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Measurement of succinyl-carnitine and methylmalonyl-carnitine on dried blood spot by liquid chromatography-tandem mass spectrometry
Clinica Chimica Acta 429 (2014) 30–33
Contents lists available at ScienceDirect
Clinica Chimica Acta jour nal homepage: www.elsevier.com/locate/clinchim
Measurement of succinyl-carnitine and methylmalonyl-carnitine on dried blood spot by liquid chromatography-tandem mass spectrometry Cristiano Rizzo a,1, Sara Boenzi b,⁎,1, Rita Inglese a, Giancarlo la Marca c,d, Maurizio Muraca a, Tegra Barreiro Martinez e, David W. Johnson f, Eleonora Zelli a, Carlo Dionisi-Vici b a
Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children’s Research Hospital, IRCCS, Rome, Italy NeuroFarba Department, University of Florence, Florence, Italy d Newborn Screening Lab, Clinic of Pediatric Neurology, AOU Meyer, Florence, Italy e Bioquimica Clinica y Istituto de Genetica Medica y Molecular, Hospital Universitario La Paz, Madrid, Spain f Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia, Australia b c
a r t i c l e
i n f o
Article history: Received 19 March 2013 Received in revised form 14 November 2013 Accepted 14 November 2013 Available online 22 November 2013 Keywords: methylmalonyl-carnitine succinyl-carnitine liquid chromatography-tandem mass spectrometry SUCLA2 SUCLG1 methylmalonic acidemia
a b s t r a c t Methylmalonic aciduria (MMA) is one of the most frequent organic acidurias, a class of diseases caused by enzymatic defects mainly involved in the catabolism of branched-chain amino acids. Recently, mild MMA and C4-dicarboxylyl-carnitine (C4DC-C) accumulation have been reported in patients carrying mutation in genes encoding the α-subunit (SUCLG1) and the β-subunit (SUCLA2) of the ADP-forming succinyl-CoA synthetase (SCS). We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify in dried blood spot the two isobaric compounds of C4DC-C, succinyl-carnitine and methylmalonyl-carnitine, to allow the differential diagnosis between classical MMA and SCS-related defects. This method, with an easy liquid-phase extraction and derivatization procedure, has been validated to demonstrate the specificity, linearity, recovery, lowest limit of quantification (LLOQ), accuracy and precision for quantitative determination of blood succinyl-carnitine and methylmalonyl-carnitine. The assay was linear over a concentration range of 0.025–10 μmol/L and achieved the LLOQ of 0.025 μmol/L for both metabolites. The average slope, intercept, and coefficient of linear regression (r2) were respectively: 0.3389 (95% confidence interval 0.2888–0.3889), 0.0113 (95% confidence interval −0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990–1.0000) for succinyl-carnitine and 0.5699 (95% confidence interval 0.5263–0.6134), 0.0319 (95% confidence interval −0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995–1.0000) for methylmalonyl-carnitine. Withinday and between-day coefficients of variation (CV) were 1.94% and 3.19% for succinyl-carnitine and 3.21%, and 2.56 for methylmalonyl-carnitine. This method is accurate and provides a new tool to differentiate patients with classical methylmalonic acidemia from those with SCS-related defects. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Methylmalonic acidurias (MMAs) are a group of genetically heterogeneous autosomal recessive disorders of methylmalonate and cobalamin Abbreviations: MMAs, Methylmalonic acidurias; SCS, succinyl-CoA synthetase; C3-C, propionyl-carnitine; C4DC-C, C4-dicarboxylyl-carnitine; C3-C/C2-C, propionyl-carnitine/ acetyl-carnitine ratio; C3-C/C16-C, propionyl-carnitine/palmitoyl-carnitine ratio; MS/MS, tandem mass spectrometry; SC, succinyl-carnitine; MMC, methylmalonyl-carnitine; TDHFA, tridecafluoroheptanoic acid; FOA, formic acid; DP, declustering potential; CXP, collision cell exit potential; CE, collision energy; LOD, limit of detection; LLOQ, lower limit of quantification; S/N, signal-to-noise ratio; SD, standard deviation; MRM, multiple reaction monitoring. ⁎ Corresponding author at: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS, P.zza S. Onofrio, 4, 00165, Roma. Tel.: +39 06 6859 2519; fax: +39 06 6859 2014. E-mail address: [email protected] (S. Boenzi). 1 These two authors contributed equally to this work. 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.016
metabolism caused by a complete (mut0) or partial (mut−) deficiency of the enzyme methylmalonyl-CoA mutase (EC 5.4.99.2), for the conversion of methylmalonyl-CoA to succinyl-CoA, or by defects in the cobalamin metabolism (CblA, CblB, CblC, CblD, CblF) [1]. Recently a few patients have been described with mild methylmalonic aciduria with deficiencies in the two subunits α and β of the mitochondrial matrix enzyme succinyl-CoA synthetase caused by SUCLG1 and SUCLA2 gene mutations. Succinyl-CoA synthetase (SCS), also called succinate ligase, is a Krebs cycle enzyme that not only converts succinyl-CoA to succinate and free Coenzyme A, but also converts ADP to ATP and GDP to GTP. The substrate specificity for ADP and GDP is determined by the β-subunits, whereas the α subunit is shared. The α-subunit is coded by the gene SUCLG1, whereas the β-subunit is encoded by SUCLA2 for the ADP specificity, and by SUCLG2 for the GDP specificity [2]. Patients with classical MMA or with SCS-related defects SUCLG1 or SUCLA2 mutation present hypotonia, muscle weakness,
C. Rizzo et al. / Clinica Chimica Acta 429 (2014) 30–33
hypoacusis, Leigh disease, lactic acidosis, polyneuropathy, mild methylmalonic aciduria and mild elevation of C4DC-C [2,3]. The diagnosis of MMAs is based on the presence of characteristic compounds in body fluids detected by organic acids analysis in urine (methylmalonic acid, 3-hydroxy-propionic acid and methylcitric acid) and blood acylcarnitine profiling (propionyl-carnitine (C3-C), C4-dicarboxylyl-carnitine (C4DC-C) and propionyl-carnitine/acetylcarnitine (C3-C/C2-C) and propionyl-carnitine/palmitoyl-carnitine (C3-C/C16-C) ratios [4,5]. The introduction of tandem mass spectrometry (MS/MS) based on rapid and simultaneous quantification of acylcarnitines and aminoacids on dried blood spot [6], has significantly increased early diagnosis of inborn errors of metabolism [7]. The commonly used method for the determination of acylcarnitines on dried blood spot quantifies the whole C4DC-C which is the sum of two different isobaric compounds: succinyl-carnitine (SC) and methylmalonyl-carnitine (MMC). In this paper we report a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to identify and quantify SC and MMC. This allows the differential diagnosis between classical MMA and SUCLA2 or SUCLG1 defects where MMC or SC was respectively elevated. Our study adds a new method to the diagnosis of MMAs and SUCLA2 or SUCLG1 defects, which may also be relevant for a second tier test when C4DC-C is found elevated in routine acylcarnitines analysis.
