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The introduction of tandem mass spectrometry (MS/MS)1 into newborn screening programs during the past 15 years has been revolutionary, and it has resulted in a marked expansion of the number of detectable metabolic disorders, from a handful to more than 30(1). The majority of these are defects of amino acid and fatty acid transport and catabolism, and most are responsive to treatment. The success of this development has prompted much interest in further expansion of newborn screening programs, particularly for lysosomal storage disorders (LSDs) that cause significant morbidity and mortality. Until recently, these disorders received low priority(2) because of the lack of effective treatment and practicable screening methodologies. Enzyme replacement and bone marrow transplant therapies have become available for several LSDs, however, and other therapies are under development(3), giving new hope for affected individuals. Furthermore, the development of viable screening methods based on measurements of lysosomal enzyme activities in dried blood spots (DBS) has paved the way for newborn screening for these disorders(4). Enzyme activities of LSDs in DBS have been measured successfully both spectrofluorometrically, using substrates that release the fluorophore 4-methylumbelliferone(5)(6), and by tandem mass spectrometry, using substrates that are, for some of these assays, more closely related to or homologous with the natural enzyme precursors(7). The MS/MS method has the advantage that it can detect multiple enzyme products simultaneously because each product has a different m/z . In this issue of Clinical Chemistry , 2 papers(8)(9) address developments with MS/MS-based enzyme assays that bring the newborn screening option for LSDs a step closer to reality. Zhang et al.(8) describe substantial methodological improvements to the MS/MS-based method developed by Li et al.(7) for multiplex assays of up to 5 lysosomal enzyme activities. This assay …

We report a new method for the diagnosis of homocystinuria and other hypermethioninemias from dried blood spots on newborn screening cards, based on isotope-dilution tandem mass spectrometry. The mean concentration of methionine in 909 unaffected newborns was 19 micromol/L (CV 44%). The variability of results was reduced when the concentration of methionine was expressed relative to that of another amino acid in the same specimen. The mean ratio of methionine to leucine plus isoleucine for these same newborn blood spots was 0.16 (CV 25%). In newborn samples from a collection categorized by a Guthrie bacterial inhibition assay as true positive, unaffected, or falsely positive for hypermethioninemias, the ratio of methionine to leucine for each true-positive specimen was at least 2.5 times greater than for respective age-matched unaffected blood specimens. The ratio for falsely positive samples did not differ from that for unaffected blood samples. We predict that the ratio of methionine to leucine plus isoleucine determined by tandem mass spectrometry will successfully detect hypermethioninemias with very low rates for false positives and false negatives.

BACKGROUND New therapies for the treatment of mucopolysaccharidoses that target the brain, including intrathecal enzyme replacement, are being explored. Quantitative analysis of the glycosaminoglycans (GAGs) that accumulate in these disorders is required to assess the disease burden and monitor the effect of therapy in affected patients. Because current methods lack the required limit of quantification and specificity to analyze GAGs in small volumes of cerebrospinal fluid (CSF), we developed a method based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). METHODS Samples of CSF (25 μL) were evaporated to dryness and subjected to methanolysis. The GAGs were degraded to uronic acid-N-acetylhexosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from heparan, dermatan and chondroitin sulfates (HS, DS and CS) were separated by UPLC and analyzed by electrospray ionization MS/MS using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. RESULTS CSF from control pediatric subjects (n = 22) contained CONCLUSIONS The method described here has potential value in monitoring patients with mucopolysaccharidoses receiving treatment targeted to the brain.

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Based on isotope-dilution tandem mass spectrometry, the method takes only a few minutes to perform and requires minimal sample preparation. The accurate assay of both phenylalanine and tyrosine in dried blood spots used for neonatal screening for phenylketonuria in North Carolina successfully differentiated infants who had been classified as normal, affected, and falsely positive by current fluorometric methods. Because the mass-spectrometric method also recognizes other aminoacidemias simultaneously and is capable of automation, it represents a useful development toward a broad-spectrum neonatal screening method.

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.

Measurements of free and total carnitine in plasma are important in the diagnosis and clinical management of patients with carnitine deficiency syndromes and certain inborn errors of metabolism (1). The most popular analytical methods are based on the enzymatic reaction in which an acetyl group is transferred from acetyl-CoA to carnitine with release of free CoA (2). A spectrophotometric assay based on this method, using a Cobas centrifugal analyzer (Roche Diagnostics), has been in use in this laboratory for the past several years (3). Limitations of the Cobas enzymatic method in our view include the initial filtration step, which requires a minimum sample volume of 300 μL, and the number and cost of the reagents required. Assay results are critically dependent on two labile reagents, the enzyme carnitine acetyltransferase and the color reagent 5,5′-dithiobis(2-nitrobenzoic acid). We have also observed that patients on valproic acid therapy sometimes give total carnitine values that are spuriously low for reasons that are not clear. Another limitation of the Cobas assay is that acylcarnitines with chain lengths >10 are lost in the filtration step (3), and the method will underestimate the total carnitine content of patients with defects in the oxidative degradation of long-chain or multiple acyl-CoAs, especially when metabolically decompensated. We have emphasized previously that the most valuable applications of the routine Cobas assay are the identification of patients with carnitine deficiency and the monitoring of patients on carnitine therapy (3). We have developed a new carnitine assay method based on isotope-dilution tandem mass spectrometry (TMS), which is inherently more straightforward and accurate than previously reported methods. This is true primarily because it has absolute molecular specificity, uses an isotope-labeled internal standard to compensate for any losses or variance resulting from the sample preparation, and has no known chemical interference. The new assay …

