Multiplex Tandem Mass Spectrometry Analysis of Novel Plasma Lyso

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Multiplex Tandem Mass Spectrometry Analysis of Novel Plasma Lyso-Gb3‑Related Analogues in Fabry Disease Michel Boutin and Christiane Auray-Blais* Service of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec J1H 5N4, Canada S Supporting Information *

ABSTRACT: Fabry disease is a multisystemic, X-linked lysosomal storage disorder caused by a deficit in α-galactosidase A enzyme activity leading to glycosphingolipid accumulation, mainly globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Recent metabolomic studies have led to the discovery of novel biomarkers related to lyso-Gb3 in plasma and urine. These biomarkers show modifications of the sphingosine moiety of the lyso-Gb3 molecule. The objectives of this study were to develop and validate a liquid chromatography-tandem mass spectrometry method for the relative quantification of novel plasma lyso-Gb3related analogues, to evaluate their levels in plasma of 74 Fabry patients and 41 healthy controls and to correlate these results with patient gender, enzyme replacement therapy treatment, and lyso-Gb3 analogue levels previously measured in urine for the same patients. As expected, the concentrations of lyso-Gb3 and its related analogues in plasma are higher in Fabry males compared to Fabry females and higher for untreated males compared to treated males. The concentration of lyso-Gb3 and its related analogues in plasma decrease significantly after the beginning of enzyme replacement therapy (ERT) treatment and remain stable for 30 months of monitored therapy in a Fabry male. In plasma, lyso-Gb3 is significantly more abundant than its related analogues, which differs from urine where the majority of the lyso-Gb3 analogues are more increased than lyso-Gb3 itself. In contrast to urine, the relative distribution of lyso-Gb3 and its analogues in plasma is similar from one individual to another in the same group of Fabry patients, irrespective of ERT. This study revealed a large discrepancy between the relative abundance of lyso-Gb3 and its analogues in urine and plasma. Further studies will thus be needed to better understand the metabolic relationship between plasma and urine lyso-Gb3-related biomarkers. abry disease (OMIM # 301500) is an X-linked lysosomal storage disorder. The deficiency of the enzyme, αgalactosidase A (α-GAL A, EC 3.2.1.22), results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) and globotriaosylphingosine (lyso-Gb3) in biological fluids and various organs.1−3 Enzyme replacement therapy (ERT) is employed to treat both males and females affected with the disease, depending on the severity of symptoms.4,5 Biomarker analysis for Fabry disease oriented toward mass6- and high-risk screening,7−9 diagnosis,10 and treatment monitoring and follow-up10−13 has been in use for several years. Urinary14 and plasma15 analogues of lyso-Gb3 have recently been detected by time-of-flight metabolomic studies and have been structurally elucidated using tandem mass spectrometry. Quantification of urinary analogues of lysoGb3 subsequently revealed that the urinary excretion of the majority of these analogues is higher than that of lyso-Gb3 itself.16 We also found that Fabry patients having the N215S cardiac variant mutation excreted abnormal levels of lyso-Gb3 and related analogues even though the excretion of urinary Gb3 was normal. The objectives of this study were to develop and validate a liquid chromatography-tandem mass spectrometry method for the relative quantification of novel plasma lyso-Gb3-

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© 2014 American Chemical Society

related analogues, to evaluate their levels in plasma of 74 Fabry patients and 41 healthy controls, and to correlate these results with patient gender, enzyme replacement therapy treatment, and lyso-Gb3 analogue levels previously measured in urine for the same patients. The chemical structures of lyso-Gb3 and the 6 sphingosine-modified analogues quantified in plasma of Fabry patients, as well as the lyso-Gb3-glycine used as the internal standard, are shown in Figure 1.

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EXPERIMENTAL SECTION Ethics Approval. This project was approved by the Research Ethics Board (REB) of the Faculty of Medicine and Health Sciences and the Centre hospitalier universitaire de Sherbrooke (CHUS), as well as the institutional REBs of each of the collaborators. Plasma Collection. Plasma EDTA specimens were collected from Fabry patients diagnosed by demonstrating marked enzyme deficiency in leucocytes or by mutation

Received: December 14, 2013 Accepted: February 28, 2014 Published: February 28, 2014 3476