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2.4. Liquid chromatography-mass spectrometry Chromatography was performed on an Agilent series 1200 pump and autosampler (Agilent Technologies Inc., Wilmington, DE, USA). The column for chromatographic separation was a Zorbax Eclipse XDBC8 column 5 μm, 4.6 × 150 mm, (Agilent Technologies, Santa Clara, CA). The mobile phase was solution A (water containing 0.5 μmol/L TDHFA) and solution B (acetonitrile containing 0.5 μmol/L TDHFA. Chromatographic separation of metabolites was obtained with gradient elution. Gradient starts from 90% A to 10% in 10 min then 3 min at 10% A. Flow rate was 1.0 mL/min. The column was maintained at room temperature. 5 μL of sample were injected into the column. The total run was 16 min. Tandem mass spectrometry experiments were carried out on an API3200 triple quadrupole mass spectrometer (Applied BiosystemsSciex, Toronto, Canada), equipped with a Turbo Ion Spray Source operating in positive ion mode with a needle potential of 5500 V; the source temperature was 550 °C. Declustering Potential (DP), Collision Cell Exit Potential (CXP) and Collision Energy (CE) were optimized by direct infusion at flow rate 10 μL/min of each analyte in the mass spectrometer. The injected solutions were 5.0 μmol/L in water/acetonitrile 50/50 v/v containing 0.05% FOA. The resulting DP was 40 V and optimal CE and CXP were found at 40 and 3 V for SC, and MMC. The following transitions were monitored: m/z 374.3 N 85.1 for SC and MMC and m/z 377.3 N 85.1 for [2H3]-SC and [2H3]-MMC.
2. Materials and methods
2.5. Standard curves for quantification
2.1. Reagents
Methanolic standard solutions of 10, 5, 1, 0.5, 0.1, 0.05 0.01 μmol/L for SC and MMC were analyzed with the same procedure of blood spot samples. The acquired data were processed using Analyst® version 1.4.2 software (Applied Biosystems-Sciex), including option for chromatographic and spectral interpretation and for quantitative information generation. Calibration curves were constructed with the Analyst Quantitation program using a linear least-square regression non-weighted. The limit of detection (LOD) was determined by progressive dilutions of calibrator solutions of each analyte and was considered as the lowest concentration for which the signal-to-noise ratio (S/N) was indicated by the Analyst software to be at least 3. The lower limit of quantification (LLOQ) was determined by preparing calibrator solutions with decreasing concentration of each analyte and was considered as the lowest concentration for which the signal-to-noise ratio (S/N) was indicated by the Analyst software to be at least 10.
SC, MMC and labelled internal standards [2H3]-SC and [2H3]-MMC, were synthesized by Dr. David Johnson (Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia). HPLC grade acetonitrile and water were purchased from Romil Ltd. (The Source Convent Drive Waterbeach Cambridge, United Kingdom). Tridecafluoroheptanoic acid (TDHFA) 98/100% and butanolic-HCl were obtained from Sigma-Aldrich (Steinheim, Germany). Formic acid (FOA) 98/100% was obtained from BDH (VWR Ltd, Poole, England).
2.2. Preparation of standard solutions We prepared [2H3]-SC and [2H3]-MMC stock solutions (Stock-1) in water 1 mmol/L in order to investigate molecular fragmentation and also to obtain calibration curves for SC and MMC. This was stored frozen at −20 °C. “Daily standard solution” containing 10 μmol/L [2H3]-SC and [2H3]-MMC was prepared by dilution of Stock-1 solution 1:100 (v/v) with methanol.
