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OBJECTIVE: The objective of this study is to develop an isotope dilution liquid chromatography tandem mass spectrometry assay to screen for hepatorenal tyrosinemia (HT) from newborn filter paper samples using pooled extracts to increase high throughput screening. DESIGN AND METHODS: Succinylacetone (SUAC), the marker for HT, was extracted from dried blood spots with the formation of the hydrazone derivative of SUAC; up to eight sample extracts were pooled and the SUAC-derivative was analyzed by mass spectrometry methods with an injection-to-injection time of one minute. If any pooled sample extract screened positive, then the samples comprising the pooled sample were assayed individually. RESULTS: Two newborn infants were identified with high levels of SUAC (7 & 23µM) and later confirmed to have HT. Three older children whose initial filter paper samples were taken at 195days to 614days of age with elevated SUAC (range 4.9-5µM) were identified; one of the three had clinical signs of HT and was placed on treatment (diagnosis of the other two are unavailable). CONCLUSION: MS/MS analysis of pooled dried blood sample extracts permits sensitive, reduced instrumental analytical time and increase high throughput screening for HT.

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OBJECTIVE: Our goal was to describe the clinical spectrum of medium-chain acyl-CoA dehydrogenase deficiency detected by routine newborn screening and assess factors associated with elevations of octanoylcarnitine in newborns and characteristics associated with adverse clinical consequences of medium-chain acyl-CoA dehydrogenase deficiency. METHODS: The first 47 medium-chain acyl-CoA dehydrogenase deficiency cases detected by the New England Newborn Screening Program were classified according to initial and follow-up octanoylcarnitine values, octanoylcarnitine-decanoylcarnitine ratios, medium-chain acyl-CoA dehydrogenase genotype, follow-up biochemical parameters, and feeding by breast milk or formula. RESULTS: All 20 patients who were homozygous for 985A-->G had high initial octanoylcarnitine values (7.0-36.8 microM) and octanoylcarnitine-decanoylcarnitine ratios (7.0-14.5), whereas the 27 patients with 0 to 1 copy of 985A-->G exhibited a wide range of octanoylcarnitine values (0.5-28.6 microM) and octanoylcarnitine-decanoylcarnitine ratios (0.8-12.7). Initial newborn octanoylcarnitine values decreased by days 5 to 8, but the octanoylcarnitine-decanoylcarnitine ratio generally remained stable. Among 985A-->G homozygotes, breastfed newborns had higher initial octanoylcarnitine values than newborns who received formula. Adverse events occurred in 5 children, 4 985A-->G homozygotes and 1 compound heterozygote with a very high initial octanoylcarnitine: 2 survived severe neonatal hypoglycemia, 1 survived a severe hypoglycemic episode at 15 months of age, and 2 died as a result of medium-chain acyl-CoA dehydrogenase deficiency at ages 11 and 33 months. CONCLUSION: Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency has detected cases with a wide range of genotypes and biochemical abnormalities. Although most children do well, adverse outcomes have not been entirely avoided. Assessment of potential risk and determination of appropriate treatment remain a challenge.

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OBJECTIVES: To develop a method for the determination of succinylacetone (SA) in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). METHODS: SA was extracted from DBS with an acetonitrile and water solution (80:20 by volume) containing formic acid and hydrazine hydrate (both at 0.1% by volume), and analyzed by MS/MS with a total run time per sample under 2 min. The reference range for SA in newborns was determined by analyzing a control group of 3199 DBS. SA was also measured in stored newborn specimens from three patients diagnosed clinically with hepatorenal tyrosinemia (HT). RESULTS: The within-run precision was

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OBJECTIVES: The treatment for phenylketonuria (PKU) includes monitoring blood phenylalanine (Phe) levels on a regular basis. To reduce inconvenience to the patient and family, blood specimens on filter paper can be obtained at home and mailed to the clinic or analytical laboratory. For this reason, we validated an 8-min isothermal and isocratic HPLC method using the Hitachi L-8800 analyzer for quantitation of Phe and tyrosine (Tyr) from dried blood specimens (DBS). DESIGN AND METHODS: The method was worked out using DBS fortified with Phe and Tyr. For method comparison, blood samples from 31 PKU patients and 5 non-PKU volunteers were analyzed as DBS by HPLC using the Hitachi L-8800 analyzer, and compared both to plasma analyzed by HPLC and DBS analyzed using tandem mass spectrometry (MS/MS). RESULTS: For HPLC analysis of DBS, the within-run precision for Phe and Tyr was < or = 5.1% and < or = 4.5%, respectively, and total precision measured over a 3-month period was < or = 7.2% and < or = 8.7%, respectively. Correlation analysis was performed using results from fresh plasma analyzed by HPLC (r = 0.988 for Phe, r = 0.964 for Tyr) and from DBS analyzed by MS/MS (r = 0.960 for Phe, r = 0.942 for Tyr). Difference plots revealed good agreement between the HPLC and MS/MS methods. CONCLUSIONS: Determination of Phe and Tyr in DBS using this HPLC technique compares well with other methods. This technique with its short analytical time is convenient for monitoring patients with PKU and might be particularly useful in centers following many patients.

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