Clinica Chimica Acta 440 (2015) 108–112
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Determination of plasma pipecolic acid by an easy and rapid liquid chromatography–tandem mass spectrometry method Michela Semeraro a,⁎, Maurizio Muraca a, Giulio Catesini a, Rita Inglese a, Francesca Iacovone a, Gloria Maria Barraco b, Melania Manco b, Sara Boenzi c, Carlo Dionisi-Vici c, Cristiano Rizzo a a b c
Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy Research Unit of Multifactorial Diseases, Obesity and Diabetes, 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
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 3 November 2014 Accepted 12 November 2014 Available online 15 November 2014 Keywords: Pipecolic acid Peroxisomal disorders Liquid chromatography–tandem mass spectrometry Pyridoxine-dependent seizures Hepatic encephalopathy
a b s t r a c t Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 μl) is extracted with 500 μl acetonitrile (ACN) containing 2 μmol/l [2H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150 × 3.0 mm; 3 μ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 μmol/l (R2 = 0.998), lower limit of detection was 0.010 μmol/l and lower limit of quantification was 0.050 μmol/l. Coefficient of variation was 3.2% intraassay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 μmol/l; range, 11.20–37.1 μmol/l) to 24 ages related healthy subjects (mean, 1.711 μmol/l; range, 0.517–3.580 μmol/l). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pipecolic acid (PA: piperidine-2-carboxylic acid) is a cyclic secondary imino acid including D- and L-enantiomers. It derives from one of the two pathways of lysine metabolism, merging at the level of αaminoadipic acid semialdehyde (AASA) [1,2,3]. In the brain, the conversion of lysine to acetoacetyl CoA is predominant via L-pipecolic acid, while in the liver and in most other vertebrate tissues, lysine is
Abbreviations: PA, pipecolic acid; ACN, acetonitrile; HPLC-MS/MS, liquid chromatography–tandem mass spectrometry; [2H5]-Phe, [2H5]-phenylalanine; AASA, α-aminoadipic acid semialdehyde; GABA, α-aminobutyric acid; GC-MS, gas chromatography–mass spectrometry; 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; RT, retention time; MRM, multiple reaction monitoring; NA, nipecotic acid; INA, isonipecotic acid. ⁎ Corresponding author at: Department of Laboratory Medicine, Bambino Gesù Children's Hospital, IRCCS, P.zza S. Onofrio, 4, 00165, Roma. Tel.: +39 0668592519; fax: +39 0668592014. E-mail address: [email protected] (M. Semeraro).
http://dx.doi.org/10.1016/j.cca.2014.11.014 0009-8981/© 2014 Elsevier B.V. All rights reserved.
metabolized via saccharopine. The exogenous source of pipecolic acid comes instead from D-lysine catabolism by intestinal bacteria and to a minor part from plant source [4]. L-Pipecolic acid is accumulated in biological fluids of patients with peroxisomal disorders, including hyperpipecolic acidemia, Zellweger syndrome, neonatal adrenoleukodystrophy or infantile Refsum disease [5]. L-Pipecolic acid is also involved in the pathway of α-aminobutyric acid (GABA) receptor agonist: it was found that both D-PA and L-PA were moderately increase in patients with liver cirrhosis and in patients with chronic hepatic encephalopathy. Although L-PA remained the predominantly circulating form, D-PA was proportionally higher in liver disease patients than in healthy individuals [4]. Finally, elevated PA is a secondary biomarker for pyridoxinedependent seizure disorder, a recessive disorder characterized by seizures in neonates or infants up to 3 years of age which respond to pharmacological doses or pyridoxine [6]. Plasma levels of PA consist of high levels of L-enantiomers and low levels of D-enantiomers (about 2%) in normal subjects [7,8]. For this reason, we only focused on the analysis and quantification of the L-form in plasma.