2.3. Sample treatment procedure Whole blood was drawn by heel prick or venipuncture and dried on a filter paper (W303). We punched one disk of 5 mm diameter containing ~ 10 μL of whole blood from each dried blood spot into a vial and mixed it with 50 μL of “Daily standard solution”. Four hundred microliters of methanol was added to precipitate proteins and to extract the metabolites. Each vial was mixed by vortex for 20 min, then centrifuged for 5 min at 10,000 rpm. The supernatant fluid was transferred into a clean vial and dried at 40 °C under nitrogen stream. Eighty microliters of butanolic-HCl was added to derivatize metabolites as butyl-esters and heated at 65 °C for 15 min. Samples were dried again at 40 °C under nitrogen stream and finally they were reconstituted with 200 μL of acetonitrile–water 50:50 (v/v) containing 0.05% FOA to obtain pH 4 which is the appropriate pH for metabolites ionization.
2.6. Sample collection of patients and controls Twenty children's blood spot, for reference values, were obtained from hospitalized neurologically healthy subjects (10 females and 10 males; age: 1 day to 18 year, mean 7 year). Blood spot samples from patients where C4DC-C was found elevated (N1 μmol/L) using routine MS/MS screening method [6] were reanalyzed with LC-MS/MS method (2 samples of 1 patient affected by methylmalonyl-CoA mutase; 6 samples of 5 patients with mutation of SUCLA2 and 3 samples of 1 patient with mutation of SUCLG1). One of SUCLA2 blood spot was a neonatal blood spot. All samples were treated as described in sample treatment procedure. Control's and patient's blood samples were collected after obtaining informed consent. Patient's blood was collected during routine outpatient evaluation following an overnight fast before receiving the first morning dose of medication. 2.7. Statistical analysis The SPSS version 11.5.1 (SPSS Inc., Chicago, US) was used as statistical software. A preliminary test (Kolmogorov–Smirnov) was performed
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C. Rizzo et al. / Clinica Chimica Acta 429 (2014) 30–33
Fig. 1. Extract ion chromatogram of m/z 374.3 N 85.1 for SC and MMC and m/z 377.3 N 85.1 for [2H3]-SC and [2H3]-MMC. Retention time for SC and [2H3]-SC was 8.45 min; retention time for MMC and [2H3]-MMC was 8.60 min.
to assess normality of distributions of SC and MMC. Descriptive statistics were presented as mean ± standard deviation (SD). Range = mean ± 2SD. 3. Results and discussion 3.1. Chromatography and mass spectra An extract ion chromatogram from a 1 μmol/L SC and MMC standard solution is shown in Fig. 1. Multiple Reaction Monitoring (MRM) was used for the detection of the specific transitions in positive ion mode for the monitoring of each molecule. Under the above described conditions, the system was efficient for the separation of the isobaric compounds SC and MMC, eluted with their respective labeled internal standard at the retention times of 8.45 and 8.60 min, respectively. 3.2. Linearity, limit of detection, precision and recovery Calibration curves were linear over concentrations from 0.025 μmol/L up to 10 μmol/L for SC and MMC, which over covers the plasma concentration ranges for both metabolites. The linearity was monitored on 7
consecutive days. The average slope, intercept, and coefficient of linear regression (r2) were respectively: 0.3389 (95% confidence interval 0.2888–0.3889), 0.0113 (95% confidence interval −0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990–1.0000) for SC and 0.5699 (95% confidence interval 0.5263–0.6134), 0.0319 (95% confidence interval −0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995– 1.0000) for MMC. LOD of the analytes was 0.01 μmol/L for SC (S/N ratio 7.8) and 0.01 μmol/L for MMC (S/N ratio 8.1). LLOQ was 0.025 μmol/L for SC (S/N ratio 13.1) and 0.025 μmol/L MMC (S/N ratio 13.6). All validation experiments were performed with pooled blood from healthy controls and the validation data of the presented method are listed in Tables 1 and 2. The intra-assay variation was assessed from 10 replicates within one day (n = 10) and inter-assay from 3 times on 10 different days (n = 30); CVs for SC and MMC in plasma were in all cases b3.2%. Recovery experiments were performed at two different concentrations: pooled blood was spiked with SC (2 and 5 μmol/L) and with MMC (2 and 5 μmol/L). The SC recovery was respectively 105.3% and 100.8% of the expected amount and the same way MMC recovery was 95.04% and 98.41%.
Table 2 Accuracy data for succinyl-carnitine and methylmalonyl-carnitine. Table 1 Precision data for the LC-MS/MS assay.
