D. Johnson
Apr 1, 1999
Citations
1
Influential Citations
30
Citations
Journal
Journal of Inherited Metabolic Disease
Abstract
An increasing number of laboratories screen for inherited metabolic diseases by electrospray tandem mass spectrometric analysis of extracts of blood spots from newborns (Chace et al 1995 ; Rashed et al 1995). The dried methanol extract of the blood spot, to which deuterium-labelled internal standards have been added, is butylated and infused into an electrospray tandem mass spectrometer. The concentrations of carnitine, acylcarnitines and amino acids are measured and compared with a normal reference range, and abnormal values are Ñagged. The standard butylation procedure involves heating the dried extract in 60 kl of anhydrous nbutanolÈ3 mol/L HCl at 65¡C for 15 min. Milder conditions a†ord incomplete butylation and the problems of interfering peaks and reduced sensitivity. Experiments in our laboratory have shown that (butylated) acylcarnitines are also converted to (butylated) carnitine in n-butanolÈ3 mol/L HCl. In separate experiments acetyl-, decanoylor octadecanoylcarnitine hydrochlorides (0.5 mmol/L) and hydrochloride (2.5 mmol/L) in n-butanolÈ3 mol/L HCl were heated L-(2H3)carnitine at 65¡C for 2 h. Five timed aliquots were removed, evaporated in a stream of nitrogen and dissolved in acetonitrileÈwaterÈformic acid (50 : 50 : 0.25). They were infused into the Ionspray of a PE Sciex API365 tandem mass spectrometer. The ratios of protonated molecular ions for butylated acylcarnitine to butylated Lwere measured from precursor ions scans of product ion m/z 85 and (2H3)carnitine plotted on a log scale against time for each acylcarnitine. From the straight-line decay graphs the half-life of each acylcarnitine in the butylating reagent was determined as well as the percentage butanolysed in 15 min. Acetylcarnitine has a half-life of 31 min and 30% butanolyses in 15 min ; decanoylcarnitine has a half-life of 125 min and 8% butanolyses in 15 min ; and octadecanoylcarnitine has a half-life of 172 min and 6% butanolyses in 15 min. Several implications derive from these observations. Some of the carnitine measured in the blood spot analysis comes from the acylcarnitines (including the deuterated internal standards). Consider a sample with low free carnitine and high acetylcarnitine. Thirty per cent of the acetylcarnitine as well as smaller amounts of higher molecular mass acylcarnitines are converted to carnitine and the lowcarnitine sample could appear to be normal. Prolonging the butylation time will not only result in a grossly inaccurate carnitine determination but will also reduce the signal intensity of the acylcarnitine ions during mass spectral analysis. Overnight butylation (15 h) will hydrolyse 97% of the high molecular mass acylcarnitines, i.e.