By Mark McCann and Mendel Tuchman,
Departments of Pediatrics and Laboratory Medicine and Pathology,
University of Minnesota, Minneapolis (U.S.A.)

Researchers at the University of Minnesota have simplified the process of monitoring the metabolic condition of patients with phenylketonuria (PKU). They developed a quick and accurate analytical method to measure phenylalanine and tyrosine from very small paper blood spots.

Mass spectra of native and isotopic phenylalanine and tyrosine; a) tyrosine, b) tyrosine (Ring-d4), c) phenylalanine, d) phenylalanine (Ring-d5)

Previously, patients have had to endure a lengthy and arduous bleeding from a lanced fingertip or heel to provide at least 300 microliters of blood into heparinized colletion tubes. The blood was then sent into the laboratory for lengthy processing, followed by a slow (1- to 2-hour-long) analysis with an automated amino acid analyzer.

Analysis Time: Less than 10 Minutes
With the new process, the patient provides just one or two drops of blood onto a filter paper and mails the dried specimen directly to the laboratory. Samples are rapidly analyzed, with no chemical preparation or derivatization, using quantitation through stable isotope dilution. Run time with APCI LC/MSD is very short – under ten minutes!

Extracted ion chromatogram monitoring a phenylketonuric patient (a) and a normal patient (b).

Small Sample Size, Accurate Determination
Using dried blood spots is particularly appealing for the analysis of phenylalanine and tyrosine, because it permits accurate and fast determinations from very small amounts of blood (6-10 µL). Since most patients are infants, sample size is of great significance.

Enhanced Patient Care
Dietary management of patients with phenylketonuria requires frequent measurements of plasma phenylalanine and tyrosine to monitor compliance with the diet and modulate dietary intake of phenylalanine to maintain plasma levels at an optimal range of 2 to 6 mg%. The development of a rapid and highly sensitive method for the quantitation of phenylalanine and tyrosine from dried blood spots enhances patient care considerably.

Easy Sample Preparation
Sample preparation is simple, and the use of an isotopic internal standard and the high resolution of selected ion monitoring enhances the reliability of the analysis.

Blood spotted on filter paper is collected by patients at home using a simple collection kit and mailed to the laboratory. A 6.4-mm blood spot disc is subjected to elution in the presence of stable isotope standards, ultrafiltered, and then injected into an HP 1100 Series LC/MSD system. The elution efficiency for phenylalanine and tyrosine is nearly 100%. Separation is achieved on a reversed-phase column with a run time of under ten minutes. Detection is by selected ion monitoring APCI LC/MSD. Tyrosine is monitored at m/z 182 and m/z 186 (ISTD), and phenylalanine at m/z 166 and m/z 171 (ISTD).

The determination of phenylalanine and tyrosine from paper blood spots by stable isotope dilution APCI LC/MSD offers a simple, fast, and accurate method for monitoring patients with PKU.

The work described here is supported by the Minnesota PKU Foundation: www.peds.umn.edu/pku