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. 2019 Jun 12;13(1):76.
doi: 10.1186/s13065-019-0591-x. eCollection 2019 Dec.

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium

Qiangqiang He  1   2   3 , Maoru Li  4   5   6 , Xuechun Wang  4 , Zhenjiang Xia  1   2   3 , Yuzhi Du  1   2 , Yan Li  1   2   3 , Lixin Wei  1   2 , Jing Shang  1   2   4   5   6
Affiliations

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium

Qiangqiang He et al. BMC Chem. .

Abstract

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 μm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (90:10 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 μg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.

Keywords: 5-Hydroxytryptamine; Bioanalytical method validation; Cell culture medium; HPLC–UV; RIN-14B cells.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Chemical structure of 5-HT
Fig. 2
Fig. 2
Representative imagines of RIN-14B cells (cultured for 48 h, Scale bar 200 μm/100 μm)
Fig. 3
Fig. 3
Flow chart of the experiment
Fig. 4
Fig. 4
5-HT synthesis and secretion by RIN-14B cells. Chromatograms of FBS free 1640 medium sample (a), cell extract sample (b), FBS free 1640 medium sample in which RIN-14B were cultured for 24 h (c), and 5-HT standard sample (d)
Fig. 5
Fig. 5
Representative chromatograms of a cell extracts sample (a), and cell culture medium sample (b), 5-HT standard sample (c), and TRP standard sample (d)
Fig. 6
Fig. 6
Superposition chromatograms of the 5-HT standard solution of 20 μg/mL and a blank sample
See this image and copyright information in PMC

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