A validated hplc-uv method and optimization of sample preparation
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Norepinephrine and serotonin are two important neurotransmitters whose variations in brain are reported to be associated with many common neuropsychiatric disorders. Yet, relevant literature on estimation of monoamines in biological samples using HPLC-UV is limited.
OBJECTIVE: The present study involves the development of a simultaneous HPLC-UV method for estimation of norepinephrine and serotonin along with optimization of the sample preparation technique.
MATERIALS AND METHODS: Chromatographic separation was achieved by injecting 20 µL of the sample after extraction into Quaternary pump HPLC equipped with C18 column using 0.05% formic acid and acetonitrile (90:10, v/v) as the mobile phase with 1 mL min(-1) flow rate. The developed method was validated as per the ICH guidelines in terms of linearity, accuracy, repeatability, precision, and robustness.
RESULTS AND DISCUSSION: The method showed a wide range of linearity (50-4000 and 31.25-4000 ng mL(-1) for norepinephrine and serotonin, respectively). The recovery was found to be in the range of 86.04-89.01% and 86.43-89.61% for norepinephrine and serotonin, respectively. The results showed low value of %RSD for repeatability, intra and inter-day precision, and robustness studies. Four different methods were used for the extraction of these neurotransmitters and the best one with maximum recovery was ascertained.
CONCLUSION: Here, we developed and validated a simple, accurate, and reliable method for the estimation of norepinephrine and serotonin in mice brain samples using HPLC-UV. The method was successfully applied to quantify these neurotransmitters in mice brain extracted by optimized sample preparation technique.
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