New Strategy for the Screening of Lysosomal Storage Disorders: The

Jun 25, 2009 - Each specific disease results in a defect of lysosomal enzyme, its cofactor, ...... to Newborn Screening of Pompe, Fabry, and Hurler Di...
16 downloads 0 Views 4MB Size
Anal. Chem. 2009, 81, 6113–6121

New Strategy for the Screening of Lysosomal Storage Disorders: The Use of the Online Trapping-and-Cleanup Liquid Chromatography/ Mass Spectrometry Giancarlo la Marca,*,† Bruno Casetta,‡ Sabrina Malvagia,§ Renzo Guerrini,§ and Enrico Zammarchi| Mass Spectrometry and Pharmacology Laboratory, A. Meyer Children’s Hospital Pediatric Neurology Unit and Laboratories, Department of Pharmacology, University of Florence, Florence, Italy, Applied Biosystems, Via Tiepolo 18, 20052 Monza, Italy, Pediatric Neurology Unit and Laboratories, A. Meyer Children’s Hospital, Florence, Department of Neurosciences, University of Florence, Florence, Italy, and Department of Pediatrics, University of Florence, Viale Pieraccini 24, 50139 Florence, Florence, Italy The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometrybased method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by “Pompe”, 6 by “Gaucher”, 12 by “Fabry”, 3 by “Niemann-Pick” A/B, and 2 by “Krabbe” diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening. The lysosomal storage diseases (LDSs) are a heterogeneous group of over 40 inherited genetic disorders. Each specific disease results in a defect of lysosomal enzyme, its cofactor, transport and membrane proteins, or protein involved in lysosomal biogenesis. A total or partial defect is characterized by the accumulation of undigested substrates from each of the many different catabolic pathways. Abnormal macromolecule storage within lysosomes * Author to whom correspondence is addressed. Phone: ++ 39-(0)55-5662988. Fax: ++ 39-(0)55-5662489. E-mail: [email protected], [email protected]. † Department of Pharmacology, University of Florence. ‡ Applied Biosystems. § Department of Neurosciences, University of Florence. | Department of Pediatrics, University of Florence. 10.1021/ac900504s CCC: $40.75  2009 American Chemical Society Published on Web 06/25/2009

