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

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

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

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

Triglyceride breakdown

Transport into mitochondria Acyl-CoA Carnitine Transporter

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

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

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

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

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

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

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

LC-MSMS

Acyl carnitine fragmentation

Precursor ion scan

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 13-53 umol/L Linearity 300 umol/L Chromsystems Neonatal Screening IQC CDC EQA DBS Scheme ERNDIM Free carnitine Scheme

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

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

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

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

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

SCADD Diagnostic peak m/z 288

MCADD - crisis Diagnostic peak m/z 344

VLCADD Diagnostic peak m/z 426

Ketotic Peaks m/z 260, 304 & 426

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

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

GA2 Diagnostic C4 – C18

b ketothiolase deficiency Diagnostic peaks m/z 300 & 318

MMA Diagnostic peaks m/z 274 & 374

PA Diagnostic peak m/z 274

IVA Diagnostic peak m/z 302

Malonic aciduria Diagnostic peak m/z 360

Increased free and short chains PMB Increased free and short chains

 Acylcarnitine MRM (butyl) MRM (underiv.] Disorder C0 218 > 85 162 > 85 PCD C2 260 > 85 204 > 85 (Glutamate) C3 274 > 85 218 > 85 MMA; PA C4 288 > 85 232 > 85 EMA;SCAD; GA2 C5:1 300 > 85 244 > 85 PA; BkT C5 302 > 85 246 > 85 IVA; GA2 C4-OH 304 > 85 248 > 85 (Ketosis) C6 316 > 85 260 > 85 GA2 (MCAD) C5-OH 318 > 85 262 > 85 Biot;IVA;BkT;3HMG C8 344 > 85 288 > 85 MCAD / [?] C3-DC 360 > 85 248 > 85 Malonic Aciduria C8-OH 360 > 85 304 > 85 (Metab Crisis) C10:1 370 > 85 314 > 85 MCAD C10 372 > 85 316 > 85 GA2 C4-DC 374 > 85 262 > 85 [MMA] C5-DC 388 > 85 276 > 85 GA1 ; (GA2) C10-OH 388 > 85 332 > 85 (Metab crisis) C12:1 398 > 85 342 > 85 [B-oxidn] C12 400 > 85 344 > 85 (B-oxidn]  

Plasticisers Diagnostic peak m/z 288

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

Cefotaxime Two peaks m/z 426 & 470

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