Newborn Screening for Hepatorenal Tyrosinemia: Tandem Mass Spectrometric Quantification of Succinylacetone Johannes Sander,1* Nils Janzen,1 Michael Peter,1 Stefanie Sander,1 Ulrike Steuerwald,1 Ute Holtkamp,1 Bernd Schwahn,2 Ertan Mayatepek,2 Friedrich K. Trefz,3 and Anibh M. Das4 Background: False-positive and false-negative results occur in current newborn-screening programs for hepa- torenal tyrosinemia, which measure tyrosine concen- trations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening. Methods: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile���water (80:20 by volume) con- taining formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a sep- arate run. Results: Of 61 344 samples, 99.6% had succinylacetone concentrations 5 mol/L. With a cutoff of 10 mol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 mol/L, respectively, and the tyrosine concentrations were 54 and 129 mol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 addi- tional known patients revealed increased succinylac- etone concentrations of 46 and 169 mol/L, respectively. Conclusions: Tandem mass spectrometric quantifica- tion directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosine- mia in newborn-screening programs. �� 2006 American Association for Clinical Chemistry Accumulation of succinylacetone (SA)5 is generally con- sidered to be pathognomonic for hepatorenal tyrosinemia (HT MIM 276700), a rare autosomal recessive metabolic disorder characterized by life-threatening progressive liver and kidney dysfunction and hepatocellular cancer. Very early diagnosis allows immediate introduction of specific treatment, with significant reduction of morbidity and mortality. Increased blood concentrations of tyrosine are neither specific nor sensitive enough to screen for HT in newborns (1, 2) it therefore seems promising to screen for SA rather than tyrosine in a routine newborn-screen- ing program. A simple method for quantifying SA in dried blood spots has recently been described by Allard et al. (3). The method is based on extraction of SA from blood spots with acetonitrile and water containing formic acid and hydrazine hydrate. Instead of deuterated SA, which is not yet available commercially, unlabeled 5,7-dioxooctanoic acid was used as an internal standard. Under the condi- tions given, hydrazine is thought to cleave covalently linked SA-protein adducts (4) and to simultaneously form a hydrazone derivative (Fig. 1), which is extracted (5). The analysis was performed by tandem mass spectrometry (MS/MS) with a total run time of 80 s per sample. 1 Screening Laboratory, Hannover, Germany. 2 Department of General Paediatrics, University Children���s Hospital, Uni- versity of Du ��sseldorf, Du ��sseldorf, Germany. 3 Department of Paediatrics, Klinikum am Steinenberg, Reutlingen, Ger- many. 4 Department of Paediatrics, Medizinische Hochschule, Hannover, Ger- many. * Address correspondence to this author at: Screening Laboratory, Han- nover, Postfach 911009, D-30430 Hannover, Germany. Fax 49-5108-9216319 e-mail j.sander@metabscreen.de. Received August 26, 2005 accepted December 22, 2005. Previously published online at DOI: 10.1373/clinchem.2005.059790 5 Nonstandard abbreviations: SA, succinylacetone HT, hepatorenal ty- rosinemia (tyrosinemia type I) MS/MS, tandem mass spectrometry and NTBC, 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (Nitisone). Clinical Chemistry 52:3 482���487 (2006) Endocrinology and Metabolism 482

Residual blood spots that had already been extracted for the analysis of acylcarnitines and amino acids were shown to be suitable for testing. It appeared, therefore, that inclusion of SA in our existing screening program could be achieved with little additional manual work. Here we report our experience using this test for newborn screening. Materials and Methods For this 16-week study, we used unselected samples from our routine screening program for inborn errors of me- tabolism and endocrinopathies, which we received from various parts of Germany. Blood collection on S&S 903 filter paper for metabolic screening in Germany is recom- mended at 36 to 72 h after birth. Sample shipping takes an average of 2.3 days (55.2 h). According to German regu- lations, reporting of results should