Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy
Despite the increasing popularity of liquid chromatography–mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantificati...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/12/9/1265 |
| _version_ | 1797490668086493184 |
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| author | Harley Robinson Jeffrey Molendijk Alok K. Shah Tony Rahman Gregory J. Anderson Michelle M. Hill |
| author_facet | Harley Robinson Jeffrey Molendijk Alok K. Shah Tony Rahman Gregory J. Anderson Michelle M. Hill |
| author_sort | Harley Robinson |
| collection | DOAJ |
| description | Despite the increasing popularity of liquid chromatography–mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids. However, the impact of complex lipid sample complexity and purity on total lipid quantification accuracy has not been investigated. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance (ATR)-FTIR and establish a simple workflow for lipidomics sample quantification. We show that both pure and complex lipids show characteristic FTIR vibrations of CH- and C=O-stretching vibrations, with a quantitative range of 40–3000 ng and a limit of detection of 12 ng, but sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification via CH stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. Taking all tested parameters together, we propose a rapid FTIR workflow for routine lipidomics sample quality and quantity assessment and tested this workflow by comparing to the total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. The uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. With low sample requirement, we anticipate this simple and rapid method will enhance lipidomics workflow by enabling accurate total lipid quantification and normalization of lipid quantity for MS analysis. |
| first_indexed | 2024-03-10T00:36:18Z |
| format | Article |
| id | doaj.art-58134eaf97e54abbb54bfe1dbb3e7b50 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T00:36:18Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-58134eaf97e54abbb54bfe1dbb3e7b502023-11-23T15:15:47ZengMDPI AGBiomolecules2218-273X2022-09-01129126510.3390/biom12091265Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared SpectroscopyHarley Robinson0Jeffrey Molendijk1Alok K. Shah2Tony Rahman3Gregory J. Anderson4Michelle M. Hill5QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, AustraliaQIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, AustraliaQIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, AustraliaDepartment of Gastroenterology & Hepatology, The Prince Charles Hospital, Rode Road, Brisbane, QLD 4032, AustraliaQIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, AustraliaQIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD 4006, AustraliaDespite the increasing popularity of liquid chromatography–mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids. However, the impact of complex lipid sample complexity and purity on total lipid quantification accuracy has not been investigated. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance (ATR)-FTIR and establish a simple workflow for lipidomics sample quantification. We show that both pure and complex lipids show characteristic FTIR vibrations of CH- and C=O-stretching vibrations, with a quantitative range of 40–3000 ng and a limit of detection of 12 ng, but sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification via CH stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. Taking all tested parameters together, we propose a rapid FTIR workflow for routine lipidomics sample quality and quantity assessment and tested this workflow by comparing to the total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. The uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. With low sample requirement, we anticipate this simple and rapid method will enhance lipidomics workflow by enabling accurate total lipid quantification and normalization of lipid quantity for MS analysis.https://www.mdpi.com/2218-273X/12/9/1265lipidsphospholipidssphingolipidstriglyceridesFTIRmass spectrometry |
| spellingShingle | Harley Robinson Jeffrey Molendijk Alok K. Shah Tony Rahman Gregory J. Anderson Michelle M. Hill Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy Biomolecules lipids phospholipids sphingolipids triglycerides FTIR mass spectrometry |
| title | Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy |
| title_full | Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy |
| title_fullStr | Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy |
| title_full_unstemmed | Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy |
| title_short | Rapid Assessment of Lipidomics Sample Purity and Quantity Using Fourier-Transform Infrared Spectroscopy |
| title_sort | rapid assessment of lipidomics sample purity and quantity using fourier transform infrared spectroscopy |
| topic | lipids phospholipids sphingolipids triglycerides FTIR mass spectrometry |
| url | https://www.mdpi.com/2218-273X/12/9/1265 |
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