Development and validation of a LC-MS/MS assay for quantifying the uptake of docosahexaenoic acid-d5 into mouse microglia
| Autor: | Joseph A. Nicolazzo, Jennifer L. Short, Yijun Pan, Yi Ling Low |
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| Rok vydání: | 2020 |
| Předmět: |
Lysis
Docosahexaenoic Acids Clinical Biochemistry Pharmaceutical Science Endogeny 01 natural sciences Analytical Chemistry Mice Liquid chromatography–mass spectrometry Tandem Mass Spectrometry Drug Discovery Animals Bradford protein assay Spectroscopy Neuroinflammation chemistry.chemical_classification Chromatography 010405 organic chemistry 010401 analytical chemistry Area under the curve food and beverages Reproducibility of Results 0104 chemical sciences chemistry Docosahexaenoic acid Calibration lipids (amino acids peptides and proteins) Microglia Polyunsaturated fatty acid Chromatography Liquid |
| Zdroj: | Journal of pharmaceutical and biomedical analysis. 191 |
| ISSN: | 1873-264X |
| Popis: | Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid with the capacity to reduce the proinflammatory response of activated microglia that occurs during neuroinflammation. The uptake of DHA into microglia is essential for it to exert its neuroprotective effects. However, quantifying the uptake of DHA into microglia is complicated by the presence of endogenous DHA interfering with any quantification technique. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was therefore developed and validated in order to assess the microglial uptake of docosahexaenoic acid-d5 (DHA-d5) as a surrogate for DHA. Using a mobile phase consisting of 90 % (v/v) acetonitrile and 10 % (v/v) water containing 2 mM ammonium acetate, a flow rate of 0.3 mL/min, and MS/MS detection in the negative ionization mode, DHA-d5 was detected at m/z transitions of 332.1/228.3/234.2, with good linearity between chromatographic area under the curve (AUC) and DHA-d5 mass (R2 = 0.999) over the range of 0.0063-0.1 ng. The precision and accuracy values for the quality control samples (0.0063, 0.025, and 0.1 ng) were less than 9.3 % and 96.6-109.8 %, respectively, and a comparison of DHA-d5 AUC when prepared in PBS or in microglial cell lysate demonstrated no significant difference between quantification of these quality control samples. Utilizing this quantification approach (with preparation of DHA-d5 calibration standards in PBS), the uptake of DHA-d5 into BV-2 microglial cells over a 15 min period was assessed, following the spiking of DHA-d5 at 50 ng/mL. Following protein normalization using a BCA protein assay, a rapid and linear uptake of DHA-d5 into BV-2 cells was observed in the first 2 min, after which a plateau in uptake was observed, in line with that reported for DHA uptake in other cell types. This novel LC-MS/MS technique can now be exploited to unravel the processes involved in microglial uptake of DHA, insights that may be used to maximize the anti-inflammatory effects of DHA in neuroinflammation. |
| Databáze: | OpenAIRE |
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