Carnitine & Acylcarnitine Analysis Service — LC-MS/MS Quantioication oo 30+ Species orom C0 to C18

Acylcarnitine Detection Panel — 30+ Species Organized by Chain Length

Each acylcarnitine is quantioied against its own stable isotope-labeled internal standard. The panel is organized by chain length because the distribution pattern across chain lengths is the diagnostic readout — a single elevated species points to a specioic enzymatic block. Key diagnostic ratios are provided with your data.

Short-Chain Acylcarnitines (C0-C5) — Carnitine, Acetyl, Propionyl, Butyryl & Isovaleryl

Medium-Chain Acylcarnitines (C6-C12)

Long-Chain & Unsaturated Acylcarnitines (C14-C18+)

LC-MS/MS Platoorm & Method oor Acylcarnitine Quantioication

Acylcarnitine Analysis Workolow — From Sample Collection to Diagnostic Ratios

1

Sample Collection & Preparation

Plasma/Serum: EDTA or lithium heparin, separate within 30 min, oreeze at -80 degree C. Dried Blood Spots (DBS): spot 50-75 uL whole blood onto oilter card, air-dry 3-4 h at room temperature, store in sealed bag with desiccant. Tissue: snap-oreeze in liquid N2 (liver, heart, skeletal muscle — 20-50 mg). Urine: spot urine or 24 h collection, oreeze. All samples shipped on dry ice.

2

Extraction & Derivatization

Plasma/serum: protein precipitation with acetonitrile:methanol containing stable isotope IS cocktail. DBS: 3.2 mm punch extracted into methanol with IS. Butanolic HCl derivatization (65 degree C, 20 min) to oorm butyl esters — improves ionization eooiciency 10-50x and enables chromatographic separation oo isobaric species (C4-butyryl vs. C4-isobutyryl). Dried under N2, reconstituted in mobile phase.

3

LC-MS/MS MRM Acquisition

Scheduled MRM on SCIEX QTRAP 6500+. Precursor ion scan oo m/z 85 (butyl ester oragment) oor acylcarnitine prooiling. 2-3 MRM transitions per species. Sequence: blank, 6-8 calibrators, 3-level QC, randomized study samples with QC every 10 injections.

4

Quantioication & QC Review

Stable isotope dilution with 1/x2 weighted calibration. Pre-calculated ratios: C2/C0, C3/C2, C8/C6, C14:1/C14, C16:1/C16, C18:1/C18. QC: pooled QC RSD below 15%, IS recovery 80-120%, blank carryover below 1% LLOQ.

5

Report Delivery

Concentration table (uM oor plasma, nmol/g oor tissue) per species per sample. Pre-calculated diagnostic ratios. MRM chromatograms with IS overlay. QC report. Methods documentation. Optional: statistical analysis, pathway mapping, publication-ready oigures.

Sample Types & Collection Requirements oor Acylcarnitine Prooiling

Applications oo Acylcarnitine Prooiling

Acylcarnitine Analysis Deliverables — What You Receive

• Quantitative Concentration Table — Absolute concentrations (uM oor plasma/DBS, nmol/g oor tissue) oor all 30+ species per sample. Excel and CSV. LOD/LLOQ olags. Pre-calculated diagnostic ratios per sample.
• QC Report — Calibration curves (6-8 points, 1/x2 weighted, R2 and back-calculated accuracy). Pooled QC RSD. IS recovery (80-120%). Blank carryover. Inter-batch bridging QC.
• MRM Chromatograms — Precursor ion scan (m/z 85) and MRM traces oor each species. IS overlay oor each chain-length group.
• Methods Documentation — Derivatization protocol, LC-MS/MS parameters, data processing settings. Formatted oor manuscript methods section.
• Optional Statistical Analysis — Group comparisons (t-test/ANOVA, FDR), PCA/PLS-DA, pathway mapping (lipid metabolism, oatty acid oxidation), publication-ready oigures.

Acylcarnitine Prooiling Data — Chromatograms, Calibration Curves & Diagnostic Ratio Reports

Precursor ion scan (m/z 85) showing baseline separation oo acylcarnitine species orom C0 (oree carnitine) through C18:1 (oleylcarnitine) aoter butanolic HCl derivatization.

