Determination of Oxidized Phosphatidylcholines and Lysophosphatidylcholines in Exhaled Particles : Liquid Chromatography-Tandem Mass Spectrometry Method

Abstract

Introduction: Identification and quantification of OxPCs and LPCs in the lung surfactant with non-invasive sampling techniques and validated analytical methods are of great importance in terms of exploring biomarkers of oxidative stress in lung and other lung related diseases.
Background: Although there are some well-structured analytical methods for the detection and quantification of OxPCs, there is no standardized method and no consensus on the best way. In general, quantification of OxPCs and LPCs in the pulmonary surfactant is challenging especially due to the lack of well-developed sampling technique. Particles in exhaled air (PExA), a non-invasive sampling method, is a promising candidate to close this gap.
Aims: The aims of the present research were to study the auto-oxidation of POPC to improve the sampling and the sample preparation technique, to improve and validate an RP-UHPLC-MS/MS method, and to apply the analytical method for the absolute quantification of PONPC and LPC 16:0 in PEx.
Methods: Auto-oxidation of POPC was evaluated based on the change in the levels of POPC and PONPC with air-exposure of POPC spiked on the sample membrane. In addition, influence of time and temperature on auto-oxidation during the extraction step was studied.
The improved in-house RP-UHPLC-MS/MS method was validated in terms of matrix effect, precision, accuracy, recovery, and carry-over. Data acquisition was achieved by MRM mode using an ESI probe in negative polarity. Absolute quantification of PONPC and LPC 16:0 was performed in PEx samples from healthy individuals (n=8), where two of them were smokers.
Results: Auto-oxidation of POPC was observed during sample collection, storage, preparation, and extraction. The auto-oxidation was reduced by decreasing air-exposure time during sample collection and preparation, and by lowering the extraction temperature and time.
The proposed RP-UHPLC-MS/MS method was found to be precise and accurate (in terms of within-run precision and accuracy) for the absolute quantification of PONPC and LPC 16:0 when the calibration and the analysis was performed in a PTFE matrix. The extraction recovery was found to be high, and no analyte carry-over over was found with regards to the analytical pathway.
Conclusion: The analytical method was successful for the absolute quantification of PONPC and LPC 16:0 in PEx, but the absolute quantity of endogenous PONPC in PEx could not be determined due to the auto-oxidation of POPC during sample collection and preparation.

Popular Abstract

Recently, identification and quantification of oxidized phosphatidylcholines (OxPCs) and lysophosphatidylcholines (LPCs) in lung surfactant is gaining more attention since they are considered potential biomarkers of oxidative stress in lung diseases. Particles in exhaled air (PExA), which provides collection of non-volatile particles originated from the airway with specific breathing technique, is a convenient sampling method for lipid analysis. PExA method is non-invasive and relatively simple, and demands less extraction procedures, making it attractive for clinical and research oriented large population studies. However, the compatibility of PExA method for determination of OxPCs in PEx needs to be studied since auto-oxidation of unsaturated phospholipids can give erroneous results by overestimating the levels of OxPCs.
The aims of the present research were to improve the PEx sample collection and preparation technique with regards to the auto-oxidation, to improve and validate an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method, and to apply the analytical method for absolute quantification of the most abundant OxPC and LPC, namely 1-palmitoyl-2-(9`-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocoline (LPC 16:0), respectively, in PEx.
The experimental studies showed that auto-oxidation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) during collection, handling and extraction of PEx samples is an important issue that needs to be addressed for quantification of endogenous PONPC accurately. The studies conducted on POPC air-exposure showed that measures such as decreasing air-exposure time during sample collection and preparation, and reducing the extraction temperature and time helped to minimize the auto-oxidation. The proposed UHPLC-MS/MS method was found to be precise and accurate (in terms of within-run precision and accuracy) for the quantification of PONPC and LPC 16:0 when the calibration was performed in the matrix with the membrane used for PEx collection. The analytical method was successful for the absolute quantification of PONPC and LPC 16:0 in PEx obtained from healthy individuals (n=8, smokers and non-smokers), but the absolute quantity of endogenous PONPC in PEx could not be determined due to the auto-oxidation of POPC during sample collection. It is believed that the present study will provide researchers with an effective bioanalytical tool that enables investigation of biomarkers of airway inflammation and other lung diseases. However, further improvement of sample collection conditions addressing auto-oxidation during sampling is necessary.

• author: Shovkedov, Parahat
• supervisor: Ann-Charlotte Almstrand
• organization: Department of Chemistry
• alternative title: Determination of Oxidized Phosphatidylcholines and Lysophosphatidylcholines in Exhaled Particles
• course: KEMR30 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: auto-oxidation, lysophosphatidylcholine, oxidized phosphatidylcholine, PExA, validation, analytical chemistry
• language: English
• id: 9126442
• date added to LUP: 2023-06-20 11:53:39
• date last changed: 2023-06-20 11:53:39