M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
Initially, total PA (D-PA and L-PA) was determined by ion-exchange chromatography on an amino acid analyzer or by HPLC using the acid ninhydrin method [9]. Several more sensitive single-ion monitoring gas chromatography–mass spectrometry (GC-MS) methods have been published for the determination of total PA in plasma and urine, usually involving solid-phase extraction followed by derivatization and analysis on an achiral GC column [10,11]. The analysis of the derivative compounds by electron-capture negative-ion GC-MS or GC with electroncapture detection had also allowed the analysis of PA in cerebrospinal fluid, where the concentrations are extremely low [12,13]. Subsequently, several GC-MS or LC-MS/MS methods were published, with or without derivatization, analyzing PA as individual enantiomers using a chiral column [7,8,14]. In this study, we present an easy and rapid liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for the quantification of PA in plasma requiring no derivatization, simple sample preparation and a short analysis time. 2. Materials and methods 2.1. Reagents L-PA, nipecotic acid (NA), isonipecotic acid (INA) and ammonium formate were purchased from Sigma-Aldrich (Steinheim, Germany). [2H5]-Phenylalanine ([2H5]-Phe) was purchased from Cambridge Isotopes. HPLC grade acetonitrile (ACN) and water were purchased from Romil Ltd. (The Source Convent Drive Waterbeach Cambridge, United Kingdom). Quality control samples were from ERNDIM. 2.2. Preparation of standard solutions Stock solutions 10 mmol/l of PA and internal standard [2H5]-Phe were prepared in water and stored at − 80 °C. The 10 μmol/l daily internal standard solution [2H5]-Phe was prepared in ACN by scalar dilution of the 10 mmol/l stock solution. 2.3. Sample treatment procedure One-hundred microliters of plasma and 500 μl ACN containing internal standard [2H5]-Phe (2 μmol/l) were added to a microfuge tube. The tube was then vortexed vigorously for 30 s and centrifuged at 10000 g for 5 min. Two hundred microliters of the clear supernatant was transferred to the wells of a 96 well-plates microplate. The microplate was covered with protective sheets to prevent solvent evaporation. Twenty microliters of extract was injected into the mass spectrometer. 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 150 × 3.00 mm 200A 3 μ Luna HILIC column (Phenomenex, Castel Maggiore, Italy). The mobile phase A was H2O containing 5 mmol/l ammonium formate and the mobile phase B was ACN/H2O (90%/10%) containing 5 mmol/l ammonium formate. Flow rate was 400 μl/min. The column was maintained at room temperature. Twenty microliters of sample were injected onto the column. Chromatographic separation of metabolites was obtained with gradient elution. Mobile phase B changed from 95% to 55% in 5 min and then remained at 55% for 2 min and finally initial condition had restored in 4 minutes. The total run time was 11 min. Tandem mass spectrometry experiments were carried out on an API3200 triple quadrupole mass spectrometer (Applied BiosystemsMDS Sciex, Toronto, Canada), equipped with a Turbo Ion Spray Source operating in positive ion mode with a needle potential of 5500 V, and the source temperature was 500 °C. Declustering potential (DP), collision cell exit potential (CXP) and collision energy (CE) were optimized
109
by direct infusion at flow rate 10 μl/min of each analyte in the mass spectrometer. The resulting DP was 32 eV, and optimal CE and CXP were found at 43 eV and 3 eV for PA and [2H5]-Phe, respectively. The following transitions were monitored in positive-ion mode using multiple reaction monitoring scan type: m/z 130.10 N 84.20 and 130.10 N 56.20 for PA and 171.20 N 125.10 for [2H5]-Phe.
2.5. Standard curves for quantification Serial dilutions of 10 mmol/l PA stock solution were used to obtain calibration points (50 μmol/l, 30 μmol/l, 20 μmol/l, 10 μmol/l, 1 μmol/l, 0.5 μmol/l, 0.1 μmol/l and 0.05 μmol/l). Calibration curve points were treated and analyzed as plasma samples. LC-MS/MS-based assays can also be affected by matrix ion suppression effects related to variation in ionization response due to matrix components coeluting with the analyte. We thus evaluated the suppression coefficient for PA by calculating the ratio of the average peak area response in spiked plasma sample to the average peak area response in ACN. The suppression coefficient of PA was 0.82, indicating acceptable matrix effects in plasma. The slopes of the calibration curves were shown to be nearly identical in ACN and plasma, indicating that ACN was a suitable surrogate matrix (Table 1). The intercept of the plasma calibration curve was larger than zero due to the endogenous PA concentrations, whereas the intercept of the ACN calibration curve was close to zero. The acquired data were processed using the Analyst® version 1.4.2 software (Applied Biosystems-Sciex), including option for chromatographic and spectral interpretation and for quantitative generation information. Calibration curves were constructed with the Analyst Quantification program using a linear least-square regression nonweighted analysis. The lower limit of detection (LLOD) was determined by progressive dilutions of calibrator solutions for each analyte, and it was considered as the lowest concentration with a signal-to-noise ratio (S/N) of at least 3, as indicated by the Analyst software. The lower limit of quantification (LLOQ) was determined by preparing calibrator solutions with decreasing concentration of each analyte and was considered at the lowest concentration with an S/N of at least 10.