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Analytical Chemistry

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human plasma stripped two times with charcoal were purchased from Bioreclamation (Hicksville, NY). The lyso-Gb3-Gly internal standard (IS) (Figure 1B) was synthesized in-house as described previously.16 Briefly, the IS consists of lyso-Gb3 coupled by an amide linkage to a glycine molecule. This IS presents extraction and ionization behaviors comparable to the lyso-Gb3 metabolite, owing to its primary amine.16 Sample Preparation. Aliquots (100 μL) of well-mixed plasma samples from Fabry patients or healthy controls were transferred to glass culture tubes containing 500 μL of H3PO4 (2% in water) and 500 μL of lyso-Gb3-Gly internal standard (10 nM in MeOH). Sample mixtures were transferred with glass pipettes to mixed-mode cation-exchange cartridges (Oasis MCX, 30 mg, 60 μm, Waters Corp., Milford, MA) conditioned successively with 1000 μL of MeOH and 1000 μL of 2% H3PO4. After loading, the cartridges were washed first with 1000 μL of 2% FA in H2O, followed by 1000 μL of 0.2% FA in MeOH. Lyso-Gb3, its related analogues, and lyso-Gb3-Gly (IS) were eluted into glass tubes with 600 μL of 2% ammonia in MeOH. Eluates were dried under a stream of nitrogen. Residues were resuspended in 100 μL of ACN 50%/FA 0.1%/H2O and transferred into glass inserts fitted into 2 mL glass vials for ultra-performance liquid chromatography (UPLC)-MS/MS analysis. For the calibration curve, the preparation was similar to that of the samples, except that the plasma aliquot used was 100 μL of plasma stripped twice with charcoal, and 20 μL of one of the following standard solutions was added: 0, 1, 10, 50, 200, 700, and 2000 nM of lyso-Gb3 in ACN 50%/FA 0.1%/H2O. No trace of lyso-Gb3 or lyso-Gb3 analogues was detected in the charcoal-stripped plasma used for the calibration curve. For the fragmentation study of the lyso-Gb3(+34) analogue, 5 plasma aliquots of 100 μL from an untreated Fabry male were prepared as previously described, except that the 5 eluates were combined, evaporated, and resuspended in 100 μL of ACN 50%/FA 0.1%/water to obtain a 5-times more concentrated sample. UPLC-MS/MS Analysis of Lyso-Gb3 and Its Analogues in Plasma. An ACQUITY UPLC I-Class system from Waters Corporation (Milford, MA) was used for the separation of metabolites before quantitative tandem mass spectrometry analysis of lyso-Gb3 and its analogues in plasma. The reversephase UPLC method used is described in Table 2. The UPLC system was coupled to a Xevo TQ-S (Waters) mass spectrometer operated in the multiple reaction monitoring (MRM) mode. Using this mode, the precursor ion is isolated in the first quadrupole, fragmented in the collision cell, and one of its specific fragment ions is isolated in the last quadrupole for detection. The fragmentation was performed by collisioninduced dissociation (CID) using argon as the collision gas. Eight MRM reactions corresponding to lyso-Gb3, its 6 analogues, and the lyso-Gb3-Gly (IS) were alternatively

Figure 1. (A) Structure of globotriaosylsphingosine (lyso-Gb3) showing the six modifications of the sphingosine chain observed in plasma. (B) Structure of lyso-Gb3-Gly used as the internal standard.

analysis. Plasma samples from healthy controls were also analyzed to establish normal reference values. Table 1 summarizes the demographic data of Fabry patients and agematched healthy controls. Fabry patients were subdivided in four groups according to gender and treatment. Treated patients were receiving ERT with either agalsidase-alfa at 0.2 mg/kg/2 weeks (Replagal, Shire Human Genetic Therapies Inc., Lexington, MA) or agalsidase-beta at 1.0 mg/kg/2 weeks (Fabrazyme, Genzyme, Cambridge, MA) at the time of the specimen collection. In order to evaluate the effect of ERT on the plasma levels of lyso-Gb3 and its related analogues, plasma samples from a male Fabry patient (age 35 yrs) were collected 6 months prior to ERT, before the beginning of ERT, and at 6, 12, 24, and 30 months after starting ERT. This patient had a 1024 C > T nonsense mutation (R342X) and α-galactosidase A enzyme activity in leucocytes of 4 nmol/h/mg of protein. The αgalactosidase A activity reference range for classic hemizygous Fabry patients ranges between 0.35 and 6.1 nmol/h/mg of proteins whereas, for healthy controls, it is 34−128 nmol/h/mg of proteins.17 During ERT, the patient received agalsidase-beta at a dosage of 1 mg/kg/infusion/14 days for the first 18 months (6 and 12 month samples) and agalsidase-alfa at 0.2 mg/kg/ infusion/14 days for the following year (24 and 30 month samples) because of a shortage of agalsidase-beta during that period. Reagents. HPLC grade methanol (MeOH) and acetonitrile (ACN) were purchased from EMD Chemicals Inc. (Darmstadt, Germany). Formic acid (FA) (99+%) was from Acros Organics (New Jersey, USA). A.C.S. grade ammonium hydroxide (NH4OH) (29%) was supplied by Fisher Scientific (Fair Lawn, NJ). A.C.S. grade o-phosphoric acid (H3PO4) (85%) and Optima grade H2O were from Fisher Scientific (Fair Lawn, NJ). Globotriaosylsphingosine (Lyso-Gb3) was bought from Matreya (Pleasant Gap, PA). Pooled human plasma and pooled Table 1. Demographics of Fabry Patients and Healthy Controls Fabry patients males number, n mean age, yrs median age, yrs (range)

