# Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS
## 1 Introduction  
The isoprenoid biosynthesis pathway (IBP) plays key roles in cellular metabolism and is responsible for the production of both sterol and non-sterol isoprenoids [1; 2]. Because the IBP is an important target in many areas of ongoing research, new inhibitors that target specific enzymes in this pathway are under development. Clinically utilized IBP inhibitors include the statins, which inhibit HMG CoA reductase and are used to treat hyperlipidemia, and the nitrogenous bisphosphonates, which inhibit farnesyl diphosphate synthase (FDPS) and are used to treat a variety of bone diseases including osteoporosis [3], metastatic bone disease [4] and myeloma bone disease [5]. In addition, there is significant interest in the therapeutic potential of inhibiting this pathway ina variety of other diseases, including cancer [2; 6; 7; 8; 9], tuberculosis [1], Parkinson's disease [10; 11; 12] and hypercholesterolemia [13]. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are utilized as the isoprenoid donors for protein farnesylation and geranylgeranylation reactions, respectively. Farnesylation and geranylgeranylation reactions are enzyme-mediated and catalyzed by farnesyl protein transferase (FIase) and geranylgeranyl protein transferases (GGTases) I and II respectively. The membrane localization and function of various proteins, including that of the Ras GTPase superfamily members is facilitated by protein isoprenylation [14]. Therefore, IsoP concentrations in cell culture, plasma and relevant tissues are of great interest in monitoring pathological conditions and modulation following subsequent therapeutic interventions. All isoprenoids share the basic C5 isoprene units, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) and are synthesized in the non-sterol, pre-squalene part of the isoprenoid biosynthesis pathway [15; 16]. The head-to-tail condensation of IPP to DMAPP results in the formation of geranyl pyrophosphate (GPP) and the further addition of another IPP produces FPP. GGPP is produced by the condensation of one FPP with one IPP molecule (Figure 1). Various analytical tools have been developed and validated for the separation and quantification of these IsoPs, including LC-fluorescence detection [17; 18; 19; 20; 21; 22], and liquid chromatography-mass spectrometry (LC-MS/MS) [23; 24; 25; 26]. LC-fluorescence improves the sensitivity and specificity of IsoP after derivatization but the derivatization process is expensive, time consuming and of limited sensitivity, which makes quantitating IsoP difficult, given that biological concentrations are typically in the pM to nM range. The high sensitivity and selectivity of LC-MS/MS can overcome most of these limitations, which makes it the method of choice for the quantification of IsoP in biological  
Figure 1: The isoprenoid biosynthetic pathway. Geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) are key intermediates in the IBP. matrices. [PERSON] et al. reported that high performance liquid chromatography (HPLC) with fluorometric detection allowed the determination of FPP in human brain tissue with a lower limit of quantitation (LLOQ) of 10 ng/mL [18]. [PERSON] et al. reported that HPLC with fluorometric detection following a derivatization process, allowed the determination of FPP and GGPP in tissues [21]. [PERSON] et al. reported the direct analysis of IsoP in HepG2 cells with an LLOQ of 10 ng/mL [25; 26]. [PERSON] et al. reported a LC-MS/MS method for detecting dansyl-labeled pentapeptide FPP with a LLOQ > 5 ng/mL in human brain tissue [24]. A LC-MS/MS method to detect FPP in human plasma was reported with a LLOQ > 0.02 ng/mL. However, this assay was unable to quantitate GGPP due to interfering peaks [23]. Therefore, a highly sensitive bioanalytical method for determining IsoP in bio-matrices is needed to assess alterations and target the effects in the IBP.