2.6. Sample collection of patients and controls For reference values, 24 plasma samples, were obtained from healthy subjects (12 females and 12 males; age: 1 month to 35 years). Five plasma samples were obtained from patients affected by peroxisomal disorders: 4 with peroxisome biogenesis disorders (PBD) and 1 with adult Refsum disease. Controls and patient blood sample were collected after obtaining informed consent. The samples were treated as described in sample treatment procedure. Quality Control and External Quality Assessment samples from ERNDIM (European Research Network for Evaluation and Improvement of Screening, Diagnosis, and Treatment of Inherited Disorders of Metabolism) were analyzed in each batch of ours to confirm the normal range of values and the abnormal levels found for the peroxisomal patients. The results obtained were within the 95% confidence interval.
Table 1 Characteristics of calibration regression line data prepared in ACN and in plasma. Analyte
Matrix
Slope
Intercept
R2
PA
ACN Plasma
0.181 0.172
0.042 0.434
0.998 0.996
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M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
2.7. Statistical analysis
We prepared a standard mixture of these molecules to check if our chromatographic method could separate them in order to avoid any their interference with the PA signal. As shown in Fig. 2, no overlap in the retention times of PA (RT 4.86 min), NA (RT 5.45 min) and INA (RT 5.83 min) was found demonstrating the efficient and specific separation of our chromatographic technique coupled with tandem mass spectrometry.
The SPSS version 11.5.1 (SPSS Inc., Chicago, US) was used as statistical software. A preliminary test (Kolmogorov–Smirnov) was performed to assess normality of distributions of PA plasma concentrations. Descriptive statistics were presented as mean ± standard deviation (SD). Range = mean ± 2 SD.
3.2. Linearity, Limit of detection, precision and recovery 3. Results and discussion Calibration curves were linear over a concentration range of 0.05 μmol/l–50 μmol/l for PA, ensuring full coverage of the plasma concentration ranges for this metabolite. Linearity was monitored on 5 consecutive days. The average slope, intercept and coefficient of linear regression (r2) were 0.181 (95% confidence interval, 0.1727–0.1879), 0.0428 (95% confidence interval, − 0.004577 to 0.09018) and 0.998 (95% confidence interval, 0.997–1), respectively. LLOD of the analytes was 0.010 μmol/l. LLOQ was 0.050 μmol/l. All validation experiments were performed with pooled blood from healthy controls and the validation data are listed in Tables 2 and 3. The intra-assay variation was assessed from 10 replicates within 1 day
3.1. Chromatography and mass spectra An extract ion chromatogram from a 1 μmol/l PA and internal standard [2H5]-Phe standard solution is shows in Fig. 1. The retention time (RT) were 4.86 min for PA and 4.45 min for internal standard [2H5]-Phe. NA and INA, two PA isobaric substances usually used as drugs, were also analyzed. NA is a piperidine-3-carboxylic acid and INA is a 4-piperidinecarboxylic acid; NA and INA mass fragmentation shown common transitions m/z 130.10 N 84.20 and 130.10 N 56.20 to PA.
XIC of +MRM (3 pairs): 130.1/84.2 amu from Sample 16 (PIP 10) of PIP200614.wiff (Turbo Spray) Intensity, cps
4.86
4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5
4.45
1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0
1
2
3
4
5
Time, min
6
7
8
9
10
Fig. 1. Extract ion chromatogram of m/z 130.1 N 84.2 and 130.1 N 56.2 for PA, 171.2 N 125.1 for [2H5]-Phe. Retention time for PA was 4.86 and for [2H5]-Phe was 4.45. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
111
XIC of +MRM (3 pairs): 130.1/84.2 amu from Sample 9 (MIX) of PIP 150114.wiff (Turbo Spray)
4.45
8.5e4 8.0e4 7.5e4
4.84
5.45
7.0e4 6.5e4
5.83
6.0e4 5.5e4 5.0e4
Intensity, cps
4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4
5000.0 0.0
1
2
3
4
5
Time, min
6
7
8
9
10
Fig. 2. Extract ion chromatogram of m/z 130.1 N 84.2 and 130.1 N 56.2 for PA, 171.2 N 125.1 for [2H5]-Phe. Retention times were as follows: PA RT = 4.84; [2H5]-Phe RT = 4.45; NA RT = 5.45; INA RT = 5.83. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
(n = 10) and inter-assay from 10 times on 3 different days (n = 30); CVs for PA in plasma were 1.6. Recovery experiments were performed at two different concentrations: pooled blood was spiked with PA (2 and 10 μmol/l). The PA recovery was respectively 93.8% and 91.6% of the expected amount.
A strong elevation of pipecolic acid was found In all patient samples (mean, 23.38 μmol/l; range, 11.20–37.10 μmol/l), when compared to controls (mean, 1.711 μmol/l; range, 0.517–3.580 μmol/l) (Table 4). 4. Conclusions
Clinical validation was obtained compared PA plasma values from 5 patients affected by peroxisomal disorders to 24 ages related healthy subjects.
