Abstract
We demonstrate the application of in-source nitrogen collision-induced dissociation (CID) that eliminates the need for ester hydrolysis before simultaneous analysis of esterified cholesterol (EC) and triglycerides (TG) along with free cholesterol (FC) from human serum, using normal phase liquid chromatography (LC) coupled to atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). The analysis requires only 50 μL of 1:100 dilute serum with a high-throughput, precipitation/evaporation/extraction protocol in one pot. Known representative mixtures of EC and TG species were used as calibrators with stable isotope labeled analogs as internal standards. The APCI MS source was operated with nitrogen source gas. Reproducible in-source CID was achieved with the use of optimal cone voltage (declustering potential), generating FC, EC, and TG lipid class-specific precursor fragment ions for multiple reaction monitoring (MRM). Using a representative mixture of purified FC, CE, and TG species as calibrators, the method accuracy was assessed with analysis of five inter-laboratory standardization materials, showing –10% bias for Total-C and –3% for Total-TG. Repeated duplicate analysis of a quality control pool showed intra-day and inter-day variation of 5% and 5.8% for FC, 5.2% and 8.5% for Total-C, and 4.1% and 7.7% for Total-TG. The applicability of the method was demonstrated on 32 serum samples and corresponding lipoprotein sub-fractions collected from normolipidemic, hypercholesterolemic, hypertriglyceridemic, and hyperlipidemic donors. The results show that in-source CID coupled with isotope dilution UHPLC-MS/MS is a viable high precision approach for translational research studies where samples are substantially diluted or the amounts of archived samples are limited.Graphical ᅟ
Highlights
Lipoproteins are dynamic lipid-protein assemblies that function as transporters of various biomolecules circulating in blood [1]
Nonane was selected because it served as an optimal liquid chromatography (LC) injection matrix and it gave ~70% analyte recovery, which was corrected by the use of isotopically labeled internal standards
Colorimetric methods performed on clinical analyzer platforms are typically required to have bias ≤3% and coefficient of variation (CV) ≤3% for total cholesterol and bias ≤5% and CV ≤5% for total glycerides relative to a standard reference materials (SRM) value assigned by a CRLMN laboratories
Summary
Lipoproteins are dynamic lipid-protein assemblies that function as transporters of various biomolecules circulating in blood [1]. The unique features of this method are the high-throughput, 2.5 min/injection LC-MS/MS run, and the enhanced TG sensitivity, achieved by generation of a single TG class-specific CID fragment as a parent ion for MS/MS detection (instead of hundreds of individual MRMs). A similar pattern of low-mass product ions could be observed in product ion scanning mode, when the diglyceride and triglyceride precursor ions were produced in Q1 using high CE ~80 V and their fragments detected in Q3, confirming their TG origin (Figure 2, right, insert).
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