MS Method for Simultaneous Determination on a Dried Blood

Technical Note pubs.acs.org/ac

LC-MS/MS Method for Simultaneous Determination on a Dried Blood Spot of Multiple Analytes Relevant for Treatment Monitoring in Patients with Tyrosinemia Type I Giancarlo la Marca,*,†,‡ Sabrina Malvagia,† Serena Materazzi,†,‡ Maria Luisa Della Bona,† Sara Boenzi,§ Diego Martinelli,§ and Carlo Dionisi-Vici§ †

Mass Spectrometry Laboratory, Clinic of Pediatric Neurology, Meyer University Children’s Hospital, Florence, Italy Department of Pharmacology, University of Florence, Florence, Italy § Division of Metabolism, Department of Pediatric Medicine, Bambino Gesù Children’s Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy ‡

S Supporting Information *

ABSTRACT: Tyrosinemia type 1 is caused by deficiency of fumarylacetoacetate hydrolase. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, causing accumulation of succinylacetone which induces severe liver and kidney dysfunction along with mutagenic changes and hepatocellular carcinoma. Treatment is based on nitisinone (NTBC), an enzymatic inhibitor which suppresses succinylacetone production. NTBC, which has dramatically changed the disease course improving liver and kidney functions and reducing risk of liver cancer, causes a side effect of the increase of tyrosine levels. Treatment is therefore based on the combination of NTBC with a protein-restricted diet to prevent the potential toxicity of excessive tyrosine accumulation. Longterm therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include succinylacetone, and a selected panel of circulating aminoacids. We have developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, succinylacetone, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run. A single assay suitable for quantitative evaluation of all biochemical markers is of great advance over conventional methods, especially in pediatric patients, since it reduces laboratory costs and blood sampling, is less invasive and particularly suitable for pediatric patients, and allows easier storage and shipping.

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is based on nitisinone (NTBC), an inhibitor of 4hydroxyphenylpyruvate dioxygenase which fully suppress SUAC production.3 This drug has dramatically changed the course of Tyr-1, improving liver and kidney functions and reducing neurologic crisis.4,5 Remarkably, patients starting NTBC therapy before the age of 2 years are, with few exceptions, at very low risk of developing HCC, whereas those who begun after this age still display a higher susceptibility for liver malignancy.6 Suppression of 4-hydroxyphenylpyruvate dioxygenase causes, as a side effect of NTBC therapy, the increase of tyrosine levels. Treatment of Tyr-1 is therefore based on the combination of NTBC with a protein-restricted diet, poor in tyrosine and phenylalanine, to prevent the potential toxicity of excessive tyrosine accumulation.4,7−10 To minimize side effects and to optimize treatment efficacy, the long-term therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include SUAC, and a selected panel

yrosinemia type 1 (Tyr-1) is caused by deficiency of fumarylacetoacetate hydrolase, the enzyme catalyzing the final step in the degradation of tyrosine. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, acetoacetate, and succinate. Unmetabolized fumarylacetoacetate is decarboxylated to succinylacetone (SUAC), and its accumulation has a toxic effect in hepatocytes and proximal renal tubal cells, causing inhibition of heme synthesis and of gluconeogenesis. Furthermore, SUAC induces oxidative damage, cell apoptosis, and mutagenic changes through DNA damage and dysfunctional gene expression.1,2 The presence of SUAC in plasma and urine, variable increase of tyrosine in plasma along with increased urinary excretion of deltaaminolevulinc acid, and markedly elevated serum concentration of alpha-fetoprotein represent the hallmarks for the diagnosis of Tyr-1.1,2 The clinical course is usually characterized by acute liver failure in infancy or by chronic liver dysfunction and renal Fanconi syndrome in late presenting cases. Phenylalanine and tyrosine restricted diet improves liver function but does not prevent progression of the hepatic disease and development of hepatocellular carcinoma (HCC) during childhood.1 To prevent the production of toxic metabolites, current treatment © 2011 American Chemical Society

Received: October 11, 2011 Accepted: December 9, 2011 Published: December 9, 2011 1184

dx.doi.org/10.1021/ac202695h | Anal. Chem. 2012, 84, 1184−1188

Analytical Chemistry

Technical Note

Figure 1. Upper panel showing the full scan MS spectrum of NTBC; the lower panel showing the precursor ion scan to identify appropriate fragment ions. The detected quantifier and qualifier MRM ions were m/z 218 and m/z 126, respectively.