An easy, accurate and precise procedure for the determination of PA in plasma based on HPLC-MS/MS has been developed and validated. Major advantages of the present method include the use of very small sample volumes (100 μl plasma), appropriate to quantify pipecolic acid concentration in a pediatric population and a rapid extraction procedure not requiring solvent evaporation and derivatization.
Table 2 Precision data for the LC/MS-MS assay.
Table 3 Accuracy data for PA.
3.3. Patients results
Intra-day (n = 10) PA Inter-day (n = 30) PA
Mean ± (SD) (μmol/l)
CV (%)
3.47 ± 0.11
3.23
3.41 ± 0.11
3.41
Mean ± (SD) (μmol/l) PA added (n = 5) 0 μmol/l 1.6 ± 0.05 2 μmol/l 3.5 ± 0.05 10 μmol/l 10.02 ± 0.27
CV (%)
Recovery (%)
3.2 1.6 2.7
Not calculated 93.8 91.6
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M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
Table 4 Concentration of PA in patients.
Pt.1 Pt. 2 Pt. 3 Pt.4 Pt. 5 Reference values
Age
Concentration (μmol/l)
1 month 9 months 3 months 12 months –
14.60 24.90 37.10 29.10 11.20 (0.52–3.580)
The chromatographic method is robust since the pipecolic acid is well separated from possible interfering exogenous substances in human plasma such as isonipecotic acid and nipecotic acid. Good linearity, quantitative recovery and precision demonstrated the feasibility and accuracy of the method for quantitative determination of PA in plasma. This procedure was successfully applied to determine the pipecolic acid in five children affected by different peroxisomal disorders and could also be used to diagnose other disorders characterized by high PA plasma levels, such pyridoxine-dependent seizures and hepatic encephalopathy. References [1] Higashino K, Tsukada K, Lieberman I. Saccharopine, a product of lysine break down by mammalian liver. Biophys Res Commun 1965;20:285–90. [2] Higashino K, Fujioka M, Aoki T, Yamamura Y. Metabolismo f lysine in rat liver. Biochem Biophys Res Commun 1967;29:95–100. [3] Higashino K, Fujioka M, Yamamura Y. The conversion of L-lysine to saccharopine and A-aminoadipate in mouse. Arch Biochem Biophys 1971;142:606–14.
[4] Fujita T, Hada T, Higashino K. Origin of D- and L-pipecolic acid in human physiological fluids: a study of the catabolic mechanism to pipecolic acid using the lysine loading test. Clin Chim Acta 1999;287:145–56. [5] Mihalik SJ, Moser HW, Watkins PA, Danks DM, Poulos A, Rhead WJ. Peroxisomal Lpipecolic acid oxidation is deficient in liver from Zellweger syndrome patients. Pediatr Res 1989;25:548–52. [6] Sadilkova K, Gospe Jr Sidney M, Si Houn H. Simultaneous determination of alphaaminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid by LCMS/MS for pyridoxine-dependent seizures and folinic acid-responsive seizures. J Neurosci Methods 2009;184:136–41. [7] Armstrong DW, Gasper M, Lee SH, Zukowski J, Ercal N. D-amino acid levels in human physiological fluids. Chirality 1993;5:375–8. [8] Armstrong DW, Zukowski J, Ercal, Gasper M. Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies. J Pharm Biomed Anal 1993;11:881–6. [9] Goverts L, Tribels F, Monnens L, Van Raay-Setlena. Pipecolic acid levels in serum and urine from neonates and normal infants: comparison with values reported in Zellweger syndrome. J Inherit Metab Dis 1985;8:87–91. [10] Kelley RI. Quantification of pipecolic acid in plasma and urine by isotope-dilution gas chromatography/mass spectrometry. In: Hommes FA, editor. Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual. New York: Wiley-Liss; 1991. p. 205–18. [11] Van Bocxlaer JF, Verhaeghe BJ, Wauters AE, De Marez WD, Thienpont LM, De Leenheer AP. Determination of pipecolic acid in serum or plasma by solid-phase extraction and isotope dilution mass spectrometry. Biomed Environ Mass Spectrom 1989;18:566–71. [12] Kok RM, Kaster L, de Jong PJM, Poll-The B, Saudubray JM, Jakobs C. Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron capture negative ion mass fragmentography. Clin Chim Acta 1987;168:143–52. [13] Zee T, Stellaard F, Jakobs C. Analysis of pipecolic acid in biological fluid using capillary gas chromatography with electron capture detection and [2H11] pipecolic acid as internal standard. J Chromatogr 1992;574:335–9. [14] Struys EA, Jackobs C. Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography. J Inherit Metab Dis 1999;22:677–8.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Determination of plasma pipecolic acid by an easy and rapid liquid chromatography–tandem mass spectrometry method Michela Semeraro a,⁎, Maurizio Muraca a, Giulio Catesini a, Rita Inglese a, Francesca Iacovone a, Gloria Maria Barraco b, Melania Manco b, Sara Boenzi c, Carlo Dionisi-Vici c, Cristiano Rizzo a a b c
Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy Research Unit of Multifactorial Diseases, Obesity and Diabetes, 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
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 3 November 2014 Accepted 12 November 2014 Available online 15 November 2014 Keywords: Pipecolic acid Peroxisomal disorders Liquid chromatography–tandem mass spectrometry Pyridoxine-dependent seizures Hepatic encephalopathy
a b s t r a c t Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 μl) is extracted with 500 μl acetonitrile (ACN) containing 2 μmol/l [2H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150 × 3.0 mm; 3 μ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 μmol/l (R2 = 0.998), lower limit of detection was 0.010 μmol/l and lower limit of quantification was 0.050 μmol/l. Coefficient of variation was 3.2% intraassay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 μmol/l; range, 11.20–37.1 μmol/l) to 24 ages related healthy subjects (mean, 1.711 μmol/l; range, 0.517–3.580 μmol/l). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pipecolic acid (PA: piperidine-2-carboxylic acid) is a cyclic secondary imino acid including D- and L-enantiomers. It derives from one of the two pathways of lysine metabolism, merging at the level of αaminoadipic acid semialdehyde (AASA) [1,2,3]. In the brain, the conversion of lysine to acetoacetyl CoA is predominant via L-pipecolic acid, while in the liver and in most other vertebrate tissues, lysine is
Abbreviations: PA, pipecolic acid; ACN, acetonitrile; HPLC-MS/MS, liquid chromatography–tandem mass spectrometry; [2H5]-Phe, [2H5]-phenylalanine; AASA, α-aminoadipic acid semialdehyde; GABA, α-aminobutyric acid; GC-MS, gas chromatography–mass spectrometry; 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; RT, retention time; MRM, multiple reaction monitoring; NA, nipecotic acid; INA, isonipecotic acid. ⁎ Corresponding author at: Department of Laboratory Medicine, Bambino Gesù Children's Hospital, IRCCS, P.zza S. Onofrio, 4, 00165, Roma. Tel.: +39 0668592519; fax: +39 0668592014. E-mail address: [email protected] (M. Semeraro).
http://dx.doi.org/10.1016/j.cca.2014.11.014 0009-8981/© 2014 Elsevier B.V. All rights reserved.
metabolized via saccharopine. The exogenous source of pipecolic acid comes instead from D-lysine catabolism by intestinal bacteria and to a minor part from plant source [4]. L-Pipecolic acid is accumulated in biological fluids of patients with peroxisomal disorders, including hyperpipecolic acidemia, Zellweger syndrome, neonatal adrenoleukodystrophy or infantile Refsum disease [5]. L-Pipecolic acid is also involved in the pathway of α-aminobutyric acid (GABA) receptor agonist: it was found that both D-PA and L-PA were moderately increase in patients with liver cirrhosis and in patients with chronic hepatic encephalopathy. Although L-PA remained the predominantly circulating form, D-PA was proportionally higher in liver disease patients than in healthy individuals [4]. Finally, elevated PA is a secondary biomarker for pyridoxinedependent seizure disorder, a recessive disorder characterized by seizures in neonates or infants up to 3 years of age which respond to pharmacological doses or pyridoxine [6]. Plasma levels of PA consist of high levels of L-enantiomers and low levels of D-enantiomers (about 2%) in normal subjects [7,8]. For this reason, we only focused on the analysis and quantification of the L-form in plasma.
