Small Molecules Quantitation

Small molecule bioanalytical quantitation can be used to measure the amount of a small molecule in complex protein mixtures, such as plasma, urine and cell extracts. LC-MS is performed using selected reaction monitoring (SRM) on an AB SCIEX 4000 QTRAP with samples ranging from clinical research to food and environmental analysis.

Yi T, Rao DA, Tang PC, Wang Y, Cuchara LA, Bothwell AL, Colangelo CM, Tellides G, Pober JS, Lorber MI. (2008) Amelioration of human allograft arterial injury by atorvastatin or simvastatin correlates with reduction of interferon-gamma production by infiltrating T cells. Transplantation. 86:719-27.

Assay Information

The principles upon which the Selected reaction monitoring (SRM)/ Multiple reaction monitoring (MRM) approaches rest are based on a very robust MS technology that has been used for many years to quantify a wide range of small molecules in clinical samples (Hankin and Murphy, 2004). This high-throughput MS method has a wide linear dynamic range of up to five orders of magnitude and also has a very high sensitivity that allows detection of ng/ml amounts of compound in complex biological matrices such as plasma, serum, or tissue protein extracts.

Small molecule compounds can be quantitated in complex mixtures using SRM/MRM on a triple quadrupole mass spectrometer. In SRM/MRM assays the first (Q1) and last (Q3) mass analyzers of a triple quadrupole mass spectrometer are used as mass filters to isolate a peptide ion and a corresponding fragment ion. The signal of the fragment ion is then monitored over the chromatographic elution time (Figure 2). The selectivity resulting from the two filtering stages, combined with the high duty cycle, results in quantitative analyses with unmatched sensitivity and specificity. The specific pairs of m/z values associated to the precursor and fragment ions selected are referred to as "transitions" and effectively constitute mass spectrometric assays that allow to identify and quantify a specific fragment molecule and, by inference, the corresponding small molecule in a complex mixture.

Application Example

Quantitation of a Chemical Messenger Linking the Gut to Brain Reward Centers

References

Hankin JA, Murphy RC (2004) Mass spectrometric quantitation of deoxyguanosine and leukotriene A4-deoxyguanosine adducts of DNA. Anal Biochem. 333:156-164.

Murray KK, Boyd RK, Eberlin MN, Langley GJ, Li L, Naito Y (2006) International Union of Pure and Applied Chemistry – Analytical Chemistry Division

Yi T, Rao DA, Tang PC, Wang Y, Cuchara LA, Bothwell AL, Colangelo CM, Tellides G, Pober JS, Lorber MI. (2008) Amelioration of human allograft arterial injury by atorvastatin or simvastatin correlates with reduction of interferon-gamma production by infiltrating T cells. Transplantation. 86:719-27.