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Abstract INTRODUCTION:
Quantification of phosphatidylethanol (PEth) homologs in whole blood for assessment of ethanol use is analytically challenging, as evidenced by the wide variability of the published LC-MS/MS methods, all of which are rather complicated. Clinical PEth tests tend to rely on a combination of extensive sample preparation and/or complicated chromatography with prolonged column washing or regeneration, which extends analysis times and lowers throughput. Some notable analytical challenges include: 1) the need for comparable extraction efficiencies of presumably soluble exogenous versus membrane-bound endogenous PEths in calibrators versus patient samples; 2) chromatographic separation of the two major clinically informative PEth homologues (PLPEth and POPEth) from each other due to potential cross-talk; 3) additional sample clean up and chromatography in order to limit cross-talk from other, far more abundant, phospholipids that produce the same fragments as the PEth analytes; 4) achieving adequate chromatographic separation and ionization efficiency for required sensitivity while maintaining short run times for high throughput. Here we demonstrate novel ways to overcome these challenges with simple sample preparation and short run times suitable for clinical analysis.
METHODS:
Extensive method development evaluating a wide breadth of mass spectrometric, liquid chromatographic, and sample preparation parameters was necessary to overcome the abovementioned challenges and develop a satisfactory clinical phosphatidylethanol method on our laboratory’s LC-MS/MS instruments.
RESULTS:
Mass spectrometric evaluation demonstrated the feasibility of using positive ionization mode (Na+ adduct) as an alternative to the previously described negative ionization mode. Retention on a C18 column and ionization of PEths were found to be exquisitely sensitive to pH and to proportion of organic solvent, respectively. Consequently, a novel LC approach using a low-to-high pH gradient in high percentage of acetonitrile allowed for initial analyte retention on a C18 column despite high proportion of organic solvent in the sample, baseline separation of the two PEth homologs, and elution of the analytes in 95% acetonitrile - resulting in excellent ionization efficiency (required for sufficient sensitivity). The pH component of this LC gradient allowed tuning the separation of PEth (pH sensitive retention) from other phospholipids (pH insensitive). Using a quaternary pump with four different mobile phases also allowed to incorporate an isopropanol column wash. An 85% isopropanol wash was found to be much superior to higher proportions of isopropanol for elution of remaining phospholipids to mitigate crosstalk. The above enabled a 4-minute analytical run time with a simple protein precipitation for sample preparation.
DISCUSSION:
None of the published PEth methods provided adequate performance on our laboratory’s LC-MS/MS instruments, necessitating extensive method development. We discovered that positive ionization using Na+ adduct resulted in similar analyte peak areas as the well-described negative ionization mode, making it a viable alternative. However, it resulted in higher background noise on our systems but may nevertheless be worthwhile exploring on other instruments. While using a low-to-high pH gradient in acetonitrile overcame chromatographic and ionization challenges, it requires tuning of the gradient to standardize retention times between columns/instruments and additional attention must be paid to preparation and storage of basic mobile phases. Using an 85% isopropanol wash versus higher percentage organic significantly reduced the time required for column washing. Ultimately, while our method requires a quaternary pump to enable pH gradient with acetonitrile elution and isopropanol wash, it allows for a simple protein precipitation sample preparation and quick 4-minute analysis time.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