Mean ± SD (μmol/L) Mean ± SD (μmol/L)
CV (%)
Intra-day (n = 10) Succinyl-carnitine Methylmalonyl-carnitine
7.630 ± 0.148 4.888 ± 0.155
1.94 3.19
Inter-day (n = 30) Succinyl-carnitine Methylmalonyl-carnitine
7.693 ± 0.247 4.948 ± 0.127
3.21 2.56
CV (%)
Recovery (%)
Succinyl-carnitine added (n = 5) 0 μmol/L 2.664 ± 0.175 2 μmol/L 4.770 ± 0.136 5 μmol/L 7.704 ± 0.269
6.59 2.86 3.49
not calculated 105.3 100.8
Methylmalonyl-carnitine added (n = 5) 0 μmol/L 0.087 ± 0.005 2 μmol/L 1.988 ± 0.081 5 μmol/L 5.008 ± 0.075
6.37 4.12 1.51
not calculated 95.04 98.41
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Table 3 Concentration of characteristic acylcarnitines obtained on patient's blood spots by MS/MS analysis compared with the new LC-MS/MS method. Acylcarnitine screening method (MS/MS)
LC-MS/MS separation method
Age
Sample number
C3
C4-DC
C3/C2
C3/C16
Succinyl-carnitine
Methylmalonyl-carnitine
pt.1 SUCLA2 pt.2 SUCLA2
5 years 3 years
pt.3 SUCLA2 pt.4 SUCLA2 pt.5 SUCLG1
10 years 2 days 9 years
pt.6 MMA
18 years
1 1 2 1 1 1 2 3 1 2
0.83 0.60 1.48 1.30 0.90 1.43 1.35 1.60 7.41 14.93 0.25–2.30
1.38 2.44 1.56 1.50 1.36 2.73 1.54 1.44 1.03 4.89 0.08–1
0.07 0.03 0.05 0.14 0.04 0.05 0.06 0.12 0.89 0.67 b0.19
2.32 0.47 1.16 1.29 0.75 1.07 1.09 1.51 27.76 35.39 b1.9
4.44 1.64 0.83 3.76 3.88 1.46 1.01 1.51 0.59 0.57 0.54–0.72
0.43 0.39 0.38 0.41 0.42 0.25 0.27 0.25 1.94 4.25 0.39–0.52
Reference values
3.3. Patients results When patient's samples obtained by classical MMA or SCS-related defects were analyzed by MS/MS method, increased levels of C4DC-C were detected as expected according to their diagnosis. Furthermore, C3-C, C3-C/C2-C and C3-C/C16-C ratios were elevated only in samples from classical MMA patient. The use of the new LC-MS/MS method allows clear differentiation of the two isobaric compounds of C4DC-C. SC was elevated only in SUCLA2 and SUCLG1 defects whereas MMC represents the characteristic compound of classical MMA alone (Table 3). Discrepancy between C4DC-C concentration values obtained by MS/MS method and the sum of the isobaric compounds SC + MMC values obtained by LC-MS/MS is explained by the use of different quantification internal standards. The internal standard used to quantify C4DC-C by the MS/MS method is labeled octanoyl-carnitine, while in our new LC-MS/MS method the two internal standards [2H3]-SC and [2H3]-MMC are used to quantify respectively SC and MMC. Similar data to those obtained in blood spots were observed in six plasma samples from three patients: two samples from one SUCLA2 patient (Pt.3), three samples from one SUCLG1 patient (Pt.5), and one from MMA patient (Pt.6). SC was increased in SUCLA2 and SUCLG1 patients (from 2.36 to 2.91 μmol/L; control values 0.10–0.80 μmol/L) whereas MMC was increased in MMA patient (2.08 μmol/L; control values 0.27–0.48 μmol/L). In order to verify correlation between SC and MMC and their related free acids, we also quantified plasma succinic acid and methylmalonic acid in 2 plasma samples from SUCLA2 patient (Pt.3) and 3 healthy controls by LC-MS/MS [4]. We found mild elevated succinic acid (6.72–7.52 μmol/L) and methylmalonic acid (1.7–1.9 μmol/L) in SUCLA2, compared to controls (succinic acid 3.7–5.1 μmol/L; methylmalonic acid not detectable). The elevation of succinic acid parallels SC whereas a slight elevation of methylmalonic acid did not parallel with MMC. Probably, higher plasma concentration of succinic acid with respect to methylmalonic acid allows a higher conjugation with carnitine in SUCLA2. Therefore, a more detailed study on correlation between methylmalonic acid and succinic acid in whole blood or plasma and MMC and SC will be very interesting for future investigations. The advantage of the new LC-MS/MS method over the MS/MS acylcarnitine analysis is the complete chromatographic separation of two isomers SC and MMC which allows a more precise and accurate quantification of the two metabolites, clarifying the diagnosis in all
cases where C4DC-C was found elevated in routine MS/MS acylcarnitines analysis. 4. Conclusions To our knowledge, this is the first time that SC and MMC involved in SUCLA2 and SUCLG1 and MMA defects have been both monitored and quantified in LC-MS/MS using a single blood spot sample. Good linearity, quantitative recovery and precision were obtained, demonstrating the accuracy of the method for quantitative determination of SC and MMC. The method is well proved and tested, and we believe that it represents an innovative contribution to the determination of SC and MMC in blood spot and therefore suitable for implementation in routine clinical acylcarnitine analysis as 2nd tier test and it could be also useful for follow up of SUCLA2, SUCLG1 defects. Acknowledgment We thank Dr. Stefano Garrone for his support in the manuscript writing. References [1] Deodato F, Boenzi S, Santorelli FM, Dionisi-Vici C. Methylmalonic and propionic aciduria. Am J Med Genet C Semin Med Genet 2006;142:104–12. [2] Van Hove JL, Saenz MS, Thomas JA, et al. Succinyl-CoA ligase deficiency: a mitochondrial hepatoencephalomyopathy. Pediatr Res 2010;68:159–64. [3] Carrozzo R, Dionisi-Vici C, Steuerwald U, et al. SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness. Brain 2007;130:862–74. [4] la Marca G, Malvagia S, Pasquini E, Innocenti M, Donati MA, Zammarchi E. Rapid 2ndtier test for measurement of 3-OH-propionic and methylmalonic acids on dried blood spots: reducing the false-positive rate for propionylcarnitine during expanded newborn screening by liquid chromatography-tandem mass spectrometry. Clin Chem 2007;53:1364–9. [5] la Marca G, Malvagia S, Casetta B, Pasquini E, Donati MA, Zammarchi E. Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: update on methods to reduce false tests. J Inherit Metab Dis 2008; 31:395–404. [6] Rashed MS, Ozand PT, Bucknall MP, Little D. Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and aminoacids profiling using automated electrospray tandem mass spectrometry. Pediatr Res 1995;38:324–31. [7] Bennett MJ, Rinaldo P, Wilcken B, Pass KA, Watson MS, Wanders WJ. Newborn screening for metabolic disorders: how are we doing, and where are we going? Clin Chem 2012;58:324–31.