leads to cellular dysfunction and cell death. LSDs generally affect any age of life but especially early childhood and have a devastating impact on the families and on public health. Affected patients, usually asymptomatic at birth, can manifest early in life various symptoms including progressive central nervous system deterioration, severe skeletal abnormalities, and multisystemic dysfunction involving heart, liver, spleen, lungs, and kidney. Multiple organ failure causes frequent hospitalizations and often leads to death in early adolescence. Over the years, approaches for treatment of some LSDs have been developed by leveraging different strategies. Bone marrow or stem cell transplantation provides an effective treatment for MPS I, MPS VI, metachromatic leukodystrophy, and Krabbe disease but only if performed when the patient is asymptomatic.1-4 Enzyme replacement therapy (ERT) has become the standard treatment for patients with Gaucher disease5,6 and, more recently, for patients with Fabry and Pompe diseases7-9 and with some mucopolysaccharidoses such as MPS I10,11 MPS VI,12,13 and MPS (1) Krivit, W. Adv. Pediatr. 2002, 49, 359–378. (2) Lim, Z. Y.; Ho, A. Y.; Abrahams, S.; Fensom, A.; Aldouri, M.; Pagliuca, A.; Shaw, C.; Mufti, G. J. Bone Marrow Transplant. 2008, 41, 831–832. (3) Peters, C.; Steward, C. G. Bone Marrow Transplant. 2003, 31, 229–239. (4) Krivit, W.; Peters, C.; Shapiro, E. G. Curr. Opin. Neurol. 1999, 12, 167– 176. (5) Barton, N. W.; Brady, R. O.; Dambrosia, J. M.; Di Bisceglie, A. M.; Doppelt, S. H.; Hill, S. C.; Mankin, H. J.; Murray, G. J.; Parker, R. I.; Argoff, C. E.; et al. N. Engl. J. Med. 1991, 324, 1464–1470. (6) Brady, R. O. Annu. Rev. Med. 2006, 57, 283–296. (7) Beck, M. Expert Opin. Investig. Drugs. 2002, 11, 851–858. (8) Parini, R.; Rigoldi, M.; Santus, F.; Furlan, F.; De Lorenzo, P.; Valsecchi, G.; Concolino, D.; Strisciuglio, P.; Feriozzi, S.; Di Vito, R.; Ravaglia, R.; Ricci, R.; Morrone, A. Clin. Genet. 2008, 74, 260–266. (9) Van den Hout, J. M.; Kamphoven, J. H.; Winkel, L. P.; Arts, W. F.; De Klerk, J. B.; Loonen, M. C.; Vulto, A. G.; Cromme-Dijkhuis, A.; Weisglas-Kuperus, N.; Hop, W.; Van Hirtum, H.; Van Diggelen, O. P.; Boer, M.; Kroos, M. A.; Van Doorn, P. A.; Van der Voort, E.; Sibbles, B.; Van Corven, E. J.; Brakenhoff, J. P.; Van Hove, J.; Smeitink, J. A.; de Jong, G.; Reuser, A. J.; Van der Ploeg, A. T. Pediatrics 2004, 113, 448–457. (10) Wraith, J. E.; Clarke, L. A.; Beck, M.; Kolodny, E. H.; Pastores, G. M.; Muenzer, J.; Rapoport, D. M.; Berger, K. I.; Swiedler, S. J.; Kakkis, E. D.; Braakman, T.; Chadbourne, E.; Walton-Bowen, K.; Cox, G. F. J. Pediatr. 2004, 144, 581–588. (11) Arora, R. S.; Mercer, J.; Thornley, M.; Tylee, K.; Wraith, J. E. J. Inherit. Metab. Dis. 2007, 30, 821.

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

6113

Figure 1. The online trapping-and-cleanup/liquid chromatography configuration.

II.14,15 Other strategies include substrate deprivation or reduction16,17 and the use of chemical chaperons.18,19 Use of Gentamicin as for the restoration of alpha iduronidase activity in some stop mutations has been reported to be effective.20 One of the critical points of the ERT is the enzymes inability to pass through the blood-brain barrier, precluding their use for treating LDSs with central nervous system involvement. Currently, novel approaches have been tested for passing across the bloodbrain barrier including the use of low density lipoproteins bundled to the therapeutic enzyme.21 The increasing availability of treatments for some of these diseases has propelled the development of new methods and techniques for rapidly establishing the diagnosis in patients with (12) Harmatz, P.; Whitley, C. B.; Waber, L.; Pais, R.; Steiner, R.; Plecko, B.; Kaplan, P.; Simon, J.; Butensky, E.; Hopwood, J. J. J. Pediatr. 2004, 144, 574–580. (13) Auclair, D.; Hein, L. K.; Hopwood, J. J.; Byers, S. Pediatr. Res. 2006, 59, 538–543. (14) Muenzer, J.; Lamsa, J. C.; Garcia, A.; Dacosta, J.; Garcia, J.; Treco, D. A. Acta Paediatr. 2002, 91, 98–99. (15) Wraith, J. E. Acta Paediatr. 2008, 97, 76–78. (16) Butters, T. D.; Dwek, R. A.; Platt, F. M. Adv. Exp. Med. Biol. 2003, 535, 219–226. (17) Butters, T. D.; Dwek, R. A.; Platt, F. M. Glycobiology 2005, 15, 43–52. (18) Matsuda, J.; Suzuki, O.; Oshima, A.; Yamamoto, Y.; Noguchi, A.; Takimoto, K.; Itoh, M.; Matsuzaki, Y.; Yasuda, Y.; Ogawa, S.; Sakata, Y.; Nanba, E.; Higaki, K.; Ogawa, Y.; Tominaga, L.; Ohno, K.; Iwasaki, H.; Watanabe, H.; Brady, R. O.; Suzuki, Y. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 5912– 5917. (19) Caciotti, A.; Donati, M. A.; d’Azzo, A.; Salvioli, R.; Guerrini, R.; Zammarchi, E.; Morrone, A. Eur. J. Paediatr. Neurol. 2008, 13, 160–164. (20) Keeling, K. M.; Brooks, D. A.; Hopwood, J. J.; Li, P.; Thompson, J. N.; Bedwell, D. M. Hum. Mol. Genet. 2001, 10, 291–299. (21) Spencer, B. J.; Verma, I. M. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 7594–7599.

