Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry

Abstract

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R(2) > 0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests.

Fig 1

Precursor ion scans of acylcarnitine in a dried serum spot and in b dried blood spot using PS–MS. Acylcarnitine calibration standards are labeled in red (C2, 5 μM; other acylcarnitines, 500 nM), and internal standards are labeled in blue (C2-d3, 1 μM; C16-d3, 400 nM; other internal standards, 200 nM)

Fig 2

Effect of different solvent compositions for acylcarnitine analysis in serum and in blood; 80 % methanol with 0.1 % formic acid (blue, circles), 80 % acetonitrile with 0.1 % formic acid (red, squares), and 80 % acetonitrile (green, triangles) were tested for a serum and b blood samples. The effect of organic solvent percentage for acylcarnitine analysis was further investigated, by varying the percentage of methanol from 60 to 100 % with 0.1 % formic acid for serum sample (c), while the percentage of acetonitrile was varied from 60 to 100 % without adding acid for the blood sample (d)

Fig 3

Effect of sample load on acylcarnitine analysis in a serum and b blood

Fig 4

Standard curves obtained for quantitative analysis of acylcarnitines in serum (top) and in blood (bottom)

Fig 5

Limit of quantitation of acylcarnitines in serum and in blood using PS–MS. The shaded area in blue indicates the majority of acylcarnitine range for the normal population. The dotted green line at the bottom is the first percentile cumulative values of normal acylcarnitine species, and the dotted red line at the top is the 99th percentile cumulative values of normal acylcarnitines, which is also the cutoff for FAO disorder diagnosis in the clinic. Blue squares show the limit of quantitation of acylcarnitines in serum, and red diamonds are the limit of quantitation of acylcarnitines in blood. The data of C2 were zoomed out 30 times, and the data from C5 to C14 were zoomed in ten times