Contents lists available at ScienceDirect
Clinica Chimica Acta jour nal homepage: www.elsevier.com/locate/clinchim
Measurement of succinyl-carnitine and methylmalonyl-carnitine on dried blood spot by liquid chromatography-tandem mass spectrometry Cristiano Rizzo a,1, Sara Boenzi b,⁎,1, Rita Inglese a, Giancarlo la Marca c,d, Maurizio Muraca a, Tegra Barreiro Martinez e, David W. Johnson f, Eleonora Zelli a, Carlo Dionisi-Vici b a
Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children’s Research Hospital, IRCCS, Rome, Italy NeuroFarba Department, University of Florence, Florence, Italy d Newborn Screening Lab, Clinic of Pediatric Neurology, AOU Meyer, Florence, Italy e Bioquimica Clinica y Istituto de Genetica Medica y Molecular, Hospital Universitario La Paz, Madrid, Spain f Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia, Australia b c
a r t i c l e
i n f o
Article history: Received 19 March 2013 Received in revised form 14 November 2013 Accepted 14 November 2013 Available online 22 November 2013 Keywords: methylmalonyl-carnitine succinyl-carnitine liquid chromatography-tandem mass spectrometry SUCLA2 SUCLG1 methylmalonic acidemia
a b s t r a c t Methylmalonic aciduria (MMA) is one of the most frequent organic acidurias, a class of diseases caused by enzymatic defects mainly involved in the catabolism of branched-chain amino acids. Recently, mild MMA and C4-dicarboxylyl-carnitine (C4DC-C) accumulation have been reported in patients carrying mutation in genes encoding the α-subunit (SUCLG1) and the β-subunit (SUCLA2) of the ADP-forming succinyl-CoA synthetase (SCS). We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify in dried blood spot the two isobaric compounds of C4DC-C, succinyl-carnitine and methylmalonyl-carnitine, to allow the differential diagnosis between classical MMA and SCS-related defects. This method, with an easy liquid-phase extraction and derivatization procedure, has been validated to demonstrate the specificity, linearity, recovery, lowest limit of quantification (LLOQ), accuracy and precision for quantitative determination of blood succinyl-carnitine and methylmalonyl-carnitine. The assay was linear over a concentration range of 0.025–10 μmol/L and achieved the LLOQ of 0.025 μmol/L for both metabolites. The average slope, intercept, and coefficient of linear regression (r2) were respectively: 0.3389 (95% confidence interval 0.2888–0.3889), 0.0113 (95% confidence interval −0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990–1.0000) for succinyl-carnitine and 0.5699 (95% confidence interval 0.5263–0.6134), 0.0319 (95% confidence interval −0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995–1.0000) for methylmalonyl-carnitine. Withinday and between-day coefficients of variation (CV) were 1.94% and 3.19% for succinyl-carnitine and 3.21%, and 2.56 for methylmalonyl-carnitine. This method is accurate and provides a new tool to differentiate patients with classical methylmalonic acidemia from those with SCS-related defects. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Methylmalonic acidurias (MMAs) are a group of genetically heterogeneous autosomal recessive disorders of methylmalonate and cobalamin Abbreviations: MMAs, Methylmalonic acidurias; SCS, succinyl-CoA synthetase; C3-C, propionyl-carnitine; C4DC-C, C4-dicarboxylyl-carnitine; C3-C/C2-C, propionyl-carnitine/ acetyl-carnitine ratio; C3-C/C16-C, propionyl-carnitine/palmitoyl-carnitine ratio; MS/MS, tandem mass spectrometry; SC, succinyl-carnitine; MMC, methylmalonyl-carnitine; TDHFA, tridecafluoroheptanoic acid; FOA, formic acid; DP, declustering potential; CXP, collision cell exit potential; CE, collision energy; LOD, limit of detection; LLOQ, lower limit of quantification; S/N, signal-to-noise ratio; SD, standard deviation; MRM, multiple reaction monitoring. ⁎ Corresponding author at: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS, P.zza S. Onofrio, 4, 00165, Roma. Tel.: +39 06 6859 2519; fax: +39 06 6859 2014. E-mail address: [email protected] (S. Boenzi). 1 These two authors contributed equally to this work. 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.016
metabolism caused by a complete (mut0) or partial (mut−) deficiency of the enzyme methylmalonyl-CoA mutase (EC 5.4.99.2), for the conversion of methylmalonyl-CoA to succinyl-CoA, or by defects in the cobalamin metabolism (CblA, CblB, CblC, CblD, CblF) [1]. Recently a few patients have been described with mild methylmalonic aciduria with deficiencies in the two subunits α and β of the mitochondrial matrix enzyme succinyl-CoA synthetase caused by SUCLG1 and SUCLA2 gene mutations. Succinyl-CoA synthetase (SCS), also called succinate ligase, is a Krebs cycle enzyme that not only converts succinyl-CoA to succinate and free Coenzyme A, but also converts ADP to ATP and GDP to GTP. The substrate specificity for ADP and GDP is determined by the β-subunits, whereas the α subunit is shared. The α-subunit is coded by the gene SUCLG1, whereas the β-subunit is encoded by SUCLA2 for the ADP specificity, and by SUCLG2 for the GDP specificity [2]. Patients with classical MMA or with SCS-related defects SUCLG1 or SUCLA2 mutation present hypotonia, muscle weakness,
C. Rizzo et al. / Clinica Chimica Acta 429 (2014) 30–33
hypoacusis, Leigh disease, lactic acidosis, polyneuropathy, mild methylmalonic aciduria and mild elevation of C4DC-C [2,3]. The diagnosis of MMAs is based on the presence of characteristic compounds in body fluids detected by organic acids analysis in urine (methylmalonic acid, 3-hydroxy-propionic acid and methylcitric acid) and blood acylcarnitine profiling (propionyl-carnitine (C3-C), C4-dicarboxylyl-carnitine (C4DC-C) and propionyl-carnitine/acetylcarnitine (C3-C/C2-C) and propionyl-carnitine/palmitoyl-carnitine (C3-C/C16-C) ratios [4,5]. The introduction of tandem mass spectrometry (MS/MS) based on rapid and simultaneous quantification of acylcarnitines and aminoacids on dried blood spot [6], has significantly increased early diagnosis of inborn errors of metabolism [7]. The commonly used method for the determination of acylcarnitines on dried blood spot quantifies the whole C4DC-C which is the sum of two different isobaric compounds: succinyl-carnitine (SC) and methylmalonyl-carnitine (MMC). In this paper we report a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to identify and quantify SC and MMC. This allows the differential diagnosis between classical MMA and SUCLA2 or SUCLG1 defects where MMC or SC was respectively elevated. Our study adds a new method to the diagnosis of MMAs and SUCLA2 or SUCLG1 defects, which may also be relevant for a second tier test when C4DC-C is found elevated in routine acylcarnitines analysis.
