Quantification of Glutamine in Dried Blood Spots and Plasma by Tandem Mass Spectrometry for the Biochemical Diagnosis and Monitoring of Ornithine Transcarbamylase Deficiency

A notable deficiency in the use of tandem mass spectrometry (MS/MS) for newborn screening is the inability to screen for urea cycle defects. The most common of these, with an incidence of 1 in 14 000 births (1), is the inherited X-linked disorder ornithine transcarbamylase deficiency (OTCD). A majority (60%) of hemizygous males risk death from hyperammonemic coma during the first week of life. The remainder, including 10% of heterozygous females, exhibit lethargy, vomiting episodes, and behavioral problems during childhood. The severity of the disorder and the potential for correction of OTCD by liver transplantation and gene therapy (2) provide adequate justification for newborn screening. OTCD patients have low blood citrulline because of reduced conversion from carbamoyl phosphate. Citrulline is one of the amino acids routinely measured in MS/MS newborn-screening programs. Unfortunately, many protein-restricted newborns also have low blood citrulline (3). A more selective amino acid metabolite for OTCD is glutamine. The derivatization procedure used in many MS/MS screening programs (4), which uses butanol–hydrogen chloride, destroys glutamine. Approximately one-half of the glutamine is converted to glutamic acid dibutyl ester and is indistinguishable from that formed from endogenous glutamic acid in the blood. The surviving glutamine butyl ester is deaminated in acidic solution to a protonated form of pyroglutamic acid butyl ester in the electrospray source of the MS/MS. Again it is not possible to distinguish this pyroglutamic acid from what is already present in the blood. As a secondary consequence, the measurements of glutamic and pyroglutamic (and by analogy, aspartic) acids in blood spots after derivatization are grossly inaccurate. MS/MS newborn-screening programs that do not derivatize amino acids avoid solvolysis of glutamine and of pyroglutamic acid to glutamic acid. During electrospray ionization-MS/MS analysis, however, glutamine is again indistinguishable from pyroglutamic acid. Resolution is possible by separation with time-consuming liquid chromatography …