healthy controls females

untreated

treated

untreated

treated

15 30 30 (17−52)

28 42 42 (20−66)

21 41 45 (21−62)

10 48 47 (31−66)

3477

males

females

15 41 36 (19−59)

26 37 36 (18−57)

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Table 2. UPLC Method for the Quantification of Lyso-Gb3 and Its Analogues in Plasma and for the Fragmentation Study of the Lyso-Gb3(+34) Isomers column

column temperature weak wash solvent strong wash solvent mobile phase A mobile phase B gradient

flow rate injection volume injection mode autosampler temperature

Table 3. Instrument Parameters for the Fragmentation Study of the Lyso-Gb3(+34) Isomers on the QTof Mass Spectrometer

Acquity UPLC BEH C18; Waters Corp. length: 50 mm internal diameter: 2.1 mm particle diameter: 1.7 μM 30 °C 50% ACN/0.2% FA/water 0.2% FA/MeOH 0.2% FA/ACN 5% ACN/0.2% FA/water 0−1 min → 100%B 1−3 min → 100−65%B 3−5.5 min → 65−50%B 5.5−7 min → 10%B 7−9 min → 100%B 0.5 mL/min 7.5 μL partial loop with needle overfill 10 °C

MS-tune parameters capillary voltage sampling cone voltage extraction cone voltage source temperature desolvation temperature cone gas flow dessolvation gas flow

MS-method parameters

3.2 kV 30 V

scan mode analyzer mode

QTof-MS(ESI+) V

5V

data format

Centroid

120 °C

trap collision energy ramp transfer collision energy mass range scan time

25−35 V

450 °C 30 L/h 700 L/h

4V 10−1000 Da 0.1 s

nmol/L, using charcoal-stripped plasma. Plasma from a highexcretor untreated Fabry male was used as a high quality control (QC) and plasma from a low-excretor untreated Fabry male was used as a low QC to evaluate intraday (n = 5) and interday (n = 5) precision. In the case of accuracy evaluation, charcoal-stripped plasma was spiked with lyso-Gb3 standard to obtain concentrations of 5 nmol/L (n = 2) and 200 nmol/L (n = 2). Those samples were analyzed as previously described. For lyso-Gb3 and its analogues, the limit of detection (LOD) was defined as 3 times the standard deviation (SD) of the response (area/area IS) (n = 5) divided by the slope of the calibration curve. The limit of quantification (LOQ) was evaluated using the same formula but with 10 times the SD. For lyso-Gb3 analogues, the sample used to evaluate LOD and LOQ was the low QC (plasma from a low-excretor Fabry male). In the case of lyso-Gb3, the low QC was diluted (1/10) with charcoalstripped plasma to evaluate LOD and LOQ with concentrations closer to LOQ. Extraction recovery was measured by spiking charcoal-depleted plasma with lyso-Gb3 to obtain concentrations of 15.7 nmol/L (low QC) (n = 3) and 163.4 nmol/L (high QC) (n = 3), respectively. Stability tests were performed using the low and high plasma QCs from the two untreated Fabry males. The two plasma samples were aliquoted and analyzed after 3, 6, and 24 h at room temperature (22 °C) (n = 2); 3, 6, 24, and 48 h at 4 °C (n = 2); and 2, 3, and 8 weeks at −20 °C (n = 2). Aliquots of the two plasma QCs at T0 (without aging) (n = 2) were left 12 h in the autosampler at 10 °C to check the stability of the samples resuspended after MCX purification. The two QCs were also used to evaluate the effect of three freeze−thaw cycles on the concentration of lyso-Gb3 and its analogues (n = 2). To evaluate the adsorption of lyso-Gb3 and its analogues on glass, low and high QCs, resuspended after MCX purification, were transferred to a glass tube using a glass pipet and vortexed. That process was repeated four times (n = 2). The same experiment was also done for transfers to plastic tubes with plastic pipet tips to evaluate the adsorption of the analytes on plastic.