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2.4. Liquid chromatography-mass spectrometry Chromatography was performed on an Agilent series 1200 pump and autosampler (Agilent Technologies Inc., Wilmington, DE, USA). The column for chromatographic separation was a Zorbax Eclipse XDBC8 column 5 μm, 4.6 × 150 mm, (Agilent Technologies, Santa Clara, CA). The mobile phase was solution A (water containing 0.5 μmol/L TDHFA) and solution B (acetonitrile containing 0.5 μmol/L TDHFA. Chromatographic separation of metabolites was obtained with gradient elution. Gradient starts from 90% A to 10% in 10 min then 3 min at 10% A. Flow rate was 1.0 mL/min. The column was maintained at room temperature. 5 μL of sample were injected into the column. The total run was 16 min. Tandem mass spectrometry experiments were carried out on an API3200 triple quadrupole mass spectrometer (Applied BiosystemsSciex, Toronto, Canada), equipped with a Turbo Ion Spray Source operating in positive ion mode with a needle potential of 5500 V; the source temperature was 550 °C. Declustering Potential (DP), Collision Cell Exit Potential (CXP) and Collision Energy (CE) were optimized by direct infusion at flow rate 10 μL/min of each analyte in the mass spectrometer. The injected solutions were 5.0 μmol/L in water/acetonitrile 50/50 v/v containing 0.05% FOA. The resulting DP was 40 V and optimal CE and CXP were found at 40 and 3 V for SC, and MMC. The following transitions were monitored: m/z 374.3 N 85.1 for SC and MMC and m/z 377.3 N 85.1 for [2H3]-SC and [2H3]-MMC.
2. Materials and methods
2.5. Standard curves for quantification
2.1. Reagents
Methanolic standard solutions of 10, 5, 1, 0.5, 0.1, 0.05 0.01 μmol/L for SC and MMC were analyzed with the same procedure of blood spot samples. The acquired data were processed using Analyst® version 1.4.2 software (Applied Biosystems-Sciex), including option for chromatographic and spectral interpretation and for quantitative information generation. Calibration curves were constructed with the Analyst Quantitation program using a linear least-square regression non-weighted. The limit of detection (LOD) was determined by progressive dilutions of calibrator solutions of each analyte and was considered as the lowest concentration for which the signal-to-noise ratio (S/N) was indicated by the Analyst software to be at least 3. The lower limit of quantification (LLOQ) was determined by preparing calibrator solutions with decreasing concentration of each analyte and was considered as the lowest concentration for which the signal-to-noise ratio (S/N) was indicated by the Analyst software to be at least 10.
SC, MMC and labelled internal standards [2H3]-SC and [2H3]-MMC, were synthesized by Dr. David Johnson (Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia). HPLC grade acetonitrile and water were purchased from Romil Ltd. (The Source Convent Drive Waterbeach Cambridge, United Kingdom). Tridecafluoroheptanoic acid (TDHFA) 98/100% and butanolic-HCl were obtained from Sigma-Aldrich (Steinheim, Germany). Formic acid (FOA) 98/100% was obtained from BDH (VWR Ltd, Poole, England).
2.2. Preparation of standard solutions We prepared [2H3]-SC and [2H3]-MMC stock solutions (Stock-1) in water 1 mmol/L in order to investigate molecular fragmentation and also to obtain calibration curves for SC and MMC. This was stored frozen at −20 °C. “Daily standard solution” containing 10 μmol/L [2H3]-SC and [2H3]-MMC was prepared by dilution of Stock-1 solution 1:100 (v/v) with methanol.
2.3. Sample treatment procedure Whole blood was drawn by heel prick or venipuncture and dried on a filter paper (W303). We punched one disk of 5 mm diameter containing ~ 10 μL of whole blood from each dried blood spot into a vial and mixed it with 50 μL of “Daily standard solution”. Four hundred microliters of methanol was added to precipitate proteins and to extract the metabolites. Each vial was mixed by vortex for 20 min, then centrifuged for 5 min at 10,000 rpm. The supernatant fluid was transferred into a clean vial and dried at 40 °C under nitrogen stream. Eighty microliters of butanolic-HCl was added to derivatize metabolites as butyl-esters and heated at 65 °C for 15 min. Samples were dried again at 40 °C under nitrogen stream and finally they were reconstituted with 200 μL of acetonitrile–water 50:50 (v/v) containing 0.05% FOA to obtain pH 4 which is the appropriate pH for metabolites ionization.