6114

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

Table 1. Most Relevant Operating Parameters for the API 2000 MRM dwell declustering collision compounds transitions times potentials energies ionization ESI, positive ion mode voltage 5200 V Turbogas heater-gun 400 °C temperature GLA-P 483.9 > 383.9 GLA-IS 488.9 > 388.9 GAA-P 497.9 > 307.9 GAA-IS 502.9 > 402.9 ASM-P 398.0 > 264.2 ASM-IS 370.0 > 264.2 GALC-P 426.0 > 264.2 GALC-IS 454.0 > 264.2 ABG-P 482.2 > 264.2 ABG-IS 510.0 > 264.2

50 msec 25 msec 50 msec 25 msec 50 msec 25 msec 50 msec 25 msec 50 msec 25 msec

80 V 80 V 80 V 80 V 60 V 60 V 20 V 20 V 20 V 20 V

20 eV 20 eV 20 eV 20 eV 25 eV 25 eV 30 eV 30 eV 30 eV 30 eV

clinical suspicion and make as soon as possible these patients undergo therapeutic treatment, if available. The ideal scenario should be to provide the treatment still at the asymptomatic phase, which is possible only in individuals with a positive family history or through newborn screening programs.22 The diagnosis of a LSD is often very complex and involves a range of time-consuming, laborious assays on urine, blood, and cultured cells with sometimes very long response times. During the past years various approaches for newborn screening of some LSDs have been developed. To our knowledge, the first published method was the Berry spot test for diagnosing mucopolysaccharidoses (MPS) in subjects with clinical suspicion.23 Then, screening tests for urine oligosaccharides24 and for (22) Meikle, P. J.; Hopwood, J. J. Eur. J. Pediatr. 2003, 162, 34–37.

