MSACL 2025 Abstract

Quantitative LC-MS/MS Assay for Ceramides in Plasma and Serum: Development, Validation, and Performance Evaluation

Amol O. Bajaj(1), Mark M. Kushnir(1,2), Preejith Vachali(1,3), Sophie Steckel(1), Gwendolyn A. McMillin(1,2), Kelly Doyle(1,2)
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA. (2) University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, Utah, USA. (3) Current affiliation: Cleveland Clinic, Robert J. Tomsich Department of Pathology and Laboratory Medicine, Cleveland, Ohio, USA.

Amol Bajaj, MS (Presenter) ARUP >> POSTER (PDF)

Presenter Bio: I am a Scientist III in R&D Mass Spectrometry at the ARUP Institute for Clinical and Experimental Pathology. I hold an MS in Biotechnology from the University of Houston Clear Lake. My primary focus is on developing innovative analytical strategies that enhance the capability of clinical laboratories to perform challenging assays. Currently, I am working on methodologies for detecting ceramides in serum/plasma, which are biomarkers for cardiovascular diseases.
I have also developed high-resolution mass spectrometric methods for the characterization and identification of microorganisms, including mycobacteria. Mycobacterium tuberculosis, responsible for one of the most devastating infectious diseases, claimed nearly 1.3 million lives in 2020 and infected approximately 10 million people. This pathogen can remain dormant within the human host, posing significant diagnostic challenges.
In addition to my work with mycobacteria, I have recently developed and validated a Thiopurine metabolites assay. Thiopurine drugs, used to treat conditions such as acute leukemia, inflammatory bowel disease (IBD), and other autoimmune diseases, are critical in managing these conditions. With IBD now a global health concern affecting over 5 million people worldwide, and autoimmune diseases like rheumatoid arthritis affecting millions in the USA, the need for precise and reliable assays has never been greater.
I am also deeply interested in the development and validation of methods for proteins and peptide biomarkers. I have successfully developed techniques for detecting intact insulin growth factors 1 (IGF1) and 2 (IGF2) in human serum, which play a crucial role in childhood growth and have anabolic effects in adults.

Relevant Financial Disclosures (within past 24 months, reported on May 20, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

BACKGROUND:
Ceramides have emerged as novel diagnostic and prognostic biomarkers for cardiovascular disease (CVD), providing additional risk prediction beyond traditional lipid measures. Several studies have highlighted their potential utility as biomarkers for insulin resistance, type 2 diabetes mellitus, severity of coronary artery disease, atherosclerosis, dyslipidemia, malignancies, and neuroinflammation. We developed a quantitative LC-MS/MS method to measure Cer 16:0, Cer 18:0, Cer 24:0, and Cer 24:1 in plasma and serum and evaluated the method’s performance.

METHODS:
Aliquots of human plasma or serum samples (40 µL) were mixed with stable isotope-labeled internal standard (IS, deuterium labeled analogs of each ceramide, 40 µL), water (100 µL) and methyl tert-butyl ether (MTBE, 400 µL). The tubes were vortexed for 20 min, and then centrifuged for 5 min at 16,000 g. The supernatants were transferred to autosampler vials, and the samples were analyzed using a 6475 LC-MS/MS, equipped with a 1290 series HPLC (Agilent). Chromatographic separation was performed using a CSH C18 column (1.7 µm, 5 cm, 100 Å, 2.1 mm ID, Waters). Quantification was performed using a six-point calibration curve (0.02–2 µmol/L for Cer 16:0 and Cer 18:0; 0.2–20 µmol/L for Cer 24:0 and Cer 24:1), monitoring two mass transitions per analyte and IS. The injection volume was 5 µL, and data acquisition was performed in positive ion mode. Evaluation of the method’s performance included assessment of precision, sensitivity, linearity, accuracy, specificity, matrix effects, dilution integrity, carryover, robustness, and correlation with validated methods performed by other laboratories. Blood collection tube types, stability, and the adsorptive losses of ceramides during the sample preparation were also evaluated. All studies with samples from human subjects were approved by the Institutional Review Board of the University of Utah (Salt Lake City, UT).

RESULTS:
We developed and evaluated analytical performance of a method for quantifying four ceramides in plasma and serum samples by LC-MS/MS. The issues related to poor retention of Cer 16:0 and Cer 18:0, and adsorptive losses of ceramides were addressed with sample preparation methods and chromatographic separation. A CSH C18 column (Waters) provided the best HPLC performance. In a sample preparation method involving solvent extraction, evaporation, and reconstitution, a variation in the slopes of the calibration curves was observed using calibrators prepared in a Mass Spec Gold Serum (Golden West Diagnostics) matrix and those prepared in a pool of patient samples. However, when the evaporation and the reconstitution steps were omitted, and the extracts were directly injected for instrumental analysis, the variation in the slopes of the regression lines were comparable, suggesting adsorptive losses of the ceramide during evaporation and reconstitution steps of sample preparation.

The assay demonstrated a total imprecision (CV) of <2-8.0% across the analytical measurement range for all the analytes included in the assay. In experiments on the evaluation of the methods’ accuracy, the observed concentrations agreed with the expected concentrations within 10% (imprecision among the replicates <5%). Slopes of linear regression lines (R2) for the agreement between the expected and the observed concentrations were 0.99 (0.996), 0.99 (0.995), 0.99 (0.995) and 0.99 (0.995) for Cer 16:0, Cer 18:0, Cer 24:0, and Cer 24:1, respectively. The lower limit of detection (LOD) was 0.01 µmol/L for Cer 16:0 and Cer 18:0, and 0.1 µmol/L for Cer 24:0 and Cer 24:1. The signal-to-noise ratio at the LOQ for the transitions of all the analytes was ≥10.
Over 600 neat patient samples (serum and plasma) have been analyzed by the assay; no peaks in the vicinity of the peaks of the analytes and the ISs were observed in the mass transitions used in the assay. Overestimation of the ceramide concentrations was observed in hemolyzed or lipemic samples, containing >300 mg/dL or >225 mg/dL of hemoglobin or intralipid, respectively. No carryover was observed after injection of samples containing 4 µmol/L for Cer 16:0 and Cer 18:0, and 40 µmol/L for Cer 24:0 and Cer 24:1. The method was compared to a validated LC-MS/MS method of another laboratory using NIST material (Metabolites in Frozen Human Plasma, SRM 1950) and patient plasma or serum pools (n=6). Median (range) for percent agreement between the developed method and the comparative method were: 132 (117-143)%, 152 (149-156)%, 101 (97-103)% and 93 (82-99)%; the R2 values for the linear regression curves were 0.978, 0.999, 0.994 and 0.987, for Cer 16:0, Cer 18:0, Cer 24:0 and Cer 24:1, respectively. Ceramide concentrations from serum (red top), EDTA plasma, and lithium heparin plasma were comparable, showing agreement within 15%.

CONCLUSION:
An LC-MS/MS method was developed for quantifying Cer 16:0, Cer 18:0, Cer 24:0, and Cer 24:1 in plasma and serum samples. The method demonstrated reliable analytical performance with adequate precision, accuracy, and robustness. We observed reasonably good inter-laboratory agreement in the measured ceramide concentrations.