M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
Initially, total PA (D-PA and L-PA) was determined by ion-exchange chromatography on an amino acid analyzer or by HPLC using the acid ninhydrin method [9]. Several more sensitive single-ion monitoring gas chromatography–mass spectrometry (GC-MS) methods have been published for the determination of total PA in plasma and urine, usually involving solid-phase extraction followed by derivatization and analysis on an achiral GC column [10,11]. The analysis of the derivative compounds by electron-capture negative-ion GC-MS or GC with electroncapture detection had also allowed the analysis of PA in cerebrospinal fluid, where the concentrations are extremely low [12,13]. Subsequently, several GC-MS or LC-MS/MS methods were published, with or without derivatization, analyzing PA as individual enantiomers using a chiral column [7,8,14]. In this study, we present an easy and rapid liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for the quantification of PA in plasma requiring no derivatization, simple sample preparation and a short analysis time. 2. Materials and methods 2.1. Reagents L-PA, nipecotic acid (NA), isonipecotic acid (INA) and ammonium formate were purchased from Sigma-Aldrich (Steinheim, Germany). [2H5]-Phenylalanine ([2H5]-Phe) was purchased from Cambridge Isotopes. HPLC grade acetonitrile (ACN) and water were purchased from Romil Ltd. (The Source Convent Drive Waterbeach Cambridge, United Kingdom). Quality control samples were from ERNDIM. 2.2. Preparation of standard solutions Stock solutions 10 mmol/l of PA and internal standard [2H5]-Phe were prepared in water and stored at − 80 °C. The 10 μmol/l daily internal standard solution [2H5]-Phe was prepared in ACN by scalar dilution of the 10 mmol/l stock solution. 2.3. Sample treatment procedure One-hundred microliters of plasma and 500 μl ACN containing internal standard [2H5]-Phe (2 μmol/l) were added to a microfuge tube. The tube was then vortexed vigorously for 30 s and centrifuged at 10000 g for 5 min. Two hundred microliters of the clear supernatant was transferred to the wells of a 96 well-plates microplate. The microplate was covered with protective sheets to prevent solvent evaporation. Twenty microliters of extract was injected into the mass spectrometer. 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 150 × 3.00 mm 200A 3 μ Luna HILIC column (Phenomenex, Castel Maggiore, Italy). The mobile phase A was H2O containing 5 mmol/l ammonium formate and the mobile phase B was ACN/H2O (90%/10%) containing 5 mmol/l ammonium formate. Flow rate was 400 μl/min. The column was maintained at room temperature. Twenty microliters of sample were injected onto the column. Chromatographic separation of metabolites was obtained with gradient elution. Mobile phase B changed from 95% to 55% in 5 min and then remained at 55% for 2 min and finally initial condition had restored in 4 minutes. The total run time was 11 min. Tandem mass spectrometry experiments were carried out on an API3200 triple quadrupole mass spectrometer (Applied BiosystemsMDS Sciex, Toronto, Canada), equipped with a Turbo Ion Spray Source operating in positive ion mode with a needle potential of 5500 V, and the source temperature was 500 °C. Declustering potential (DP), collision cell exit potential (CXP) and collision energy (CE) were optimized
109
by direct infusion at flow rate 10 μl/min of each analyte in the mass spectrometer. The resulting DP was 32 eV, and optimal CE and CXP were found at 43 eV and 3 eV for PA and [2H5]-Phe, respectively. The following transitions were monitored in positive-ion mode using multiple reaction monitoring scan type: m/z 130.10 N 84.20 and 130.10 N 56.20 for PA and 171.20 N 125.10 for [2H5]-Phe.
2.5. Standard curves for quantification Serial dilutions of 10 mmol/l PA stock solution were used to obtain calibration points (50 μmol/l, 30 μmol/l, 20 μmol/l, 10 μmol/l, 1 μmol/l, 0.5 μmol/l, 0.1 μmol/l and 0.05 μmol/l). Calibration curve points were treated and analyzed as plasma samples. LC-MS/MS-based assays can also be affected by matrix ion suppression effects related to variation in ionization response due to matrix components coeluting with the analyte. We thus evaluated the suppression coefficient for PA by calculating the ratio of the average peak area response in spiked plasma sample to the average peak area response in ACN. The suppression coefficient of PA was 0.82, indicating acceptable matrix effects in plasma. The slopes of the calibration curves were shown to be nearly identical in ACN and plasma, indicating that ACN was a suitable surrogate matrix (Table 1). The intercept of the plasma calibration curve was larger than zero due to the endogenous PA concentrations, whereas the intercept of the ACN calibration curve was close to zero. The acquired data were processed using the Analyst® version 1.4.2 software (Applied Biosystems-Sciex), including option for chromatographic and spectral interpretation and for quantitative generation information. Calibration curves were constructed with the Analyst Quantification program using a linear least-square regression nonweighted analysis. The lower limit of detection (LLOD) was determined by progressive dilutions of calibrator solutions for each analyte, and it was considered as the lowest concentration with a signal-to-noise ratio (S/N) of at least 3, as indicated by the Analyst software. The lower limit of quantification (LLOQ) was determined by preparing calibrator solutions with decreasing concentration of each analyte and was considered at the lowest concentration with an S/N of at least 10.
2.6. Sample collection of patients and controls For reference values, 24 plasma samples, were obtained from healthy subjects (12 females and 12 males; age: 1 month to 35 years). Five plasma samples were obtained from patients affected by peroxisomal disorders: 4 with peroxisome biogenesis disorders (PBD) and 1 with adult Refsum disease. Controls and patient blood sample were collected after obtaining informed consent. The samples were treated as described in sample treatment procedure. Quality Control and External Quality Assessment samples from ERNDIM (European Research Network for Evaluation and Improvement of Screening, Diagnosis, and Treatment of Inherited Disorders of Metabolism) were analyzed in each batch of ours to confirm the normal range of values and the abnormal levels found for the peroxisomal patients. The results obtained were within the 95% confidence interval.
