(M0930-09-49) Comparative Study of Protein Precipitation, Solid-Phase Extraction and Phospholipid Removal for Simultaneous Quantification of Serotonin, Dopamine, and Norepinephrine by LC-MS/MS in the Mouse Brain

Purpose: Monoamine (MA) neurotransmitters are important bioactive substances in mammalian organisms, which participate in several physiological functions, including emotion, learning, and memory. In addition, MAs play a crucial role in several neurological disorders, including stroke, and contribute significantly to the pathogenic mechanisms of the disease. In this study, we developed and validated a method for simultaneous quantification of the three main MAs, serotonin (5-HT), dopamine (DA), and norepinephrine (NE), in mouse cerebral cortical homogenates using highly sensitive liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The developed method may contribute to a better understanding of the pathogenic mechanisms of neurological disorders and pharmaceutical research directed towards the development of new drugs.

Methods: We compared three different methods for extraction of analytes from brain homogenates: (1) Protein Precipitation (PPT), (2) Solid-Phase Extraction (SPE) and (3) Phospholipid Removal (PLR) as a sample clean-up technique to reduce the ionization suppression induced by the matrix effect. The MAs were detected and quantified by LC-MS/MS with positive electrospray ionization, which operates in a multiple reaction monitoring mode. The recovery of the MAs was compared for all three methods. The quantitative analysis of 5-HT, DA and NE was executed using a reversed-phase Raptor Biphenyl column (100 × 2.1 mm, 2.7µm) coupled to an API-QTRAP 5500 (AB SCIEX) mass spectrometer under a gradient mode, using a 0.2% formic acid in water and 0.2% formic acid in methanol at a flow rate of 0.2 mL/min. The method was validated by determining the linearity, intra and inter-day precision and accuracy, recovery, matrix effect, carry-over and stability in cerebral cortical samples obtained from CD-1 mice. The Analyst software performed data acquisition and processing (version 1.7). Calibration curves of the MAs were established by plotting the peak area ratios of each MA to IS (internal standard) versus MA concentrations.
In addition, the basal levels of the MAs were compared in samples obtained from male and female adult mice. Lastly, the basal levels of MAs in adult mouse brains (8-9 weeks) were compared with that of old CD-1 mice (12-18 months).

Results: We observed some matrix effect (ion suppression or ion enhancement) in the case of the PPT method; however, the recovery range for the MAs was 90-100%. PLR recovery for the MAs was between 50 and 70%. Correspondingly, SPE recovery for the MAs was between 80 to 92%, and we observed less matrix effect. The recovery for the MAs improved in the case of SPE compared to PLR but failed to reach the level of the PPT method. Based on these findings, we proceeded with the PPT method to afford the highest recovery and accurate determination of endogenous MA concentrations in the brain.
The linearity for 5-HT and NE was achieved at concentrations ranging from 1.953 – 1000 ng/ml and DA at 7.812 – 2000ng/ml with an r² value > 0.99. The method exhibited good intra and inter-day precisions (RSD < 15%) with good accuracy (85-115%). The mean recovery ranged from 92% to 102%. All three MAs were well separated within 6 mins runtime. Other factors like matrix effect, carry-over and stability were consistent with the requirements of regulatory agencies. No significant differences were observed in the basal levels of the MAs in both genders for old and young animals. Nevertheless, we noticed a significant decrease in the basal level of the 5-HT old female group compared to the young female group (p < 0.0469).

Conclusion: Our findings indicate that the PPT method is best suited for simultaneous evaluation of MAs levels in the mouse brain among the three tested approaches. No substantial differences were detected in the basal levels of MAs in the brains of adult and old male and female mice.

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Acknowledgements: No conflict of interest.


Comparison of basal levels of MAs in adult mouse brains (8-9 weeks) with old CD-1 mice (12-18 months)


Table 1: Calibration Curve of MAs in the mice brain by LC-MS/MS
Table 2: Extraction stability assays for the MAs


Table 3: Matrix effect and Recovery
Table 4: Inter-Intra Day (Accuracy & Precision)