Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry

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Abstract

Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a 13C4-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 μM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 μM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.

Introduction

Hepatorenal tyrosinemia (tyrosinemia type-I; HT1) is a rare autosomal recessive metabolic disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the terminal step in tyrosine (tyr) degradation [1], [2], [3], [4], [5], [6]. Toxic metabolites upstream of the enzyme block, namely, fumarylacetoacetate (FAA) and maleylacetoacetate are converted into succinylacetoacetate (SAA) which decarboxylates to give succinylacetone (SA; 4,6-dioxoheptanoic acid), which is elevated in body fluids of HT1 patients. It is believed that these toxic agents are the cause of the extensive clinical, pathological and neurological manifestations leading to liver cirrhosis, renal failure, hypophosphataemic rickets, hepatocellular carcinoma and death in early childhood [1], [7], [8]. HT1 is considered a treatable metabolic disease provided it is detected early in life and treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; Nitisinone™), the only alternative available for liver transplantation [9].

Biochemical findings associated with HT1 such as elevated α1-fetoprotein, hepatic transaminases, tyr, methionine (met), delta-aminolevulinic acid, 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate and 4-hydroxyphenylacetate are non-specific markers and are elevated in a number of other liver diseases in childhood [3], [4]. SA is not known to occur as an intermediate in any other metabolic pathway; therefore it is considered a specific diagnostic marker for HT1. Determination of FAH activity in human liver, lymphocytes and fibroblasts is restricted to patients with elevated SA due to the large number of cases presenting with non-specific clinical and biochemical findings [10], [11].

SA in plasma, urine and amniotic fluid was determined by gas chromatography–mass spectrometry (GC–MS) [12], [13], [14], [15], [16]. Traditionally, urine was the preferred matrix for diagnosis and treatment follow up for HT1 cases. This is due to the polarity of SA that has a relatively high renal clearance where the levels are about three times higher in urine than in plasma [14]. Recently, we described a liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) for the analysis of SA in urine [17]. This method involved the use of 15N-labeled 5(3)-methyl-3(5)-isoxazole propionic acid as internal standard. Urine samples from patients suspected with HT1 were converted to the isoxazole derivative by hydroxylamine HCl, extracted from the matrix followed by derivatization to the butyl ester. Although the method was sufficiently sensitive for urine, it was not sensitive enough to allow us to determine SA in dried blood spots (DBS).

The introduction of MS–MS-based methods for newborn and selective screening through the analysis of blood spots for amino acids (AAs) and acylcarnitines (ACs) for a multitude of inherited disorders made the DBS specimens the most frequently received sample in biochemical genetics laboratories due to the convenience of blood transport on filter paper plus the fact that it can be used readily for both biochemical and molecular testing [18]. Actually, in our previous report most of the 12 new HT1 cases were suspected and later confirmed due to the finding of high met (197–864 μM; cut-off 63 μM) and high tyr (207–681 μM; cut-off 200 μM) in DBS specimens by MS–MS analysis [17].

In the literature, there are two reports describing the analysis of SA in DBS. Schulze et al. reported a second-tier spectrophotometric method for the indirect determination of SA in DBS based on inhibition of delta-aminolevulinate dehydratase enzyme (ALA-D) by SA [19]. Allard et al. presented a primary screening method for HT1 by ESI–MS–MS analysis of SA from blood spots that were already extracted for AAs and ACs analysis [20].

In the work presented here we sought to develop a sensitive approach for the determination of SA that would allow us to reach a definitive diagnosis for clinically or biochemically suspected HT1 cases from the same DBS specimens that are used for AAs and ACs determination. This approach may eventually eliminate the need for urine analysis.

Section snippets

Chemicals

(13C4)Succinic anhydride (99 at.%) was purchased from Isotec. 4,6-Dioxoheptanoic acid (SA), thionyl chloride, t-butylacetoacetate were obtained from Sigma–Aldrich. 5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (dansylhydrazine, Dns-H) was purchased from Fluka Chemie. Trifluoroacetic acid (TFA), methanol, HPLC grade acetonitrile, HPLC grade ethylacetate and ammonium acetate were obtained from Fisher. Butanolic HCl was from Regis Technologies. Water used throughout this study was prepared by

Internal standard

To the best of our knowledge, this is the first report on the synthesis of stable-isotope 13C4-SA internal standard. This isotope is more stable compared to its deuterium antipode which may undergo exchange during sample preparation which involves heating in acidic medium and has advantages over our previously reported 15N-labeled 5(3)-methyl-3(5)isoxazole propionic acid used as IS in previous reports [14], [15], [17]. The main advantage is that the 13C4-SA differs by 4 mass units from SA and

Conclusion

We report a new approach to the determination of SA, the only reliable and accepted hallmark of HT1. This LC–MS–MS method allows for determination of SA in DBS as well as in urine specimens with a short analytical time. Based on our retrospective and prospective experiments the analysis of SA in DBS can clearly distinguish between normal and affected infants. The method is convenient that it allows for reaching a definitive diagnosis from the same DBS used for patient screening and may be used

Acknowledgment

This study was funded by a grant from Prince Salman Center for Disability Research. The authors are grateful for Gulf Bioanalytical Co. for providing the LC–MS–MS system employed.

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