Isoprenoids are a large family of compounds (more than 55,000) comprising numerous products used as fragrances, insecticides, nutraceuticals and pharmaceuticals 
. Supply of these molecules has been limited by scarce plant resources from which they were originally extracted. Production by chemical synthesis is uneconomical due to the complex structure of these products 
. Microbial metabolic engineering has been intensively explored in the past decade for isoprenoid production and gram per liter production levels has been achieved for certain isoprenoid precursors 
Despite the structural diversity of isoprenoids, they are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Both metabolites are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway () 
. Engineering of the DXP pathway (also known as MEP pathway) in Escherichia coli (E. coli)
results in significant increase in the biosynthesis of IPP/DMAPP 
. Four enzymes in the DXP pathway (dxs, idi, ispD and ispF) were identified combinatorially to be rate limiting 
and overexpressions of the genes encoding these enzymes were empirically optimized to maximize isoprenoid yields. It is also known that excessive overexpression of these enzymes can inhibit isoprenoid production 
. Recently, we demonstrated that strong overexpression of metabolic enzymes (dxs, idi, ispD and ispF) did not affect the expression of isoprenoid transcripts which was hypothesized to contribute to the decrease in lycopene production 
. To better understand the mechanisms underlying the inverse correlation of lycopene production with overexpression of the DXP genes, a direct measurement of the metabolites is desirable.
Metabolites and enzymes related to the 1-Deoxy-D-xylulose 5-Phosphate pathway.
Quantification of the metabolites of the heterologous mevalonate pathway in engineered E. coli
allowed the identification of pathway limiting steps and generated rational gene targets for circumventing production inhibition 
. Attempts have been made previously to measure methylerythritol cyclodiphosphate (MEC), a metabolite of the DXP pathway, in cell extracts using a semi-quantitative and labor intensive method involving 31
. DMAPP and IPP, products of the DXP and MVA pathways, have also been previously quantified by mass-spectrometry 
. As yet, there is no direct method to simultaneously quantify all DXP pathway metabolic intermediates ().
To address the unmet need of a method for the simultaneous quantification of DXP metabolites, we have developed an integrated preanalytical solid phase extraction (SPE) procedure with the use of ultra performance liquid chromatography mass spectrometry (UPLC-MS). The SPE procedure exploits the unique physiocochemical properties of the DXP intermediates to selectively enrich these compounds from biological samples. The performance of this SPE coupled UPLC-MS protocol was then demonstrated with synthetic standards in E. coli extracts, and was found to be superior in analytical sensitivity and ease-of-use. With the developed method, MEC was found to be unexpectedly effluxed from E. coli, by yet to be characterized mechanisms, which significantly reduced the production of isoprenoids. The overexpression of ispG was predictably found to result in lower MEC efflux and increased isoprenoid production. This study has developed invaluable tools for rational engineering of the DXP pathway for overproduction of isoprenoids.