1. Acyl carnitine analysis: Pitfalls & Problems
Rachel Webster
Birmingham Children’s Hospital

2. Carnitine Quaternary ammonium compound
Biosynthesised from lysine and methionine
Liver and kidney
Transports fatty acids from cytosol into mitochondria
Facilitates the production of energy from fat

3. Dietary fat Major component of dietary fat is triglycerides 1 glycerol
3 fatty acids

4. Energy production Preferentially use carbohydrates
Glucose
Glycogen
Hypoglycaemia (fasting, illness, infection)
Fat metabolism
Mitochondrial oxidation of fatty acids provides upto 80% of total requirement
Protein metabolism
Last resort
Periods of excessive starvation

5. Triglyceride breakdown

6. Transport into mitochondria
Acyl-CoA
Carnitine
Transporter

8. Acyl co-A dehydrogenase species
SCAD
C4-C6
MCAD
C4-C12
LCAD
C8-C20
VLCAD
C12-C24

9. Energy yield Fat Carbohydrate
106 ATP
1 molecule of C16 palmitate
Carbohydrate
36 ATP
1 molecule of glucose
 Why we only need a small amount of fat in our diets

10. Defects Carnitine deficiency CPT-1 deficiency CPT-2 deficiency
CACT (carnitine transporter defect)
VLCADD
LCADD
MCADD
SCADD
Plus many more!!!
 all differing acyl carnitine profiles

11. Free and acyl carnitine analysis
Native (underivatised) acyl carnitines
Butylated derivatives
Carboxylic acid group is esterified
Both fragment to yield a common m/z 85 daughter ion

12. BCH Practice Paired DBS and plasma CIL NSK-B IS kit Derivatise Report
Quantitative free carnitine (plasma)
Qualitative acyl carnitine interpretation (plasma & DBS)
Quantitate any relevant species
Underivatised
Urgent samples
Unusual peaks

13. BCH Practice DBS and Plasma
Acute scenario
DBS
Better overview of long-term status
Some disorders are better represented in different sample types
GA-1
HMG CoA Lyase deficiency

14. Sample preparation 3mm DBS 10ul plasma 200ul IS c stable isotopes
30min elution
Dry
Protein crash
Derivatised
Underivatised
Butanol HCL
Dry
Direct flow injection +ve ESI MSMS

15. LC-MSMS

16. Acyl carnitine fragmentation

17. Precursor ion scan

18. BCH Practice Acyl carnitines Free carnitine quantitation
Parents m/z 85 scan
Currently generating age-related reference ranges
Free carnitine quantitation
MRM 218 > 85
Ref range umol/L
Linearity 300 umol/L
Chromsystems Neonatal Screening IQC
CDC EQA DBS Scheme
ERNDIM Free carnitine Scheme

19. Internal Standard - Deriv
C0d9
C2d3
C16d3
C5d9
C14d9
C4d3
C3d3
C8d3

20. Advantages of derivatisation
Increased mass compared to underivitised
avoids low mass contaminants
solvent adducts
Less affected by ‘isobaric conflicts’
dicarboxylic acylcarnitines C3DC
hydroxycarboxylic acylcarnitines [OH]C4
Better ionisation of dicarboxylics
2 COOH gps
Double derivitisation
Increased positivity  excellent for +ve ESI
Culture established worldwide
published data
better understanding of analysis

21. Underiv - ?Malonyl/OHBut
Patient 1
m/z 248
Patient 2

22. Deriv - ? Malonyl/OHBut Patient 1 Patient 2
m/z 360 ie malonyl carnitine C3DC
Patient 1
m/z 304 ie hydroxy butyryl carnitine
Patient 2

24. Disadvantages to derivatisation
For big batches (screening)…time, effort, cost and acid corrosion……!!!
More steps to method - potential for more errors
Hydrolysis during derivatisation
loss of acylcarnitines
increase in free carnitine
Isobaric conflict
Acetylcarnitine and glutamate m/z 260…esp DBS
dicarboxylic acylcarnitines and hydroxyacylcarnitines
[OH]C8
[OH]C10
 ‘pseudo-glutaryl carnitinaemia’ in MCADD

26. SCADD
Diagnostic peak m/z 288

27. MCADD - crisis
Diagnostic peak m/z 344

28. VLCADD
Diagnostic peak m/z 426

29. Ketotic
Peaks m/z 260, 304 & 426

30. GA1 DBS vs Plasma - Deriv
Diagnostic peak m/z 388

31. GA1 Plasma Deriv vs Underiv
Diagnostic peak m/z 388
Diagnostic peak m/z 275

32. GA2
Diagnostic
C4 – C18

33. b ketothiolase deficiency
Diagnostic peaks m/z 300 & 318

34. MMA
Diagnostic peaks m/z 274 & 374

35. PA
Diagnostic peak m/z 274

36. IVA
Diagnostic peak m/z 302

37. Malonic aciduria
Diagnostic peak m/z 360

38. Increased free and short chains
PMB
Increased free and short chains

39.  Acylcarnitine MRM (butyl) MRM (underiv.] Disorder
C > > 85 PCD
C > > 85 (Glutamate)
C > > 85 MMA; PA
C > > 85 EMA;SCAD; GA2
C5: > > 85 PA; BkT
C > > 85 IVA; GA2
C4-OH > > 85 (Ketosis)
C > > GA2 (MCAD)
C5-OH > > 85 Biot;IVA;BkT;3HMG
C > > 85 MCAD / [?]
C3-DC > > 85 Malonic Aciduria
C8-OH > > 85 (Metab Crisis)
C10: > > 85 MCAD
C > > 85 GA2
C4-DC > > 85 [MMA]
C5-DC > > 85 GA1 ; (GA2)
C10-OH > > 85 (Metab crisis)
C12: > > 85 [B-oxidn]
C > > 85 (B-oxidn]

40. Plasticisers
Diagnostic peak m/z 288

41. Additional peaks Benzoate m/z 332 Phenylbutyrate m/z 336
Cefotaxime m/z 470 & 426

42. Cefotaxime
Two peaks
m/z 426 & 470

43. Conclusions Isobaric compounds Deriv vs underiv Plasma vs DBS
Which ever method run routinely must be ready to run other way for confirmation
Plasma vs DBS
Plasticisers