Table 1 Characteristics of calibration regression line data prepared in ACN and in plasma. Analyte
Matrix
Slope
Intercept
R2
PA
ACN Plasma
0.181 0.172
0.042 0.434
0.998 0.996
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2.7. Statistical analysis
We prepared a standard mixture of these molecules to check if our chromatographic method could separate them in order to avoid any their interference with the PA signal. As shown in Fig. 2, no overlap in the retention times of PA (RT 4.86 min), NA (RT 5.45 min) and INA (RT 5.83 min) was found demonstrating the efficient and specific separation of our chromatographic technique coupled with tandem mass spectrometry.
The SPSS version 11.5.1 (SPSS Inc., Chicago, US) was used as statistical software. A preliminary test (Kolmogorov–Smirnov) was performed to assess normality of distributions of PA plasma concentrations. Descriptive statistics were presented as mean ± standard deviation (SD). Range = mean ± 2 SD.
3.2. Linearity, Limit of detection, precision and recovery 3. Results and discussion Calibration curves were linear over a concentration range of 0.05 μmol/l–50 μmol/l for PA, ensuring full coverage of the plasma concentration ranges for this metabolite. Linearity was monitored on 5 consecutive days. The average slope, intercept and coefficient of linear regression (r2) were 0.181 (95% confidence interval, 0.1727–0.1879), 0.0428 (95% confidence interval, − 0.004577 to 0.09018) and 0.998 (95% confidence interval, 0.997–1), respectively. LLOD of the analytes was 0.010 μmol/l. LLOQ was 0.050 μmol/l. All validation experiments were performed with pooled blood from healthy controls and the validation data are listed in Tables 2 and 3. The intra-assay variation was assessed from 10 replicates within 1 day
3.1. Chromatography and mass spectra An extract ion chromatogram from a 1 μmol/l PA and internal standard [2H5]-Phe standard solution is shows in Fig. 1. The retention time (RT) were 4.86 min for PA and 4.45 min for internal standard [2H5]-Phe. NA and INA, two PA isobaric substances usually used as drugs, were also analyzed. NA is a piperidine-3-carboxylic acid and INA is a 4-piperidinecarboxylic acid; NA and INA mass fragmentation shown common transitions m/z 130.10 N 84.20 and 130.10 N 56.20 to PA.
XIC of +MRM (3 pairs): 130.1/84.2 amu from Sample 16 (PIP 10) of PIP200614.wiff (Turbo Spray) Intensity, cps
4.86
4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5
4.45
1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0
1
2
3
4
5
Time, min
6
7
8
9
10
Fig. 1. Extract ion chromatogram of m/z 130.1 N 84.2 and 130.1 N 56.2 for PA, 171.2 N 125.1 for [2H5]-Phe. Retention time for PA was 4.86 and for [2H5]-Phe was 4.45. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
M. Semeraro et al. / Clinica Chimica Acta 440 (2015) 108–112
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XIC of +MRM (3 pairs): 130.1/84.2 amu from Sample 9 (MIX) of PIP 150114.wiff (Turbo Spray)
4.45
8.5e4 8.0e4 7.5e4
4.84
5.45
7.0e4 6.5e4
5.83
6.0e4 5.5e4 5.0e4
Intensity, cps
4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4
5000.0 0.0
1
2
3
4
5
Time, min
6
7
8
9
10
Fig. 2. Extract ion chromatogram of m/z 130.1 N 84.2 and 130.1 N 56.2 for PA, 171.2 N 125.1 for [2H5]-Phe. Retention times were as follows: PA RT = 4.84; [2H5]-Phe RT = 4.45; NA RT = 5.45; INA RT = 5.83. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
(n = 10) and inter-assay from 10 times on 3 different days (n = 30); CVs for PA in plasma were 1.6. Recovery experiments were performed at two different concentrations: pooled blood was spiked with PA (2 and 10 μmol/l). The PA recovery was respectively 93.8% and 91.6% of the expected amount.
A strong elevation of pipecolic acid was found In all patient samples (mean, 23.38 μmol/l; range, 11.20–37.10 μmol/l), when compared to controls (mean, 1.711 μmol/l; range, 0.517–3.580 μmol/l) (Table 4). 4. Conclusions
Clinical validation was obtained compared PA plasma values from 5 patients affected by peroxisomal disorders to 24 ages related healthy subjects.
