Samantha Herbick explores the analysis of acylcarnitines for differential diagnosis.
Carnitine is a quaternary ammonium compound that is required for fatty acid β-oxidation. In the body, it can exist either in free form or bind with fatty acids to form acylcarnitines. Many different organic acid and fatty acid oxidation disorders can be diagnosed using acylcarnitine profiles in the blood or plasma. Acylcarnitines are especially important in newborn infant testing because – if proper treatment is prescribed early – it can greatly reduce the risk of long-term effects.
Restek previously developed a rapid screening method for acylcarnitines using a Raptor HILIC-Si EXP guard column cartridge.[1] That method can improve productivity by allowing combined screening of acylcarnitines and amino acids without derivatisation, but it does not provide chromatographic separation of several compounds that are used for differential diagnosis. Although high-throughput screening methods are valuable, the method developed here takes a different approach and focuses on the chromatographic separation of several key acylcarnitines that are used for identifying different disorders.
Simple, Derivatisation-Free Sample Preparation
To simplify and speed up sample preparation, this method was developed without derivatisation. Aliquots of plasma (100 µL) were fortified with internal standard, vortexed for 10 seconds, and then incubated for 10 minutes at ambient temperature. Following incubation, 300 µL of methanol was added to each sample, and the samples were vortexed briefly and centrifuged for 10 minutes at 4,000rpm. Finally, 100 µL of the supernatant was added to 900 µL of mobile phase A (0.1% formic acid in water), and then samples were vortexed prior to LC-MS/MS analysis on a Raptor ARC-18 column.
Preliminary experiments determined that plasma was not a suitable analyte-free matrix for standards and QC samples due to the endogenous acylcarnitine levels. Multiple matrices were investigated, and 100 µg/mL bovine serum albumin (BSA) in water was found to be the best surrogate matrix. Calibration and QC solutions were then made across a 25-5,000 ng/mL concentration range and processed following the same procedure that was used for the plasma samples.
Method Evaluation Results
Simultaneous LC-MS/MS analysis of 25 underivatised acylcarnitines was achieved on a Raptor ARC-18 column under the conditions shown in Fig. 1. This method provides chromatographic separation of multiple isobars that are essential for differential diagnosis. For example, C4-butyryl-L-carnitine was separated from C4-isobutyryl-L-carnitine, which allows butyryl-CoA dehydrogenase deficiency to be distinguished from isobutyryl-CoA dehydrogenase deficiency. Good separation of C5-valeryl-L-carnitine, C5-isovaleryl-L-carnitine, and 2-methyl-butyryl-L-carnitine was also obtained. The separation of these critical isobars was easily achieved in a 9-minute run, which allows higher sample throughput compared to the 20+ minute runs observed in some literature for this type of isobaric separation.
To evaluate method precision, accuracy, and linearity, C5-valeryl-L-carnitine was chosen as a representative analyte due to its mid-range polarity and presence in a group of isomers. Accuracy and precision were evaluated using BSA-based QC samples that were analysed over three days (Table I). Method accuracy was demonstrated by recovery values being within 15% of the nominal concentrations for all QC samples. Precision was shown by the %RSD values being ≤14% for all QC samples. In addition, linearity was assessed, and C5-valeryl-L-carnitine showed acceptable linearity with an R2 value of 0.9952 or greater (1/x2 weighted linear regression) over the course of testing.
To assess the suitability of the surrogate matrix, fortified plasma samples were prepared from three different lots of plasma and analysed using calibrators prepared in BSA. First, the endogenous concentration of C5-valeryl-L-carnitine was quantitated for each lot. Then, standard addition was used to spike the samples with an additional 1000 ng/mL. All three lots of plasma showed acceptable results that were less than 11% different from the expected values for both intra- and interday repeatability studies.
Conclusion
The acylcarnitines LC-MS/MS analysis developed here provides a quick, efficient approach for the preparation and analysis of underivatized acylcarnitines in plasma samples. Separation of critical pairs was achieved in a fast, 9-minute run, allowing high-throughput analysis that can support differential diagnosis. Method performance testing demonstrated acceptable method precision, accuracy, and linearity.
Samantha Herbick is an advanced scientist in LC solutions at Restek.
*Fig. 1. Column: Raptor ARC-18 (cat.# 9314A12); Dimensions: 100 mm x 2.1 mm ID; Particle Size: 2.7 μm; Guard Column: Raptor ARC-18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9314A0252); Temp.: 35 °C. Diluent: 0.1% Formic acid in water; Conc.: 100 ng/mL; Inj. Vol.: 3 μL. Mobile Phase: A. 0.1% Formic acid in water, B. 0.1% Formic acid in acetonitrile:isopropanol (90:10). Gradient (%B): 0.00 min (2%); 1.00 min (2%); 4.00 min (12%); 6.00 min (100%); 7.50 min (100%); 8.00 min (2%); 9.00 min (stop). Flow: 0.6 mL/min; Detector: MS/MS; Interface: ESI+; Instrument: UHPLC. Note: Complete sample preparation steps and ion transitions are available at www.restek.com by searching for application note CFAN4029-UNV under Resources. Peak ID: 1. C0-L-Carnitine; 2. C2-Acetyl-L-carnitine; 3. Methyl-malonyl-L-carnitine; 4. C3-Propionyl-L-carnitine; 5. 3-Hydroxyisovaleryl-L-carnitine; 6. C4-Isobutyrl-L-carnitine; 7. C4-Butyryl-L-carnitine; 8. 3-Methylcrotonyl-L-carnitine; 9. C5:1-Tigyl-L-carnitine; 10. 2-Methylbutyryl-L-carnitine; 11. C5-Isovaleryl-L-carnitine; 12. C5-Valeryl-L-carnitine; 13. C6-Hexanoyl-L-carnitine; 14. C7-Heptanoyl-L-carnitine; 15. C8-Octanoyl-L-carnitine; 16. C10-Decanoyl-L-carnitine; 17. C14:2-Tetradecadienoyl-L-carnitine; 18. C12-Lauroyl-L-carnitine; 19. C14:1-Tetradecanoyl-L-carnitine; 20. C14-Myristoyl-L-carnitine; 21. C18:2 Linoleoyl-L-carnitine; 22. C16:1 Palmitolelyl-L-carnitine; 23. C16-Palmitoyl-L-carnitine; 24. C18:1 Oleoyl-L-carnitine; 25. C18 - Stearoyl-L-carnitine
References
[1]R. Alagandula, Improved screening method for acylcarnitines & amino acids in dried blood spots by LC-MS/MS. www.restek.com/articles/improved-screening-method-for-acylcarnitines-and-amino-acids-in-dried-blood-spots-by-ls-msms