Background: The isotope-labeled intravenous glucose tolerance test (IVGTT) combined with computer modeling is widely used to derive parameters related to glucose metabolism in vivo. Most of these methods involve use of either 2H2-labeled or 13C1-labeled d-glucose as a tracer with GC-MS to measure the isotope enrichment. These methods are challenging, both technologically and economically. We have developed a novel approach that is suitable for labeled-IVGTT studies involving a large cohort of individuals. Methods: The tracer, d-[13C6]glucose, is a low-cost alternative with the significant advantage that the sixth isotope of natural glucose has virtually zero natural abundance, which facilitates isotopomer analysis with Results: With labeled-IVGTT data from 10 healthy male individuals, the values for insulin sensitivity, glucose effectiveness, and the plasma clearance rate estimated with the 2-pool minimal model compared well with values obtained via traditional methods. Conclusions: The relative simplicity and robustness of the new method permit the preparation and analysis of up to 48 samples/day, a throughput equivalent to 2 complete IVGTT experiments, and this method is readily adaptable to existing 96 well–format purification and analytical systems.

The concept of screening newborns for inherited metabolic disorders was the brainchild of Robert Guthrie, an upstate New York microbiologist with a passion to prevent the devastating and irreversible neurologic damage sustained by victims of untreated phenylketonuria (PKU). The solution he developed was a simple and inexpensive bacterial inhibition assay for phenylalanine in blood (1). He also invented a unique method of specimen collection, in which peripheral blood was collected from a newborn’s pricked heel onto a special cotton fiber filter-paper known as a “PKU card” or “Guthrie card”. After the blood had dried, the specimen was mailed to a laboratory that would identify any child at risk for PKU from a concentration of phenylalanine above an age-matched control limit. Further diagnostic testing was then required to determine whether the disease was present. Treating affected children with a phenylalanine-depleted diet, started within the first month of life, was effective in preventing mental retardation. Unable to persuade the state of New York to conduct newborn screening with the test, Guthrie convinced Massachusetts that the cost of screening the entire population of newborns and treating affected children with the special diet was less than the cost to society of untreated PKU cases. Thus, in 1963 began state-mandated newborn screening for PKU (2). The test was gradually adopted by other states and eventually by countries all over the world. The success of PKU screening in dried blood spots (DBS), which identifies ∼200 new cases annually in the United States alone, prompted the addition of tests for other disorders that fit the PKU paradigm. By the …

We report a new method for the diagnosis of homocystinuria and other hypermethioninemias from dried blood spots on newborn screening cards, based on isotope-dilution tandem mass spectrometry. The mean concentration of methionine in 909 unaffected newborns was 19 micromol/L (CV 44%). The variability of results was reduced when the concentration of methionine was expressed relative to that of another amino acid in the same specimen. The mean ratio of methionine to leucine plus isoleucine for these same newborn blood spots was 0.16 (CV 25%). In newborn samples from a collection categorized by a Guthrie bacterial inhibition assay as true positive, unaffected, or falsely positive for hypermethioninemias, the ratio of methionine to leucine for each true-positive specimen was at least 2.5 times greater than for respective age-matched unaffected blood specimens. The ratio for falsely positive samples did not differ from that for unaffected blood samples. We predict that the ratio of methionine to leucine plus isoleucine determined by tandem mass spectrometry will successfully detect hypermethioninemias with very low rates for false positives and false negatives.

We report a new method for the diagnosis of maple syrup urine disease (MSUD) from dried blood spots on newborn screening cards based on tandem mass spectrometry (MS-MS). The mean +/- SD concentration of Leu plus Ile in normal newborns was 151 +/- 47 mumol/L (n = 1096); for Val, 131 +/- 58 mumol/L (n = 791). SDs were lower when the concentrations of these amino acids were expressed relative to that of Phe. The mean ratio for Leu + Ile to Phe was 2.5 +/- 0.49; for Val to Phe, 2.18 +/- 0.51. These results compare well with values previously reported in the literature. With these criteria, samples from a collection categorized by a bacterial inhibition assay as normal or falsely positive for MSUD were normal by MS-MS [(Leu + Ile): Phe5.0]. Samples from confirmed MSUD patients were categorized as abnormal [(Leu+Ile): Phe9.0] by MS-MS.

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Based on isotope-dilution tandem mass spectrometry, the method takes only a few minutes to perform and requires minimal sample preparation. The accurate assay of both phenylalanine and tyrosine in dried blood spots used for neonatal screening for phenylketonuria in North Carolina successfully differentiated infants who had been classified as normal, affected, and falsely positive by current fluorometric methods. Because the mass-spectrometric method also recognizes other aminoacidemias simultaneously and is capable of automation, it represents a useful development toward a broad-spectrum neonatal screening method.

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis ofor = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.