An easy, accurate and precise procedure for the determination of PA in plasma based on HPLC-MS/MS has been developed and validated. Major advantages of the present method include the use of very small sample volumes (100 μl plasma), appropriate to quantify pipecolic acid concentration in a pediatric population and a rapid extraction procedure not requiring solvent evaporation and derivatization.
Table 2 Precision data for the LC/MS-MS assay.
Table 3 Accuracy data for PA.
3.3. Patients results
Intra-day (n = 10) PA Inter-day (n = 30) PA
Mean ± (SD) (μmol/l)
CV (%)
3.47 ± 0.11
3.23
3.41 ± 0.11
3.41
Mean ± (SD) (μmol/l) PA added (n = 5) 0 μmol/l 1.6 ± 0.05 2 μmol/l 3.5 ± 0.05 10 μmol/l 10.02 ± 0.27
CV (%)
Recovery (%)
3.2 1.6 2.7
Not calculated 93.8 91.6
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Table 4 Concentration of PA in patients.
Pt.1 Pt. 2 Pt. 3 Pt.4 Pt. 5 Reference values
Age
Concentration (μmol/l)
1 month 9 months 3 months 12 months –
14.60 24.90 37.10 29.10 11.20 (0.52–3.580)
The chromatographic method is robust since the pipecolic acid is well separated from possible interfering exogenous substances in human plasma such as isonipecotic acid and nipecotic acid. Good linearity, quantitative recovery and precision demonstrated the feasibility and accuracy of the method for quantitative determination of PA in plasma. This procedure was successfully applied to determine the pipecolic acid in five children affected by different peroxisomal disorders and could also be used to diagnose other disorders characterized by high PA plasma levels, such pyridoxine-dependent seizures and hepatic encephalopathy. References [1] Higashino K, Tsukada K, Lieberman I. Saccharopine, a product of lysine break down by mammalian liver. Biophys Res Commun 1965;20:285–90. [2] Higashino K, Fujioka M, Aoki T, Yamamura Y. Metabolismo f lysine in rat liver. Biochem Biophys Res Commun 1967;29:95–100. [3] Higashino K, Fujioka M, Yamamura Y. The conversion of L-lysine to saccharopine and A-aminoadipate in mouse. Arch Biochem Biophys 1971;142:606–14.
[4] Fujita T, Hada T, Higashino K. Origin of D- and L-pipecolic acid in human physiological fluids: a study of the catabolic mechanism to pipecolic acid using the lysine loading test. Clin Chim Acta 1999;287:145–56. [5] Mihalik SJ, Moser HW, Watkins PA, Danks DM, Poulos A, Rhead WJ. Peroxisomal Lpipecolic acid oxidation is deficient in liver from Zellweger syndrome patients. Pediatr Res 1989;25:548–52. [6] Sadilkova K, Gospe Jr Sidney M, Si Houn H. Simultaneous determination of alphaaminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid by LCMS/MS for pyridoxine-dependent seizures and folinic acid-responsive seizures. J Neurosci Methods 2009;184:136–41. [7] Armstrong DW, Gasper M, Lee SH, Zukowski J, Ercal N. D-amino acid levels in human physiological fluids. Chirality 1993;5:375–8. [8] Armstrong DW, Zukowski J, Ercal, Gasper M. Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies. J Pharm Biomed Anal 1993;11:881–6. [9] Goverts L, Tribels F, Monnens L, Van Raay-Setlena. Pipecolic acid levels in serum and urine from neonates and normal infants: comparison with values reported in Zellweger syndrome. J Inherit Metab Dis 1985;8:87–91. [10] Kelley RI. Quantification of pipecolic acid in plasma and urine by isotope-dilution gas chromatography/mass spectrometry. In: Hommes FA, editor. Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual. New York: Wiley-Liss; 1991. p. 205–18. [11] Van Bocxlaer JF, Verhaeghe BJ, Wauters AE, De Marez WD, Thienpont LM, De Leenheer AP. Determination of pipecolic acid in serum or plasma by solid-phase extraction and isotope dilution mass spectrometry. Biomed Environ Mass Spectrom 1989;18:566–71. [12] Kok RM, Kaster L, de Jong PJM, Poll-The B, Saudubray JM, Jakobs C. Stable isotope dilution analysis of pipecolic acid in cerebrospinal fluid, plasma, urine and amniotic fluid using electron capture negative ion mass fragmentography. Clin Chim Acta 1987;168:143–52. [13] Zee T, Stellaard F, Jakobs C. Analysis of pipecolic acid in biological fluid using capillary gas chromatography with electron capture detection and [2H11] pipecolic acid as internal standard. J Chromatogr 1992;574:335–9. [14] Struys EA, Jackobs C. Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography. J Inherit Metab Dis 1999;22:677–8.
