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. 2022 Aug 2;12(8):716.
doi: 10.3390/metabo12080716.

A Selective and Sensitive LC-MS/MS Method for Quantitation of Indole in Mouse Serum and Tissues

Vineet Joshi  1 , Yashpal S Chhonker  1 , Dhruvkumar Soni  1 , Kelly C Cunningham  2 , Derrick R Samuelson  2 , Daryl J Murry  1   3
Affiliations

A Selective and Sensitive LC-MS/MS Method for Quantitation of Indole in Mouse Serum and Tissues

Vineet Joshi et al. Metabolites. .

Abstract

Indole is an endogenous substance currently being evaluated as a biomarker for ulcerative colitis, irritable bowel syndrome, Crohn’s disease and non-alcoholic fatty liver disease. A novel, selective, and sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for quantitation of indole concentrations in mouse plasma and tissues. Samples were prepared by protein precipitation using ice-cold acetonitrile (ACN) followed by injecting the extracted analyte to LC-MS/MS system. Indole was separated using Synergi Fusion C18 (4 µm, 250 × 2.0 mm) column with mobile phase 0.1% aqueous formic acid (A) and methanol (B) using gradient flow with run time 12 min. The mass spectrometer was operated in atmospheric pressure chemical ionization (APCI) positive mode at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combinations of 118.1 > 91.1 m/z for indole and 124.15 > 96.1 m/z for internal standard (IS) indole d7. The MS/MS response was linear over the range of indole concentrations (1−500 ng/mL). The validated method was applied for quantitation of indole concentrations range in mouse lungs (4.3−69.4 ng/g), serum (0.8−38.7 ng/mL) and cecum (1043.8−12,124.4 ng/g). This method would help investigate the role of indole as a biomarker and understand its implications in different disease states.

Keywords: LC-MS/MS; biomarker; indole; indole biodistribution.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MS/MS product ion spectra of indole (A) in positive APCI mode [M + H], shows prominent fragments peak at m/z 91.1, 65.05, and 45.10 at CE-30 units and IS (B) shows prominent fragments peak at m/z 96.05 at CE-30 units.
Figure 2
Figure 2
Representative MRM ion-chromatograms of (a) blank mouse lung homogenate using the conditions for indole detection, (b) indole spiked in mouse serum at 10 ng/mL (rt. 5.8 min), (c) indole mouse lungs homogenate study sample (rt. 5.8 min, Id: AI-2), (d) blank mouse lung homogenate using the conditions for indole-d7 detection, (e) indole-d7 spiked in mouse lung homogenate (rt. 5.8 min, 200 ng/mL) and (f) indole-d7 spiked in mouse study lung homogenate sample (Id: AI-2).
Figure 3
Figure 3
Representative MRM ion-chromatograms of (a) blank mouse serum using the conditions for indole detection, (b) indole spiked in mouse serum at 10 ng/mL (retention time, rt. 5.8 min), (c) indole mouse study serum sample (rt. 5.8 min, Id: AI-1_9-17-20), (d) blank mouse serum using the conditions for indole-d7 (IS) detection, (e) indole-d7 spiked in mouse serum (rt. 5.8 min, 200 ng/mL) and (f) indole-d7 spiked in mouse study serum sample (Id: AI-1_9-17-20).
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