# Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS
## 3 Results and Discussion  
### Chromatographic and Mass Spectrometric Conditions Optimization  
The mass spectrometric conditions for detection of IsoP and IS were optimized in the electrospray ionization mode (ESI). To achieve a high selectivity and sensitivity for this method, we optimized our method by comparing electrospray ionization in the negative mode and an atmospheric pressure chemical ionization (APCI) source. ESI in the negative mode was found to offer an improved intensity for the analytes compared to the APCI mode (data not shown). During method optimization, the mass spectra for GPP, FPP, GGPP and IS revealed peaks at \(m/z\) 313.1, 381.2, 449.2 and 356.2, respectively as deprotonated molecular ions, \([\mathrm{M}-\mathrm{H}]^{-}\). The fragmentation of analytes and IS were auto-optimized via a product ion search of approximately 1000 ng/mL of stock solution for each analyte. The most abundant precursor > product ions for GPP, FPP, GGPP and IS were found to be \(m/z\) 313.1\(\rightarrow\)79.1, 381.2\(\rightarrow\)79.1, 449.2\(\rightarrow\)79.1 and 356.2\(\rightarrow\)79.1, respectively (Figure 2). Chromatographic conditions were optimized to separate all IsoPs of interest from interference with a desirable peak shape and signal intensity using an ACCQ-TAG Ultra C18, 100 mm \(\times\) 2.1 mm I.D., 1.7 \(\upmu\)m, column (Waters). Different solvent systems such as MeCN and MeOH with various buffers including ammonium carbonate and ammonium bicarbonate with different pH ranges, composition and different flow rates were evaluated. The suitability and robustness of the method were evaluated using different varieties of reverse phase HPLC columns ranging from 50 mm to 150 mm in length (data not shown). The less hydrophobic IsoPs including MVA and IPP eluted early and independently of the mobile phase pH. NH\({}_{4}\)OH (0.1%) was used as an aqueous and organic mobile phase modifier as a basic pH mobile phase resulted in better peak shape and a longer retention of the more hydrophobic IsoPs including GPP, FPP and GGPP. Under our final LC-MS/MS conditions, all IsoPs of interest were resolved from each other as well co-eluting isobaric interferences for target analytes in less than 10 min. IsoPs are endogenous compounds and are isobaric with very similar physio-chemical properties. Endogenous compounds not only share the same mass, but also may have the same fragmentation ion (79 is common ion representing the phosphate group), resulting in the same MRM transitions. In addition, interfering peaks could arise from other unknown endogenous matrix components. Therefore, MS/MS specificity is not always adequate for separation of all IsoPs, and chromatographic resolution is required for their separation and accurate quantification. For example, FPP and GGPP had unknown endogenous co-eluting peaks including the same MRM transition but we were able to separate them chromatographically (Figure 3). Matrix effects became problematic while performing quantification of endogenous analytes including IsoPs. Therefore, it is important to prepare calibration curves in the same or equivalent matrices as of the study samples to minimize the matrix effects. Various approaches have been reported to solve the problem of endogenous background in blank matrices for the construction of calibration curves including background subtraction [32; 33], the standard addition method [34], stripping the matrix with charcoal [35; 36], and the use of surrogate matrices or stable-isotope labeled standards [37; 38; 39; 40; 41; 42]. Figure 2: MS/MS product ion spectra of (**a**) GPP, (**b**) FPP, (**c**) GGPP and (**d**) RAM1147 (internal Standard). By comparing various calibration approaches, we found that depleting endogenous analytes in human plasma by using activated charcoal was the most accurate and convenient method for this application. Therefore, blank plasma was prepared by stripping plasma from the endogenous IsoP using activated charcoal. The charcoal stripping conditions were optimized to maximize IsoP depletion from plasma.