The instrument variation for CL and MLCL in a control bloodspot extract was 13% and 44%, respectively.
A repeated extraction step increased the extraction yield by approximately 15% for CL and 25% for MLCL, which indicated that recovery from a bloodspot with the procedure is <85% for CL and <75% for MLCL.
After the first successful detection of MLCL and CL in bloodspots, it became clear that quantification of both analytes was difficult given their low levels and the inability to reliably introduce an internal standard in the dried bloodspot.
Therefore the actual value of this ratio is not the molar ratio of the MLCL and CL species in the bloodspot sample; it is a practical parameter that expresses the combined effect of two separate relevant indicators.
The high inter--and intraassay variation found for the MLCL/CL ratio (both about 50%) can be explained by the fact that in a bloodspot measurement either MLCL (in controls) or CL levels (in BTHS) are close to the detection limit.