# Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS
## 3 Results and Discussion
### Application of the Method for Samples Analysis  
The applicability of our method was demonstrated by the quantitative profiling of endogenous IsoP in six human pancreatic cancer cell lines: AsPC1, MiaPaca, BxPC3, Panc1, S2013 and Capan-1 (Table 5). GPP levels were below the limit of quantitation in Capan-1 and MiaPaca cell lines and could not be quantified. In order to find correlations among the different cancer cell lines, the mean concentrations (given in nmol/10\({}^{6}\) cells) and standard deviation of isoprenoid concentrations were recalculated in relation to the number of cells in each analyzed sample. A wide variation of the intracellular concentration for each targeted isoprenoid among the different cancer cell lines was observed. The highest concentrations of GPP and FPP were observed in the Panc1 cell lines.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & \multicolumn{3}{c}{**% Stability (Mean \(\pm\) SD)**} \\ \cline{2-4}  & **Auto-Sampler (4 \({}^{\circ}\)C, 36 h)** & **Long-Term (\(-\)80 \(\pm\) 5 \({}^{\circ}\)C, 30 days)** & **Bench-Top (20 \({}^{\circ}\)C, 8 h)** \\ \hline
**GPP** & 96.4 \(\pm\) 4.6 & 91.3 \(\pm\) 7.8 & 103.4 \(\pm\) 8.6 \\
**FPP** & 93.5 \(\pm\) 9.6 & 102.5 \(\pm\) 9.5 & 98.5 \(\pm\) 9.1 \\
**GGPP** & 104.4 \(\pm\) 6.7 & 93.6 \(\pm\) 7.3 & 109.2 \(\pm\) 5.2 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Mean stability of the GPP, FPP and GGPP at different storage conditions in human plasma.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & \multicolumn{2}{c}{**Carcoalated Human Plasma**} & \multicolumn{2}{c}{**Buffer**} \\ \cline{2-5}  & **LQC** & **HQC** & **LQC** & **HQC** \\ \hline
**GPP** & 55.1 \(\pm\) 4.2 & 59.1 \(\pm\) 5.9 & 85.1 \(\pm\) 9.1 & 81.5 \(\pm\) 9.9 \\
**FPP** & 63.8 \(\pm\) 5.9 & 64.2 \(\pm\) 4.4 & 87.8 \(\pm\) 10.5 & 84.8 \(\pm\) 8.9 \\
**GGPP** & 40.2 \(\pm\) 4.4 & 47.3 \(\pm\) 5.5 & 74.5 \(\pm\) 8.7 & 88.5 \(\pm\) 5.7 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Mean extraction recoveries (percentages, mean \(\pm\) SD, n = 3) of GPP, FPP and GGPP from human plasma and cultured cancer cells.  
We also analyzed human plasma samples to measure the endogenous concentrations of individual IsoPs. The plasma concentrations of GPP, FPP and GGPP were 0.14 \(\pm\) 0.02, 0.25 \(\pm\) 0.09 and 1.66 \(\pm\) 0.54 ng/mL in human plasma, respectively. The obtained plasma concentrations of FPP (0.25 \(\pm\) 0.09 ng/mL) in this study were lower than the previously reported value of 1.61 \(\pm\) 0.40 ng/mL, which may be due to sample variability [24]. Previously, others have not reported human plasma GGPP levels because of the presence of interfering peaks [24]. Our results demonstrate marked variation in the basal concentrations of FPP, GPP and GGPP in human plasma and human pancreatic adenocarcinoma cell lines. The relationship between isoprenoid pool sizes and tumor characteristics is currently not well understood and further research is needed to define this relationship as well as to determine the impact of isoprenoid pool sizes on the response to therapies targeting this pathway.