of circulating aminoacids related to tyrosine metabolism and liver function (i.e., tyrosine, phenylalanine, and methionine). In the last years, several assays were developed to detect NTBC in plasma using HPLC, NMR spectroscopy, liquid chromatography coupled to tandem mass spectrometry (LCMS/MS), and capillary electrophoresis.11−14 Very recently, NTBC quantification has been reported on dried blood spot (DBS) based assays which showed a good correlation with values obtained on corresponding plasma samples.15,16 However, all these assays allow the quantification of NTBC, but they do not detect the entire metabolite panel necessary for treatment monitoring for which additional clinical chemistry tests and blood sampling are required. A single assay on DBS suitable for quantitative evaluation of all biochemical markers would be of great advantage, especially in pediatric patients, to reduce laboratory costs and blood sampling. On the basis of this assumption, we developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, SUAC, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run.

available from commercial sources and used without any further purification. Sample Preparation. Blood spots were kept in sealed plastic bags at 4 °C with desiccant until analysis. A 3.2-mm diameter disk was punched out from each DBS on the filter paper (903, Whatman GmbH, Dassel Germany) and extracted by addition of 0.2 mL of acetonitrile/water (70:30, v/v) and 0.05% formic acid containing the internal standards for tyrosine (13C6-Tyr, 5 μmol/L), methionine (2H3-Met, 5 μmol/L), phenylalanine (13C6-Phe, 5 μmol/L), and succinylacetone (13C4-SUAC, 0.1 μmol/L). Samples were put in an orbital shaker and kept at 37 °C for 25 min. The pooled samples were spiked with varying levels of target markers and used to perform the analytical method validation. We tested 15 DBS from 6 patients with confirmed Tyrosinemia type I to whom NTBC was administered. The age of patients ranged from 6 days to 15 years. The dose of NTBC was 1 mg/kg twice daily. The entire procedure was approved by the Ethical Committee of the Bambino Gesù Hospital, and patients’ parents signed an informed consent. Validation Procedures. The concentration of NTBC, tyrosine, phenylalanine, methionine, and SUAC in each sample was determined using a calibration curve prepared in duplicate by spotting on filter paper spiked human control blood to obtain concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 200.0, and 1000 μmol/L. Calibration curves were constructed using the observed peak area vs nominal concentrations of the analyte. Intraday variation was assessed from ten replicates of seven different concentrations analyzed during the same day

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EXPERIMENTAL SECTION Materials. Nitisinone powder (Orfadin) with a purity of 99.8% (w/w) was supplied by Swedish Orphan AB, (Stockholm, Sweden). Stock solution of 10 mg/L was made in acetonitrile/water (70:30 v/v). Successive dilutions were made using HPLC grade water. All solutions were stored in a freezer at −20 °C. All chemicals and solvents were of the highest purity 1185



Technical Note

Since long-term stability of amino acids in dried blood spots has been widely reported in literature,17 we assessed the effect of temperature on DBS samples for NTBC stored under various conditions over 1 month. Table 1 shows that specimens stored for 1 month at room temperature, +4 °C, and −20 °C were highly stable.

using a daily prepared calibration curve. Interday variation was assessed by analyzing identical sets of samples on ten different days over a 1 month period. The limit of determination (LOD) and the limit of quantization (LOQ) for NTBC and SUAC was defined by the signal-to-noise (S/N) ratio approach, measuring the chromatographic response of the analyte as 3 and 10 times the baseline noise, respectively. Average recovery of all analytes from DBS samples was calculated by comparing responses with those obtained by direct injection of the same amount of drug at two different concentrations (5.0 and 100.0 μmol/L). The short-term NTBC stability study on DBS samples was evaluated up to 1 month after storage at room temperature, −20°C, and 4 °C. Mass Spectrometry. An API 4000 triple quadrupole instrument (ABI-SCIEX, Toronto, Canada) equipped with the TurboIonSpray source operated in multiple reaction monitoring (MRM) under positive ion mode was used for the analysis. The spraying needle voltage was set to 5400 V, and turbo gas flow was set at 5 L/min of air heated at 450 °C (nominal heating-gun temperature). The optimized parameters and transitions monitored in MRM experiments are reported in Table S-1, Supporting Information. MS and MS/MS spectra were recorded by connecting the Harvard infusion pump directly to the ion sources. The quantitation experiments were undertaken using an Agilent Series 1100 instrument (Agilent Technologies Waldbronn, Germany) equipped with a binary pump delivery system (G1376A) and a robotic autosampler (G1377A), both being fully controlled from the API 4000 data system. Liquid chromatography was performed using an Agilent Poroshell 120 EC-C18 2.7 μm, 2.1 × 50 mm HPLC column (Agilent Technologies Waldbronn, Germany). Column flow was 0.5 mL/min using an aqueous solution of 85% acetonitrile containing 0.05% formic acid. The eluent from the column was directed to the TurboIonSpray probe without split ratio. Five microliters of the extracted sample were injected for the LC-MS/MS experiments. System control and data acquisition were performed with Analyst 1.4.1 software (AB Sciex, Foster City, CA, USA) including the “Explore” option (for chromatographic and spectral interpretation) and the “Quantitate” option (for quantitative information generation). Calibration curves were constructed with the Analyst Quantitation program using a linear least-squares regression nonweighted.