sphingolipidoses25,26 have been proposed as useful diagnostic tools to aid clinicians. Other approaches are aimed at the identification of lysosomal proteins such as lysosome-associated membrane proteins (LAMPs) and SAP-C, that are already altered in affected babies during the first days of life corresponding to the presymptomatic phase.27-30 However, these methods are disadvantageous since the reported large number of false-positive and false-negative results.31 Even the immunological assay system may be used to perform multiple tests.32 This method is based on a multiplexed immune-quantification of the different implicated lysosomal proteins.27,28,33-35 However, since in some affected individuals nonfunctional proteins can be quantitatively normal, this approach is not suitable in these specific cases.32,36 Chamoles et al.37-39 have demonstrated that many lysosomal enzymes are still active in rehydrated dried blood spots. Direct multiplex assay of lysosomal enzymes in dried blood spots by tandem mass spectrometry has been developed based on these observations.36,40 The use of mass spectrometric techniques has exhibited advantages over fluorometric or spectrophotometric assays in the simultaneous quantification of several markers.36 Based on the pioneering works of Chamoles and Gelb, several experimental approaches have been developed to provide a newborn screening program for LSDs using enzymatic assay followed by fluorimetric or tandem mass spectrometric methods in order to detect the product of one specific enzyme41-45 or by (23) Berry, H. K. Clin. Biochem. 1987, 20, 365–371. (24) Humbel, R.; Collart, M. Clin. Chim. Acta 1975, 60, 143–145. (25) Philippart, M.; Sarlieve, L.; Meurant, C.; Mechler, L. J. Lipid Res. 1971, 12, 434–441. (26) Berra, B.; di Palma, S.; Primi, D. A. Biochem. Exp. Biol. 1977, 13, 79–83. (27) Meikle, P. J.; Brooks, D. A.; Ravenscroft, E. M.; Yan, M.; Williams, R. E.; Jaunzems, A. E.; Chataway, T. K.; Karageorgos, L. E.; Davey, R. C.; Boulter, C. D.; Carlsson, S. R.; Hopwood, J. J. Clin. Chem. 1997, 43, 1325–1335. (28) Hua, C. T.; Hopwood, J. J.; Carlsson, S. R.; Harris, R. J.; Meikle, P. J. Clin. Chem. 1998, 44, 2094–2102. (29) Ranierri, E.; Gerace, R. L.; Ravenscroft, E. M.; Hopwood, J. J.; Meikle, P. J. Southeast Asian J. Trop. Med. Public Health 1999, 30, 111–113. (30) Meikle, P. J.; Ranieri, E.; Ravenscroft, E. M.; Hua, C. T.; Brooks, D. A.; Hopwood, J. J. Southeast Asian J. Trop. Med. Public Health 1999, 30, 104– 110. (31) Meikle, P. J.; Ranieri, E.; Simonsen, H.; Rozaklis, T.; Ramsay, S. L.; Whitfield, P. D.; Fuller, M.; Christensen, E.; Skovby, F.; Hopwood, J. J. Pediatrics 2004, 114, 909–916. (32) Meikle, P. J.; Grasby, D. J.; Dean, C. J.; Lang, D. L.; Bockmann, M.; Whittle, A. M.; Fietz, M. J.; Simonsen, H.; Fuller, M.; Brooks, D. A.; Hopwood, J. J. Mol. Genet. Metab. 2006, 88, 307–314. (33) Fuller, M.; Lovejoy, M.; Brooks, D. A.; Harkin, M. L.; Hopwood, J. J.; Meikle, P. J. Clin. Chem. 2004, 50, 1979–1985. (34) Fuller, M.; Brooks, D. A.; Evangelista, M.; Hein, L. K.; Hopwood, J. J.; Meikle, P. J. Mol. Genet. Metab. 2005, 84, 18–24. (35) Umapathysivam, K.; Hopwood, J. J.; Meikle, P. J. Clin. Chem. 2001, 47, 1378–1383. (36) Gelb, M. H.; Turecek, F.; Scott, C. R.; Chamoles, N. A. J. Inherit. Metab. Dis. 2006, 29, 397–404. (37) Chamoles, N. A.; Blanco, M.; Gaggioli, D. Clin. Chim. Acta 2001, 308, 195–196. (38) Chamoles, N. A.; Blanco, M.; Gaggioli, D.; Casentini, C. Clin. Chim. Acta 2002, 317, 191–197. (39) Niizawa, G.; Levin, C.; Aranda, C.; Blanco, M.; Chamoles, N. A. Clin. Chim. Acta 2005, 359, 205–206. (40) Li, Y.; Scott, C. R.; Chamoles, N. A.; Ghavami, A.; Pinto, B. M.; Turecek, F.; Gelb, M. H. Clin. Chem. 2004, 50, 1785–1796. (41) Spada, M.; Pagliardini, S.; Yasuda, M.; Tukel, T.; Thiagarajan, G.; Sakuraba, H.; Ponzone, A.; Desnick, R. J. Am. J. Hum. Genet. 2006, 79, 31–40. (42) Chien, Y. H.; Chiang, S. C.; Zhang, X. K.; Keutzer, J.; Lee, N. C.; Huang, A. C.; Chen, C. A.; Wu, M. H.; Huang, P. H.; Tsai, F. J.; Chen, Y. T.; Hwu, W. L. Pediatrics 2008, 122, 39–45.