recorded. The MRM transitions and the mass spectrometer parameters are summarized in Supporting Information Table S1. All the mass spectrometry parameters were optimized to obtain the highest possible sensitivity for lyso-Gb3 and its analogues. The divert valve of the mass spectrometer was programmed to discard the UPLC effluent before (0 to 2 min) and after (5 to 9 min) the elution of the analytes to prevent system contamination. The concentrations of lyso-Gb3 and its analogues were evaluated using the TargetLynx 4.1 software (Waters). Lyso-Gb3-Gly was used as the internal standard for the quantification of lyso-Gb3 and analogues. The lyso-Gb3 calibration curve was quadratic with a 1/x weighing function, and the origin was excluded. The lyso-Gb3 calibration curve was also used for the quantification of the lyso-Gb3 analogues since standards of these analogues are not commercially available. Quadrupole Time-of-Flight (QTof)-MS Fragmentation Study of the Lyso-Gb3(+34) Analogue. The MRM ion chromatogram of lyso-Gb3(+34) (m/z 820.45) analogue shows two major peaks separated by approximately 1 min. These two molecules were fragmented on our Synapt QTof-MS mass spectrometer (Waters) equipped with an ACQUITY UPLC (Waters) for structural elucidation, using the 5-times concentrated plasma sample prepared from an untreated Fabry male patient. The UPLC method used for the separation was the same as the one used for the quantification of lyso-Gb3 and its analogues (Table 2). The mass spectrometry parameters for the fragmentation study are summarized in Table 3. Method Validation. To evaluate the matrix effect on the lyso-Gb3 and lyso-Gb3 analogue levels, 50 μL aliquots of plasma from an untreated Fabry male were each combined with 50 μL of one of the following plasma controls: (1) pooled plasma stripped twice with charcoal from Bioreclamation (the plasma used for the calibration curve); (2) pooled plasma from bioreclamation; (3) plasma from a 20 year-old healthy woman; (4) plasma from a 40 year-old healthy woman; (5) plasma from a 24 year-old healthy man; and (6) plasma from a 44 year-old healthy man. All 6 samples were prepared and analyzed as previously described. The lyso-Gb3 linearity response was assessed by preparing a standard curve with 7 concentrations, ranging from 0 to 400

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RESULTS AND DISCUSSION UPLC-MS/MS Analysis of Lyso-Gb3 and Its Analogues in Plasma. Figure 2 presents MRM chromatograms for lysoGb3, its 6 related analogues, and the lyso-Gb3-Gly internal standard (IS) in the plasma of an untreated Fabry male. For each MRM transition, the fragment ion reflecting the highest sensitivity was selected. In the case of lyso-Gb3 and lysoGb3(−28) and (−2) analogues, the fragment ion corresponds 3478

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metabolomic study in plasma owing to their low abundance level. The peak corresponding to lyso-Gb3(+50) presents a peak splitting. The same situation occurred during the metabolomic study performed on urine of Fabry patients.14 The peak splitting was attributed to different structural isomers generated by the addition of hydroxyl groups to the sphingosine chain of this lyso-Gb3 analogue. In the case of the lyso-Gb3(+34) analogue, two major peaks, separated by approximately 1 min, were observed on its MRM chromatogram. Both were present in Fabry patients but not in healthy controls. Only the most abundant peak, at a retention time (RT) of 4.06 min, was revealed during the metabolomic study performed on plasma.15 The second peak at RT 3.08 min was fragmented to elucidate its structure. QTof-MS Fragmentation Study of the Lyso-Gb3(+34) Analogue. The two peaks, separated by approximately one minute, in the MRM chromatogram corresponding to lysoGb3(+34), were analyzed and fragmented on a UPLC-QTofMS system. The chromatographic method used was the same as that used for MRM quantitation in order to obtain similar retention times. A sample 5 times more concentrated than the one used for MRM quantitation was needed to obtain good quality fragmentation spectra. Figure 3A presents the extracted

Figure 2. MRM chromatograms of lyso-Gb3, its six related analogues, and lyso-Gb3-Gly, used as the internal standard (IS), in a plasma sample from an untreated Fabry male. The peaks corresponding to the molecules analyzed are shaded. The MRM transitions are expressed as “precursor ion > fragment ion”.