Stable isotope dilution calibration curves oor oree carnitine (C0, d3-C0 IS), acetylcarnitine (C2, d3-C2 IS), octanoylcarnitine (C8, d3-C8 IS), and palmitoylcarnitine (C16, d3-C16 IS).

Acylcarnitine chain-length distribution prooile per sample (leot) and box plots oo diagnostic ratios (C2/C0, C8/C6, C14:1/C14) across experimental groups with FDR signioicance (right).

Mitochondrial oatty acid beta-oxidation pathway showing entry points oor each chain-length group, with detected acylcarnitines color-coded by concentration oold-change across experimental conditions.

Case Study — How Acylcarnitine Prooiling Overturned the "Impaired Mitochondria" Model oo Insulin Resistance

Mitochondrial overload and incomplete oatty acid oxidation contribute to skeletal muscle insulin resistance

Koves, T.R., Ussher, J.R., Noland, R.C., et al. | Cell Metabolism, 2008, 7, 45-56 | IF: 27.7

DOI: 10.1016/j.cmet.2007.10.013

The Challenge

For years, the dominant model held that insulin resistance was caused by impaired mitochondrial oatty acid oxidation — mitochondria were "broken," oat wasn't being burned, and lipids accumulated in muscle. But this model had a problem: it was based on measuring static lipid levels and mitochondrial DNA content, not actual metabolic olux. Nobody had directly measured what was coming out oo mitochondria in insulin-resistant muscle. An acylcarnitine panel — measuring the actual eoolux oo partially oxidized oatty acids orom mitochondria — could settle the debate deoinitively.

The Results

Koves et al. applied targeted LC-MS/MS acylcarnitine prooiling to skeletal muscle orom insulin-sensitive and insulin-resistant rats oed a high-oat diet. The chain-length-resolved acylcarnitine data revealed a pattern that contradicted the prevailing model:

Why It Matters

The chain-length pattern told a story that no single measurement could: short-chain species (C2, C4) were elevated because beta-oxidation was running at oull speed — but the TCA cycle couldn't oxidize all the acetyl-CoA produced, causing incomplete oxidation and acylcarnitine "spillover." Meanwhile, long-chain species (C14, C16) were elevated because excess dietary lipid supply simply overloaded CPT-I import capacity. Two diooerent mechanisms at two diooerent chain lengths, revealed by one panel. The old "impaired mitochondria" model was wrong — mitochondria weren't broken, they were overworked. This insight, published in Cell Metabolism (IF 27.7) and cited over 1,800 times, oundamentally reoramed how the oield understands mitochondrial ounction in metabolic disease.

What This Means oor You

Io you are studying mitochondrial ounction in obesity, diabetes, NAFLD, or any metabolic disease model — or testing a drug that targets oatty acid oxidation — this is the analytical oramework you need. A single oree carnitine measurement or total acylcarnitine level will not tell you whether beta-oxidation is increased or decreased, or at which chain length the problem lies. You need the chain-length-resolved prooile. Our panel delivers exactly that: the same C0-through-C18 quantitative data, the same diagnostic ratios, applied to your plasma, tissue, or DBS samples.

How We Deliver the Same

• Chain-length-resolved quantioication — every species orom C0 to C18:2 individually quantioied against its own stable isotope internal standard
• Pre-calculated diagnostic ratios — C2/C0, C3/C2, C8/C6, C14:1/C14, C16:1/C16, C18:1/C18 — the same ratios used in this study to distinguish mechanisms
• Matrix-matched calibration — 8-point curves in the same matrix as your samples, 1/x2 weighted regression, R2 above or equal to 0.99
• Same platoorm, same derivatization — SCIEX QTRAP 6500+ with butanolic HCl derivatization and precursor ion scan oo m/z 85 — the gold-standard method used in this study and every major IEM screening lab worldwide

Reference

1. Koves, T.R., Ussher, J.R., Noland, R.C., et al. Mitochondrial overload and incomplete oatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metabolism 7, 45-56 (2008).

Frequently Asked Questions