tandem mass spectrometry to quantify multiple enzymes simultaneously.46 With the availability of a treatment for an even growing number of LSDs the development of a multiple test applicable to neonatal screening has become important for the early diagnosis and for improving the health outcomes in affected individuals. Herein, we report a new and simplified tandem mass spectrometry-based method to perform multiple enzyme analysis on dried blood spots. This simple method takes only 4 min as analysis run time and does not involve any time-consuming sample preparation step after the enzymatic reaction. Many authors used online extraction coupled to LC-MS/MS especially in drug assay applications.47,48 As far as we know, this is the first use of this analytical approach to detect some LSDs for screening purposes. EXPERIMENTAL SECTION Standards and Chemicals. All substrates (S) and internal standards (IS) were manufactured at Genzyme Pharmaceutical, Liestal, Switzerland and are available through the Newborn Screening Branch at the Centers for Disease Control and Prevention (Atlanta, Georgia). Reconstitution buffers for each vial, containing both S and IS, were performed according to the manufacturer’s protocol according to recent published procedures.49 All chemicals and solvents were of the highest purity available from commercial sources and were used without any further purification. DBS Collection. Blood collection for expanded newborn screening by tandem mass spectrometry in Tuscany is recommended between 48 and 72 h of life. Blood samples were obtained by heel stick, spotted on filter paper (903, Schleicher & Schuell), dried, and sent by courier to the screening center.50 Dried blood spots (DBS) from healthy adult blood donors were prepared by spotting 25 µL of whole blood on filter paper. Dried blood spots (DBS) from a healthy adult blood donor were prepared by spotting 100 µL of whole blood on filter paper for the study of the stability of enzyme activity and the repeatability tests. Blood spots were dried for a period of at least 2 h at room temperature (RT) and were stored at 4 °C until analysis in sealed plastic bags containing desiccant and a humidity indicator card dried. Whole blood from donors was collected after they gave informed consent. All experiments were conducted in compliance with institutional review board guidelines. In all affected patients, the disease had been diagnosed previously with established biochemical and clinical procedures. Sample Preparation. The enzymatic reactions were carried out separately. Five 3.2 mm blood spots (containing about 3.3-3.4 (43) Wang, D.; Wood, T.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Clin. Chem. 2007, 53, 137–140. (44) Dajnoki, A.; Mu ¨ hl, A.; Fekete, G.; Keutzer, J.; Orsini, J.; Dejesus, V.; Zhang, X. K.; Bodamer, O. A. Clin. Chem. 2008, 54, 1624–1629. (45) Blanchard, S.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Clin. Chem. 2008, 54, 2067–2070. (46) Zhang, X. K.; Elbin, C. S.; Chuang, W. L.; Cooper, S. K.; Marashio, C. A.; Beauregard, C.; Keutzer, J. M. Clin. Chem. 2008, 54, 1725–1728. (47) Ripolle´s, C.; Marı´n, J. M.; Lo´pez, F. J.; Sancho, J. V.; Herna´ndez, F. Rapid Commun. Mass Spectrom. 2009, 23, 1841–1848. (48) Corona, G.; Casetta, B.; Sandron, S.; Vaccher, E.; Toffoli, G. Rapid Commun. Mass Spectrom. 2008, 22, 519–525. (49) De Jesus, V. R.; Zhang, X. K.; Keutzer, J.; Bodamer, O. A.; Mu ¨ hl, A.; Orsini, J. J.; Caggana, M.; Vogt, R. F.; Hannon, W. H. Clin. Chem. 2009, 55, 158– 164. (50) la Marca, G.; Malvagia, S.; Casetta, B.; Pasquini, E.; Donati, M. A.; Zammarchi, E. J. Inherit. Metab. Dis. 2008, 31, 769.

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

6115

Figure 2. Multiple extracted ion chromatograms (XICs) of DBS from a healthy newborn.