Table 1. Stability of NTBC on a Dried Blood Spot at Different Temperatures expected concentration (μmol/L)

storage temperature (°C)

mean (n = 3) samples stored for 1 month

DS

CV%

accuracy

5 100 5 100 5 100

RT RT +4 +4 −20 −20

4.96 100.55 5.11 99.8 5.09 100.96

0.13 2.66 0.21 4.03 0.19 2.36

2.62 2.64 4.11 4.03 3.73 2.34

99.2 100.55 102.2 99.8 101.8 100.96

The method precision was evaluated by inter- and intraday repeatability. Seven different concentrations of spike (0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0 μmol/L) were run ten times in 1 day, resulting in a intraday repeatability expressed as a CV below 13% for all values and all analytes. As a representative, the NTBC interday repeatability obtained in ten separate assays for 2 weeks was better than 9.5%. The obtained results are summarized in Table S-2, Supporting Information.

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DISCUSSION AND CONCLUSIONS The main purpose of our study was the development of a method to quantify in DBS the entire panel of analytes relevant for treatment monitoring of tyrosinemia type I; therefore, we compared DBS results with simultaneous plasma samples. To calculate the plasma volume contained in DBS, hematocrit was measured in 15 simultaneous blood samples obtained from 6 patients. Furthermore, the accuracy of the method was estimated by comparing DBS values to the corresponding plasma values using the population’s mean hematocrit instead of individual hematocrit. The relationship between DBS and plasma values over a wide range of concentrations seems to be acceptable. The comparative data in the two matrixes indicate that results obtained from DBS are suitable to estimate the concentrations of NTBC and of relevant amino acids. We also observed an appropriate fit between plasma values and DBS values obtained by correction for mean hematocrit levels adjusted for age as reported in Figure 2. The equation used to calculate plasma concentration from DBS samples is as follows: DBSconc × (100/ 100 − HT) = Plasmaconc. Chromatographic conditions and mass spectral parameters were adjusted in order to provide a good and fast performance of the assay since specificity is provided by the MS/MS measurement (Figure 3). In conclusion, a rapid, reliable, and sensitive MS/MS method has been developed and fully validated for the simultaneous determination in DBS of NTBC and relevant biomarkers of Tyr-1. The method will be helpful in processing a large number of samples for patient’s treatment monitoring at follow-up. Use of DBS for sampling requires little volume of blood spotted and dried on a card, with great advantages over conventional plasma sampling being less invasive, particularly in the pediatric population, and allowing easier storage and shipping.

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RESULTS Multiple reaction-monitoring (MRM) mode was used with two transitions for NTBC and one for all other analytes (Figure 1). The LOD was 0.1 μmol/L (or 32.9 ng/mL) and 0.1 μmol/L (or 15.5 ng/mL) for both NTBC and SUAC. The LOQ was 0.25 μmol/L (or 82.2 ng/mL) and 0.3 μmol/L (or 46.5 ng/ mL), respectively. The specificity of the method was investigated. No interference was observed in the chromatogram, even if matrix components interfere reducing the signal intensity. No deterioration in column efficiency was observed after the analysis of 100 DBS samples. The average recovery of NTBC and SUAC exceeded 99% for both tested concentrations. The recovery for tyrosine, phenylalanine, and methionine ranged from 95.3% to 97.1%. A linear response was found for all analytes with wide linear ranges. The correlation coefficient (R2) for each analyte was greater than 0.997 as determined by least-squares analysis. 1186



Technical Note

Figure 2. Scatter plots showing a high correlation between analyte concentrations obtained on a dried blood spot corrected for hematocrit vs plasma values. No significant differences were observed between matrixes, even though methionine resulted in the worst correlation factor (R2).

Figure 3. Top panel showing selected ion chromatograms from a patient DBS sample. The bottom panel shows the MRM transitions chosen to quantify NTBC.

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ASSOCIATED CONTENT

S Supporting Information *

AUTHOR INFORMATION

Corresponding Author

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

*Address: Department of Pharmacology, University of Florence, Viale Pieraccini, 24, Florence, Italy 50134. Phone: + 1187



Technical Note

+ 39-(0)55-5662988. Fax: ++ 39-(0)55-5662489. E-mail: [email protected] or [email protected].

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ACKNOWLEDGMENTS C.D.-V. and S.B. have been supported by the Project CCM from the Italian Ministry of Health “Costruzione di percorsi diagnostico-assistenziali per le malattie oggetto di screening neonatale allargato”; we thank the Association “la Vita è un Dono” for supporting the fellowship of D.M.

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MS Method for Simultaneous Determination on a Dried Blood

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