with an root mean squared difference of 0.74% between the two measurements. The inset shows that the frequency distribution of residuals was not Gaussian.

Figure 4. Frequency distribution of the raw isotope ratio for 4450 measurements of a ¹⁴C standard over the period of two years shows a 2.7% standard deviation in a nearly normal distribution

Figure 5. Repeatability in accelerator mass spectrometry analysis of metabolite profiles by UPLC is shown for two LC runs completed on separate days

The mass of equivalent drug was determined from the ¹⁴C concentration, the specific labeling of the compound and the molecular weight. Error bars represent propagated errors due to measurement of both the fraction and the added carrier compound.

Figure 6. Frequency distribution of a 0.1-Modern carrier compound has a standard deviation of 5.4%, due primarily to counting statistics limited by impatient operators. A tail toward high values indicates occasional contamination with higher ¹⁴C

Figure 7. The main plot shows the measured ¹⁴C concentrations versus time of measurements over a year’s period for a ‘hot’ international comparison material

The measurements have a standard deviation of 2.8% and are well represented by a Gaussian distribution function with a 2.6% width.

Figure 8. Atmospheric ¹⁴C concentration as reflected in tree rings and other plant material

The ‘bomb spike’ of 1963 has become an essential tracing tool for carbon movement throughout oceans, soils, and humans. Predose samples of subject plasma will closely correlate with the atmospheric concentration.

Figure 9. Frequency distribution plot of ¹⁴C concentration in blood serum for 103 humans during late 2002 shows a peak at the expected 1.08 Modern with a 1.2% standard deviation in the Gaussian fit

Figure 10. Accelerator mass spectrometry (AMS) recovery fractions of labeled compounds in both a biological fluid (urine) and a carrier compound are shown in the lower frame which includes direct comparison of liquid scintillation counting data with the AMS recovery (diamonds) and the dilution of a ‘hot’ urine into further urine (closed circles)

AMS recovery of ¹⁴C-atrazine in carrier compound is shown in open circles. The upper frame shows the standard deviation of these data sets as a function as assayed ¹⁴C.

Figure 11. Assigned precision in the quantitation of LC eluent fractions is plotted against the amount of ¹⁴C in the fraction

The carrier carbon had an average value of 0.111 ± 0.005 Modern (3.4%). An uncertainty in the single fraction measure was also assumed at 3.4%.

Figure 12. Frequency distributions of ¹⁴C concentrations in serum and urine samples of predose human subjects at multiple clinic sites show the expected tight distribution around the atmospheric level for the serum; but urine concentrations are lower with large variability

Figure 13. Devation of data points from the interpolated value of neighboring data from the serum kinetic curves of 32 humans is shown as a function of the time post dose, in which the sera varied from 40 Modern to natural ¹⁴C levels

The right frame shows that the frequency distribution of the deviations has a normal core and enhanced tails. CV: Coefficient of variation.

Figure 14. Quantitation of UPLC eluent fractions of a parent compound and its metabolites are shown by the solid line

The cumulative sum of the quantified fractions (dashed line) is compared with the direct measure of the labeled compound in the injectate. The LLOQ was found as ten-times the uncertainty in the ¹⁴C concentration of the added carrier compound.