2.6. Sample collection of patients and controls Twenty children's blood spot, for reference values, were obtained from hospitalized neurologically healthy subjects (10 females and 10 males; age: 1 day to 18 year, mean 7 year). Blood spot samples from patients where C4DC-C was found elevated (N1 μmol/L) using routine MS/MS screening method [6] were reanalyzed with LC-MS/MS method (2 samples of 1 patient affected by methylmalonyl-CoA mutase; 6 samples of 5 patients with mutation of SUCLA2 and 3 samples of 1 patient with mutation of SUCLG1). One of SUCLA2 blood spot was a neonatal blood spot. All samples were treated as described in sample treatment procedure. Control's and patient's blood samples were collected after obtaining informed consent. Patient's blood was collected during routine outpatient evaluation following an overnight fast before receiving the first morning dose of medication. 2.7. Statistical analysis The SPSS version 11.5.1 (SPSS Inc., Chicago, US) was used as statistical software. A preliminary test (Kolmogorov–Smirnov) was performed
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Fig. 1. Extract ion chromatogram of m/z 374.3 N 85.1 for SC and MMC and m/z 377.3 N 85.1 for [2H3]-SC and [2H3]-MMC. Retention time for SC and [2H3]-SC was 8.45 min; retention time for MMC and [2H3]-MMC was 8.60 min.
to assess normality of distributions of SC and MMC. Descriptive statistics were presented as mean ± standard deviation (SD). Range = mean ± 2SD. 3. Results and discussion 3.1. Chromatography and mass spectra An extract ion chromatogram from a 1 μmol/L SC and MMC standard solution is shown in Fig. 1. Multiple Reaction Monitoring (MRM) was used for the detection of the specific transitions in positive ion mode for the monitoring of each molecule. Under the above described conditions, the system was efficient for the separation of the isobaric compounds SC and MMC, eluted with their respective labeled internal standard at the retention times of 8.45 and 8.60 min, respectively. 3.2. Linearity, limit of detection, precision and recovery Calibration curves were linear over concentrations from 0.025 μmol/L up to 10 μmol/L for SC and MMC, which over covers the plasma concentration ranges for both metabolites. The linearity was monitored on 7
consecutive days. The average slope, intercept, and coefficient of linear regression (r2) were respectively: 0.3389 (95% confidence interval 0.2888–0.3889), 0.0113 (95% confidence interval −0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990–1.0000) for SC and 0.5699 (95% confidence interval 0.5263–0.6134), 0.0319 (95% confidence interval −0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995– 1.0000) for MMC. LOD of the analytes was 0.01 μmol/L for SC (S/N ratio 7.8) and 0.01 μmol/L for MMC (S/N ratio 8.1). LLOQ was 0.025 μmol/L for SC (S/N ratio 13.1) and 0.025 μmol/L MMC (S/N ratio 13.6). All validation experiments were performed with pooled blood from healthy controls and the validation data of the presented method are listed in Tables 1 and 2. The intra-assay variation was assessed from 10 replicates within one day (n = 10) and inter-assay from 3 times on 10 different days (n = 30); CVs for SC and MMC in plasma were in all cases b3.2%. Recovery experiments were performed at two different concentrations: pooled blood was spiked with SC (2 and 5 μmol/L) and with MMC (2 and 5 μmol/L). The SC recovery was respectively 105.3% and 100.8% of the expected amount and the same way MMC recovery was 95.04% and 98.41%.
Table 2 Accuracy data for succinyl-carnitine and methylmalonyl-carnitine. Table 1 Precision data for the LC-MS/MS assay.
