10.3.1.3 Matrix Effect of Biological Samples MSC uses solid scintillators to
convert radioactivity decay energy into photons. Biological samples, such as
plasma, urine, and feces, which contain significant amounts of proteins, salts,
and small organic chemicals, may coelute with radiolabeled analytes, and be
present on the bottom of the wells after solvent is removed. These components
could affect the performance of MSC through color quenching, or other
mechanisms. The matrix effect of biological samples on the performance of a
TopCount was recently evaluated (Bruin et al., 2006; Zhu et al., 2005b).
Extracts or concentrates of plasma (Fig. 10.4d), urine (Fig. 10.4e), and HLM
incubation solution (Fig. 10.4c) showed no effect on the measurement of
radioactivity in any fractions except for those corresponding to the retention
time of the HPLC void volume in the chromatograms of the HLM incubation
solution and urine. Unlike the plasma sample, the urine and HLM samples
were not treated with solid phase extraction and, therefore, proteins, salts, and
other polar components were not removed from these samples. Elution of these
components in the void volume and deposition in the 96-well plates likely acted
as a physical barrier between the radiolabeled compound and the solid
scintillators, resulting in lower counting efficiency. Lower CPM values were
also observed in radiochromatograms of fecal extract (fractions 84–88, Fig.
10.4f). In a manner similar to that caused by salts eluting at the solvent front,
this might be due to a physical interaction of endogenous components with the
scintillators rather than color quenching because these fractions did not display
intense color. Compared to the MicroBeta counter (Wallace et al., 2004), the
matrix effects of urine and fecal samples on TopCount performance appeared
to be minimal, suggesting that TopCount is better for analyzing these types of
samples.
The quenching observed in the fecal sample analysis was proportional to the
amount of fecal extract injected. Therefore, it could be minimized to an
accepted level while not sacrificing sensitivity by limiting the amount of sample
injected. Based on data from a study with^14 Cit was recommended that
injection of human samples (equivalent to original volume or mass) should be
1 mL for liver microsomal incubations and plasma,2 mL for urine, and
50 mg for feces. In addition, conditions must be set such that polar
————————————————————————————"
FIGURE 10.4 The effect of biological matrices on TopCount performance (Zhu et al.,
2005b). Extracted or concentrated biological samples were injected onto HPLC and
eluted with solvents A (water) and B (acetonitrile) up to 80 % solvent B. A [^14 C] labeled
drug was infused into the HPLC effluent using a post column Tee. The mixed effluent
was collected into 96-well plates for TopCount analyses or test tubes for LSC analysis at
four fractions per min for 10 min counting. (a) TopCount control, no injection of a
biological sample; (b) LSC control, no injection of a biological sample; (c) HLM
(equivalent to 1 mL HLM incubation); (d) human plasma (equivalent to 1 mL plasma);
(e) human urine (equivalent to 2 mL urine); (f) human feces (equivalent to 100 mg feces).
The up and down lines in each figure represent15 % of the mean values.
296 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES