Cholesterol Metabolism Pathway Profiling by LC–MS/MS

Why Measure Cholesterol Intermediates And Oxysterols

Measuring "cholesterol" alone often answers only whether a change happened—not why it happened. In cholesterol homeostasis research, multiple processes can drive the same endpoint shift:

• Biosynthesis modulation (e.g., pathway activation/suppression)
• Oxidation-derived signaling (oxysterols) and stress-related remodeling
• Esterification and storage (cholesteryl esters) linked to lipid droplet dynamics
• Transport/efflux remodeling impacting sterol redistribution across compartments

By combining cholesterol + intermediates + oxysterols/CE, you can build a defensible pathway narrative—especially in drug/genetic perturbation, time-course, and multi-matrix studies.

Cholesterol Analysis Solutions

Free & Total Cholesterol Quantification

Measure both free and total cholesterol to capture baseline and intervention effects across samples.

Cholesterol Biosynthesis Intermediates Panel

Profile key precursors like lanosterol and desmosterol to reveal cholesterol synthesis dynamics and pathway shifts.

Oxysterols Quantification Panel

Quantify signaling-active oxysterols such as 24-OHC and 27-OHC to explore oxidation and lipid regulation pathways.

Cholesteryl Esters (CE) Profiling

Assess CE species to investigate cholesterol storage, esterification, and lipid droplet remodeling.

Not sure which combination fits your study design? Share your matrix, perturbation plan, and targets—our scientists will recommend a pathway-first configuration.

Complete Target List

Panels are customizable by matrix and study design (add/remove targets upon request). Target availability and reporting mode may vary by matrix.

Quick module pairing (research context):

• Synthesis mechanism: Core + Intermediates
• Signaling/oxidation: Core + Oxysterols
• Storage remodeling: Core + CE

Study Design Support

We help plan pathway-oriented studies to reduce rework and improve interpretability across cholesterol biosynthesis, oxysterol signaling, and CE remodeling.

Common Study Formats

• Drug perturbation (synthesis/efflux/esterification modulators)
• Genetic perturbation (KO/KD/CRISPR in sterol regulation genes)
• Time-course and dose-response designs
• Multi-matrix studies (cells → tissues → plasma)

If you're unsure what's feasible in your matrix or expected range, contact us for a quick consult.

Quantitative Cholesterol Metabolism Analysis Process

Key Instrument Parameters for Cholesterol Quantification

We support optional platform configurations based on your targets, matrix, and required performance. Below are representative examples—final instrumentation and settings are selected per project.

Representative Instrumentation Options

• UHPLC systems: Agilent 1290 UHPLC System (example) / equivalent UHPLC platforms
• High-Sensitivity Triple Quadrupole MS: Agilent 6495C Triple Quadrupole (example) / equivalent triple quad systems
• Acquisition mode: MRM (Multiple Reaction Monitoring)
• Ionization: APCI and/or ESI (method-dependent)

Key Parameters

If your project involves challenging isomers or oxidation-prone targets, we can incorporate additional method considerations (e.g., separation strategy and handling controls) within the method summary.

Agilent 1290 UHPLC System (Figure from Agilent)

Agilent 6495C Triple Quadrupole (Figure from Agilent)

Quality Control and Performance

Sterols and oxysterols can be challenging due to isomeric similarity, matrix effects, and oxidation artifacts. We emphasize QC outputs you can verify in the final report.

QC Elements Included (Typical)

• Blanks and carryover checks
• Calibration curve review (when applicable)
• Internal standard monitoring (response/recovery)
• Batch QC samples and/or technical replicates (project-dependent)
• Below-LLOQ flagging rules (when applicable)

Optional Enhancements (Upon Request)

• Expanded replicate strategy for precision assessment
• Oxysterol-focused stability/oxidation-control handling plan
• Custom acceptance criteria aligned with study goals

Deliverables: What You Receive From Cholesterol Pathway Analysis

Standard Deliverables

• Quantitative results table per sample (units, missing-value rules, below-quantitation flags as applicable)
• QC summary (batch notes, internal standard response overview, blank/carryover conclusions, replicate consistency when applicable)
• Method summary (sample preparation overview, LC–MS/MS mode, quantification strategy, processing rules)

Isomer Separation Chromatograms

Baseline UHPLC–MRM separation of key sterol isomers (7α/7β-OHC; desmosterol/7-DHC) with RT labels.

Calibration Curves and LLOQ

Multi-analyte calibration curves with fitted regression and LLOQ markers for cholesterol, intermediates, and oxysterols.

Matrix Recovery / Matrix Effect

ISTD-normalized recovery across plasma/serum, tissue, and cells with a 70–130% acceptance band.

Batch QC Stability

Pooled QC stability across injection order for representative targets, with median CV% summary inset.

Applications of Cholesterol Profiling

Metabolic Pathway Mechanisms

Identify biosynthesis vs signaling vs storage-driven changes

Drug Perturbation Studies

Evaluate pathway response to synthesis/efflux/esterification modulators

Genetic Perturbation Experiments

Map sterol node changes after KO/KD/CRISPR edits

Oxidative Stress Biology

Track oxysterol shifts linked to lipid oxidation and signaling

Lipid Droplet And Storage Remodeling

Profile CE remodeling associated with esterification dynamics

Multi-Omics Integration

Link sterol pathway readouts with transcriptomics/proteomics phenotypes

Sample Requirements and Submission Guide

Recommended Sample Amount

Handling and Shipping (Best Practices)

• Store at -80°C when possible and minimize freeze–thaw cycles
• Avoid extended room-temperature exposure; for oxysterol-focused projects, reduce light/air exposure where feasible
• Ship on dry ice with leak-proof packaging and a sample manifest

More guidelines?

FAQs for Cholesterol Pathway Analysis Service