µL of blood each if from newborns, about 2.8-3.0 µL if from adults) were punched from each DBS sample into a 96-well plate. Each of the five wells were added by 20 µL of specific assay cocktail for Gaucher disease (ABG), Fabry disease (GLA), Pompe disease (GAA), Krabbe disease (GALC), and Niemann-Pick disease type A and B (ASM), respectively. The plates, covered with lids and aluminum foil, were placed in a water bath set at 37 °C for 20-24 h. Incubation with a saturated, humid atmosphere helped to reduce or avoid evaporation. After incubation, all five reaction mixtures were quenched with 60 µL of methanol containing 0.1% formic acid, combined together and centrifuged (ALC PK 120 R, DJB Labcare, Newport Pagnell, England) to remove any suspended paper particles (2500 rpm for 3 min). Blank reactions in duplicate were performed for each enzyme assay by using 3.2 mm diameter disks from blank filter paper. All enzyme activities were calculated by subtracting the blank contribution. Instrumentation. A method has been developed on an API 2000 Tandem Mass Spectrometer (Applied Biosystems - Foster City CA, USA) equipped with the TurboSpray source. Subsequently the daily routine work has been done on an API 3200 (Applied Biosystems, Foster City, CA, USA) equipped with the ESI ionization probe fitted on the Turbo-V source. For the online trapping and-cleanup/liquid chromatography configuration, an Agilent 1100 LC-binary pump and an Agilent 1100 thermostatted autosampler were used. In order to operate the Agilent 1100 binary pump as two independent pumping heads, 6116

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

internal plumbing has been modified by removing the mixing-T and leaving the dumper on the channel A. The trapping and cleanup procedure was centered on a Perfusion column POROS R1/20 2 × 30 mm (Applied Biosystems, Foster City, CA, USA). Separation chromatography was performed through a Metachem Polaris C18 3 µm, 2 × 50 mm (Metachem, Lake Forest, CA, USA) (Figure 1). The two operations are articulated through the following steps. - Upon the injection, the sample is cleaned through the Perfusion column with an aqueous solution containing 2% methanol and 0.05% formic acid and delivered by channel A of the binary pump at 1.2 mL/min for 1 min. - With the activation of the valve, the Perfusion column is connected in line with the C18 column and both are flowed by 300 µL/min of methanol supplied by the channel B of the binary pump. - With the switching-back of the valve, occurring at 3 min, the Perfusion column is re-equilibrated with an aqueous solution containing 2% methanol and 0.05% formic acid and delivered by channel A of the binary pump at 1.2 mL/min for 1 min. - At 4 min, running is completed, and the system is ready for the next injection. For the quantitation measurements, the tandem mass spectrometer was operated in multiple reaction monitoring (MRM) mode by exploiting the transitions reported in Table 1.

Figure 3. Example of the effect of a high DP value (left panel) and a low DP value in ABG product chromatogram. DP at 80 V caused an “in-source” breakdown of the substrate with loss of sugar moiety. An undue appearance of the product at RT 3.3 min was evident. 3.3 min corresponded to RT of ABG substrate (ion pair transition (644 > 482), Figure S-4).

The amount of product generated by the enzyme assay was calculated from the ion abundance ratio between the enzymatic reaction product (P) and the associated internal standard (P/

IS), multiplied by the amount of added IS, and divided by the response factor ratio of P and IS. The enzyme activity expressed in units of “µmol/h/L whole blood” was then calculated from Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

6117

Table 2. Lysosomal Enzyme Activities in DBSa healthy newborns n ) 5000 GAA ABG GLA ASM GALC a

healthy adults n ) 300

heterozygotes

affected patients

median

high value

low value

median

high value

low value

high value

low value

n

high value

n

11.56 11.65 11.95 2.43 1.41

27.38 47.41 36.6 7.43 5.64

2.31 2.2 1.13 0.56 0.50

6.56 14.4 21.2 1.78 3.38

21.88 38.33 49.02 5.55 10.07

1.86 1.72 5.71 0.42 1.01

4.85 8.89 5.87

2.72 3.4 1.01

10 4 18

0.13 0.45 0.40 0.08 0.20

10 6 12 4 2

Ranges are expressed as µmol/h/L blood.

