2. Academic Validation
  3. Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS

Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS

  • Molecules. 2018 Dec 11;23(12):3275. doi: 10.3390/molecules23123275.
Yashpal S Chhonker 1,  Staci L Haney 2,  Veenu Bala 3,  Sarah A Holstein 4 5,  Daryl J Murry 6 7
Affiliations

Affiliations

  • 1 Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
  • 2 Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
  • 3 Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur, Rajasthan 313001, India. [email protected].
  • 4 Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
  • 5 Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
  • 6 Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
  • 7 Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA. [email protected].
PMID: 30544938 DOI: 10.3390/molecules23123275
Abstract

Isoprenoids (IsoP) are an important class of molecules involved in many different cellular processes including Cholesterol synthesis. We have developed a sensitive and specific LC-MS/MS method for the quantitation of three key IsoPs in bio-matrices, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). LC-MS/MS analysis was performed using a Nexera UPLC System connected to a LCMS-8060 (Shimadzu Scientific Instruments, Columbia, MD) with a dual ion source. The electrospray ionization source was operated in the negative MRM mode. The chromatographic separation and detection of analytes was achieved on a reversed phase ACCQ-TAG Ultra C18 (1.7 µm, 100 mm × 2.1 mm I.D.) column. The mobile phase consisted of (1) a 10 mM ammonium carbonate with 0.1% ammonium hydroxide in water, and (2) a 0.1% ammonium hydroxide in acetonitrile/methanol (75/25). The flow rate was set to 0.25 mL/min in a gradient condition. The limit of quantification was 0.04 ng/mL for all analytes with a correlation coefficient (r2) of 0.998 or better and a total run time of 12 min. The inter- and intra-day accuracy (85⁻115%) precision (<15%), and recovery (40⁻90%) values met the acceptance criteria. The validated method was successfully applied to quantitate basal concentrations of GPP, FPP and GGPP in human plasma and in cultured Cancer cell lines. Our LC-MS/MS method may be used for IsoP quantification in different bio-fluids and to further investigate the role of these compounds in various physiological processes.

Keywords

LC-MS/MS; farnesyl pyrophosphate; geranyl pyrophosphate; geranylgeranyl pyrophosphate.

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