Bloodspot assay using HPLC-tandem mass spectrometry for detection of Barth syndrome

Barth syndrome (BTHS) is a serious X-linked, metabolic, multisystem disorder characterized by cardiomyopathy, neutropenia, myopathy, and growth delay. Because early diagnosis and appropriate treatment are of key importance for the survival of affected boys, we developed a biochemical BTHS screening method based on analysis of the monolysocardiolipin:cardiolipin ratio in bloodspots.

We performed chloroform/methanol extraction on quarter-inch punches of dried bloodspots on Guthrie cards from BTHS patients and controls. Extracts were dried (60 degrees C, N(2)) and reconstituted in CHCl(3)/methanol/H(2)O [50:45:5 vol/vol/vol, 0.1% NH(3) (25%)]. HPLC-tandem mass spectrometry analysis was performed with a normal-phase HPLC column and multiple reaction monitoring transitions for monolysocardiolipin (MLCL) and cardiolipin (CL) with a total run time of 10 min. The ratio of MLCL and CL was used as screening parameter.

All BTHS patients (n = 31) had monolysocardiolipin:cardiolipin ratios >0.40 and all controls (n = 215) had monolysocardiolipin:cardiolipin ratios <0.23. Using a cutoff point of 0.30, a blind test of 206 samples (199 controls, 7 BTHS) had sensitivity and specificity of 100%. Bloodspots could be stored at 4 degrees C or room temperature for >1 year without affecting the test outcome. Three neonatal Guthrie cards of BTHS patients taken 3.6 to 5.8 years previously were correctly identified as positive for BTHS.

HPLC-tandem mass spectrometry analysis of dried bloodspots is an unambiguous screening test for BTHS with potential for rapid screening of neonates suspected of having BTHS, making remote and retrospective diagnosis accessible for a disease that is almost certainly underdiagnosed.