to the respective sphingosine moiety with the loss of a water molecule (−H2O). For lyso-Gb3(+16) and the lyso-Gb3-Gly internal standard, it corresponds to the sphingosine moiety with the loss of two water molecules (−2H2O). Finally, for the lyso-Gb3(+18), (+34), and (+50) analogues, it corresponds to the respective sphingosine moiety. The chromatographic gradient was programmed to separate the peaks of the analytes from the interferences detected in the same MRM channels for a plasma pool from healthy controls. The elution of the chromatographic peaks was also extended for a sufficiently long period to minimize ion suppression without losing the peak shape. The metabolomic study previously performed on plasma samples of Fabry patients15 revealed 4 new lyso-Gb3 analogues (lyso-Gb3(−2), (+16), (+18), and (+34)) as biomarkers characteristic of Fabry disease. A similar metabolomic study performed on urine of Fabry patients also revealed the lysoGb3(−28), (−12), (+14), and (+50) analogues as Fabry disease biomarkers.14 The MRM methods performed on triplequadrupole mass spectrometers are much more sensitive than the Tof-MS full-scan methods used to discover new biomarkers. For this reason, the presence of the biomarkers previously detected in urine, but not in plasma during the course of previous metabolomic studies, was verified in plasma using MRM. The lyso-Gb3(−28) and (+50) analogues were detected, and this is the first time, to our knowledge, that these molecules are reported in plasma. These two lyso-Gb3 analogues were probably not detected during the MS-Tof

Figure 3. (A) Extracted ion chromatogram of the lyso-Gb3(+34) analogue (m/z 820.45) in the plasma sample of an untreated Fabry male, showing the presence of two structural isomers. (B and C) present similar fragmentation profiles for the two structural isomers. Collision energy ramp: 25−35 V. Gal = galactose, Glc = glucose.

ion chromatogram corresponding to lyso-Gb3(+34) (m/z 820.45) showing the two peaks at 3.14 and 4.19 min previously observed during the MRM analysis. Figure 3B and C show the fragmentation spectrum corresponding to the peaks at retention times of 3.14 and 4.19 min, respectively. The two fragmentation spectra are similar indicating that the two peaks correspond to structural isomers of lyso-Gb3(+34). For the quantitative analysis, these latter peaks were integrated together and their areas added together. 3479

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Method Validation. The matrix effect affecting the analysis of lyso-Gb3 and its analogues was evaluated by mixing 50 μL of plasma from an untreated Fabry male (high QC) with 6 different plasma specimens from healthy controls, including the plasma pool two times charcoal depleted used as matrix for the calibration curve. The measured concentrations of lyso-Gb3 and its analogues were similar for all the six samples, with relative standard deviations (RSD) less than 3.2%, indicating a very low matrix effect on the reproducibility of the results obtained. The lyso-Gb3 calibration curve (0.2 to 400 nmol/L, n = 7) was linear with a mean coefficient of regression (R2) of 0.9988 (n = 5). Since standards are not commercially available for the lyso-Gb3 analogues, the response factor of lyso-Gb3 was used to quantify these molecules. Similar response factors were expected for lyso-Gb3 analogues compared to lyso-Gb3 owing to the fact that each of these molecules has a single primary amine function where the ionization occurs. However, the fragmentation patterns of the lyso-Gb3 analogues differ slightly from one to the other.14,15 Therefore, even if the fragment used for quantitation of each lyso-Gb3 analogue corresponds to the most abundant sphingosine related fragment, small differences in response factors might be expected between lyso-Gb3 and its analogues. Table 4 summarizes the intra- and interday assay precision measured for lyso-Gb3 and its analogues using low and high QCs. All were below 15% variation.

Table 5. Limit of Detection (LOD) and Limit of Quantification (LOQ) for Lyso-Gb3 and Its Related Analogues

a

precision (CV%) intraday n = 5

interday n = 5

analytes

L

H

L

H

L

H

lyso-Gb3 lyso-Gb3(−28) lyso-Gb3(−2) lyso-Gb3(+16) lyso-Gb3(+18) lyso-Gb3(+34) lyso-Gb3(+50)

19.15 0.52 5.34 1.57 2.54 4.16 2.20

196.23 8.83 49.48 22.86 30.35 41.23 7.51

0.4 8.3 2.6 7.0 3.2 5.0 4.2

0.6 2.8 1.5 2.3 1.7 1.6 4.1

7.9 14.9 7.7 10.5 7.3 7.8 9.7

4.0 2.0 3.1 3.0 10.3 5.0 3.0

LOD (nM)a

LOQ (nM)a

lyso-Gb3 lyso-Gb3(−28) lyso-Gb3(−2) lyso-Gb3(+16) lyso-Gb3(+18) lyso-Gb3(+34) lyso-Gb3(+50)

0.23 0.06 0.29 0.22 0.14 0.24 0.09

0.77 0.21 0.97 0.72 0.47 0.79 0.30

Concentration evaluated using the response factor of lyso-Gb3.

samples were considered stable when the coefficients (CV%) of variation for lyso-Gb3 and its analogues were