be completed within 72 h after blood collection, leaving only 1 day to perform all laboratory work and data reporting. Routine neonatal metabolic screening for amino acids and acylcarnitines in our laboratory is therefore done on 4 different MS/MS instruments (MS/MS microTM and Quatro LCTM Waters/ Micromass Inc.), and 2 additional tandem mass spectrom- eters used for scientific purposes are available for back- up. With laboratory work starting at 0700 in the morning, this instrumentation allows us to finish sample prepara- tion and the complete analytical run for up to 1000 samples per day by late afternoon. Final results for more than 90% of newborns are available by the end of the first week of life. For this study, SA was extracted from residual blood spots (3.2 mm) with 100 L of a solution of acetonitrile and HPLC-grade water (80:20 by volume) containing, per liter, 1 mL of formic acid, 15 mmol of hydrazine hydrate (1 mL), and 100 nmol of 5,7-dioxooctanoic acid as de- scribed by Allard et al. (3). Stability of the extract was excellent with 10% degradation of the derivative over the course of 60 h. Analytical preparations require some precaution (use of a fume hood and gloves) because hydrazine is a second-class carcinogen. The microtitration plates were agitated gently and incubated at 37 ��C cov- ered with aluminum foil. After 45 min, the extract was transferred to a second plate, which was covered with aluminum foil, for MS/MS analysis. The blood spots had earlier been extracted with 100 L of absolute methanol for the measurement of amino acids and acylcarnitines. Use of residual spots not only saved sample material but also reduced sample preparation time considerably. Quantitative results did not differ in residual spots com- pared with direct preparation, but the analytical back- ground was considerably less in residual spots, giving higher sensitivity. Quantification of SA was done in separate runs after the regular metabolic screening pro- gram on 2 of the routine instruments. This allowed us to use our laboratory���s analytical capacity during a time when it was otherwise not needed. For calibrators and quality-control samples, EDTA whole blood was fortified with 2, 5, 10, 20, 50, and 100 mol/L SA, and 25 L of each calibrator or quality- control sample was spotted on S&S filter cards and subsequently dried at ambient temperature overnight. Samples were then stored at 4 ��C. The donor blood had no detectable SA. We calculated the recovery from the cali- brator blood spots by repeating the analysis 6 times and comparing the results with those of the respective aque- ous solutions. The results are shown in Table 1. Results for SA were generated in positive ion mode with cone energy set at 20 V, collision voltage at 10 eV, and dwell time at 9.1 s. The injection volume was 30 L. The analysis was performed in multiple-reaction monitor- ing mode. Chemicals were of analytical grade: acetoni- trile, methanol, and formic acid were from Merck and SA, 5,7-dioxooctanoic acid (internal standard), and hydrazine hydrate were from Sigma-Aldrich. We measured 2 mass- specific transitions for SA (m/z 155.23137.1 and 155.23109.1) and 1 transition for 5,7-dioxooctanoic acid (m/z 169.33151.2 Fig. 2). We also measured the amino acids in multiple-reaction monitoring mode. Samples were prepared according to standard methods (6, 7). Briefly, blood spots 3.2 mm in diameter were extracted with 200 L of methanol contain- ing the deuterated internal standards as described previ- ously (8). After the solvent was evaporated, the amino acids were butylated by incubation with 50 L of bu- tanol���HCl at 65 ��C for 15 min, after which the sample was again dried. The residue was dissolved in acetonitrile��� water (80:20 by volume). During the 16-week study, we prospectively analyzed 61 344 unselected neonatal blood samples. Two affected children (patients 1 and 2) were identified. Two addi- tional samples were analyzed retrospectively, these were Fig. 1. Reaction scheme of the formation of a hydrazone from SA. Table 1. Precision of SA quantification and recovery. Concentration, mol/L CV, % Mean (SD) recovery, % Intraassay Interassay 2 10 13 80 (15) 10 6.1 10 75 (9.5) 50 4.6 5.8 72 (5.6) Clinical Chemistry 52, No. 3, 2006 483