are generally utilized for the calculation of relative radioactivity abundance. If
counting efficiencies of HPLC effluent fractions across the entire HPLC run are
consistent, or vary within an accepted range, the relative abundance of
radioactivity peaks calculated from CPM values will not be significantly
different from those of DPM values. In the following sections sensitivity,
precision, and accuracy of radioactivity profiling by HPLC–TopCount are
described. In addition, biological matrix effects on HPLC–MSC performance
are discussed.
10.3.1.1 Sensitivity The limit of detection of radioactivity counting can be
calculated using the following Equation 10.1 (Zhu et al., 2005b), where LD is
the limit of detection expressed in DPM,Bis the background radioactivity
expressed in DPM,Tis counting time (min) andEis counting efficiency.
LD¼ 2 : 71 =TEþ 4 : 65ffiffiffiffiffiffiffiffiffiffiffiffi
B=TEp
ð 10 : 1 ÞA primary factor contributing to the superior sensitivity of off-line
scintillation counting techniques is the greater counting time available. In
HPLC–MSC analysis, the counting time is typically around 10 min, which is
approximately 30-to 60-fold longer than that in HPLC–RFD analysis (Table
10.1). The low background radioactivity (1–2 DPM) of MSC is another factor
contributing to its superior sensitivity (Zhu et al., 2005b). The limit of detection
for^14 Con a TopCount instrument with 10 min counting was favored to be
approximately 5 DPM (Table 10.1). This was approximately twofold better
than LSC, and 50–100-fold better than RFD (Zhu et al., 2005b). A comparison
of the radiodetection sensitivity of MSC, LSC, and RFD is illustrated in Fig.
(a) LC/UVLC/UV MicroplateMSMSC(b) LC/UVLC/UV MicroplateMSMSCRecovered
radioactivityLC/MSFIGURE 10.3 Configuration of HPLC with an off-line MSC with or without coupling
with a mass spectrometer.
294 APPLICATIONS OF LIQUID RADIOCHROMATOGRAPHY TECHNIQUES