Mean ± SD (μmol/L) Mean ± SD (μmol/L)
CV (%)
Intra-day (n = 10) Succinyl-carnitine Methylmalonyl-carnitine
7.630 ± 0.148 4.888 ± 0.155
1.94 3.19
Inter-day (n = 30) Succinyl-carnitine Methylmalonyl-carnitine
7.693 ± 0.247 4.948 ± 0.127
3.21 2.56
CV (%)
Recovery (%)
Succinyl-carnitine added (n = 5) 0 μmol/L 2.664 ± 0.175 2 μmol/L 4.770 ± 0.136 5 μmol/L 7.704 ± 0.269
6.59 2.86 3.49
not calculated 105.3 100.8
Methylmalonyl-carnitine added (n = 5) 0 μmol/L 0.087 ± 0.005 2 μmol/L 1.988 ± 0.081 5 μmol/L 5.008 ± 0.075
6.37 4.12 1.51
not calculated 95.04 98.41
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Table 3 Concentration of characteristic acylcarnitines obtained on patient's blood spots by MS/MS analysis compared with the new LC-MS/MS method. Acylcarnitine screening method (MS/MS)
LC-MS/MS separation method
Age
Sample number
C3
C4-DC
C3/C2
C3/C16
Succinyl-carnitine
Methylmalonyl-carnitine
pt.1 SUCLA2 pt.2 SUCLA2
5 years 3 years
pt.3 SUCLA2 pt.4 SUCLA2 pt.5 SUCLG1
10 years 2 days 9 years
pt.6 MMA
18 years
1 1 2 1 1 1 2 3 1 2
0.83 0.60 1.48 1.30 0.90 1.43 1.35 1.60 7.41 14.93 0.25–2.30
1.38 2.44 1.56 1.50 1.36 2.73 1.54 1.44 1.03 4.89 0.08–1
0.07 0.03 0.05 0.14 0.04 0.05 0.06 0.12 0.89 0.67 b0.19
2.32 0.47 1.16 1.29 0.75 1.07 1.09 1.51 27.76 35.39 b1.9
4.44 1.64 0.83 3.76 3.88 1.46 1.01 1.51 0.59 0.57 0.54–0.72
0.43 0.39 0.38 0.41 0.42 0.25 0.27 0.25 1.94 4.25 0.39–0.52
Reference values
3.3. Patients results When patient's samples obtained by classical MMA or SCS-related defects were analyzed by MS/MS method, increased levels of C4DC-C were detected as expected according to their diagnosis. Furthermore, C3-C, C3-C/C2-C and C3-C/C16-C ratios were elevated only in samples from classical MMA patient. The use of the new LC-MS/MS method allows clear differentiation of the two isobaric compounds of C4DC-C. SC was elevated only in SUCLA2 and SUCLG1 defects whereas MMC represents the characteristic compound of classical MMA alone (Table 3). Discrepancy between C4DC-C concentration values obtained by MS/MS method and the sum of the isobaric compounds SC + MMC values obtained by LC-MS/MS is explained by the use of different quantification internal standards. The internal standard used to quantify C4DC-C by the MS/MS method is labeled octanoyl-carnitine, while in our new LC-MS/MS method the two internal standards [2H3]-SC and [2H3]-MMC are used to quantify respectively SC and MMC. Similar data to those obtained in blood spots were observed in six plasma samples from three patients: two samples from one SUCLA2 patient (Pt.3), three samples from one SUCLG1 patient (Pt.5), and one from MMA patient (Pt.6). SC was increased in SUCLA2 and SUCLG1 patients (from 2.36 to 2.91 μmol/L; control values 0.10–0.80 μmol/L) whereas MMC was increased in MMA patient (2.08 μmol/L; control values 0.27–0.48 μmol/L). In order to verify correlation between SC and MMC and their related free acids, we also quantified plasma succinic acid and methylmalonic acid in 2 plasma samples from SUCLA2 patient (Pt.3) and 3 healthy controls by LC-MS/MS [4]. We found mild elevated succinic acid (6.72–7.52 μmol/L) and methylmalonic acid (1.7–1.9 μmol/L) in SUCLA2, compared to controls (succinic acid 3.7–5.1 μmol/L; methylmalonic acid not detectable). The elevation of succinic acid parallels SC whereas a slight elevation of methylmalonic acid did not parallel with MMC. Probably, higher plasma concentration of succinic acid with respect to methylmalonic acid allows a higher conjugation with carnitine in SUCLA2. Therefore, a more detailed study on correlation between methylmalonic acid and succinic acid in whole blood or plasma and MMC and SC will be very interesting for future investigations. The advantage of the new LC-MS/MS method over the MS/MS acylcarnitine analysis is the complete chromatographic separation of two isomers SC and MMC which allows a more precise and accurate quantification of the two metabolites, clarifying the diagnosis in all
cases where C4DC-C was found elevated in routine MS/MS acylcarnitines analysis. 4. Conclusions To our knowledge, this is the first time that SC and MMC involved in SUCLA2 and SUCLG1 and MMA defects have been both monitored and quantified in LC-MS/MS using a single blood spot sample. Good linearity, quantitative recovery and precision were obtained, demonstrating the accuracy of the method for quantitative determination of SC and MMC. The method is well proved and tested, and we believe that it represents an innovative contribution to the determination of SC and MMC in blood spot and therefore suitable for implementation in routine clinical acylcarnitine analysis as 2nd tier test and it could be also useful for follow up of SUCLA2, SUCLG1 defects. Acknowledgment We thank Dr. Stefano Garrone for his support in the manuscript writing. References [1] Deodato F, Boenzi S, Santorelli FM, Dionisi-Vici C. Methylmalonic and propionic aciduria. Am J Med Genet C Semin Med Genet 2006;142:104–12. [2] Van Hove JL, Saenz MS, Thomas JA, et al. Succinyl-CoA ligase deficiency: a mitochondrial hepatoencephalomyopathy. Pediatr Res 2010;68:159–64. [3] Carrozzo R, Dionisi-Vici C, Steuerwald U, et al. SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness. Brain 2007;130:862–74. [4] la Marca G, Malvagia S, Pasquini E, Innocenti M, Donati MA, Zammarchi E. Rapid 2ndtier test for measurement of 3-OH-propionic and methylmalonic acids on dried blood spots: reducing the false-positive rate for propionylcarnitine during expanded newborn screening by liquid chromatography-tandem mass spectrometry. Clin Chem 2007;53:1364–9. [5] la Marca G, Malvagia S, Casetta B, Pasquini E, Donati MA, Zammarchi E. Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: update on methods to reduce false tests. J Inherit Metab Dis 2008; 31:395–404. [6] Rashed MS, Ozand PT, Bucknall MP, Little D. Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and aminoacids profiling using automated electrospray tandem mass spectrometry. Pediatr Res 1995;38:324–31. [7] Bennett MJ, Rinaldo P, Wilcken B, Pass KA, Watson MS, Wanders WJ. Newborn screening for metabolic disorders: how are we doing, and where are we going? Clin Chem 2012;58:324–31.