the amount of product further divided by the used amount of blood and by the incubation time. We assumed 3.4 µL of whole blood in each 3.2 mm punch of dried blood spot. The above calculations were embedded in the functionalities of the mass-spectrometer software for an automatic processing during the routine operation. RESULTS AND DISCUSSION The relatively large buffer amount needed for sustaining the enzymatic reaction is an issue since it can affect the electrospray ionization by signal suppression. Indeed, up to now, all the published methodologies foresee some manipulations on the sample after the incubation in order to have a clean extract, suitable for the mass spectrometric measurement. In the present approach, samples were directly cleaned-up and automatically extracted by using online a Perfusion column (POROS R1/20 2 × 30 mm). Chromatography by a Metachem Polaris C18 3 µm, 2 × 50 mm (Metachem, Lake Forest, CA, USA) allowed the separation of ABG, GLA, GAA, ASM, and GALC products and related IS as eluted from the Perfusion column. Figure 2 shows a typical LC-MS/MS ion chromatogram from normal DBS obtained by using the proposed method and where five products and their respective IS are evidenced. The chromatographic run is less than 4 min. Coextracted components did not cause any interference or quenching on the signals. GAA and GLA presented the same RT for enzyme activity products and labeled IS. Instead, RTs for ASM, GALC, and ABG were different because IS were homologous molecules. It is noteworthy to point out as some of the operating parameters of the mass spectrometer are of paramount importance. Since most of the substrates are embedding in their structure a sugar moiety (except ASM), which is intended to be cleaved off by the enzyme, particular care must be taken in optimizing the declustering potential (DP) parameter. Indeed a too high DP value can cause an “in-source” breakdown of the substrate with an undue appearance of the product and with the subsequent appearance of an additive peak at the MRM measurement (Figure 3). For addressing this risk, DP optimization must not be carried through just the infusion of the cocktail but by injecting it in the configured LC experiment and by collecting the MRM signals on the same product-analyte with different DP values (Figure S-1). The parameters specifically optimized for each product are subsequently embedded in the parameter setting as in Table 1, where each product (P) and its related internal standard (IS) is monitored at its optimized parameter value without compromise. The choice of a methanol/0.1% formic acid solution for stopping the enzymatic reactions was made by considering the chromato6118

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

graphic column conditions. Final sample dilution rate and injection volume (5 µL) were selected in order to avoid any overloading of the chromatographic columns even after a high number of sample injections. So far the method demonstrated robust performances regardless of the salt amount or any other interfering component concentration in the specimen. No deterioration in column efficiency was observed after the analysis of more than 5300 DBS samples. To ascertain the sensitivity and the specificity of the method, we measured the activity of five enzymes on DBS samples from 300 consenting healthy volunteers (spanning different ages) and from 5000 newborns in order to establish reference values. For each enzyme assay imprecision was calculated using 5 punches from 1 healthy adult. The assay has been repeated for 12 days. The intraday imprecision CVs were 2.89%, 6.65%, 3.79%, 8.27%, and 1.31% for the GAA, ABG, GLA, ASM, and GALC assay, respectively. The interdays imprecision CVs were 9.77%, 6.87%, 5.56%, 15.74%, and 5.44%, respectively. The limits of detection (LOD, signal-to-noise (S/N) ratio of blank >3) of GAA, ABG, GLA, ASM, and GALC assay were 0.08, 0.29, 0.27, 0.04, and 0.06 µmol/ h/L in whole blood, respectively. The LODs of each assay were at least 30% lower than the maximum observed disease activity. The stability of enzyme activity from one healthy adult on DBS was measured in triplicates during a 180-day period at room temperature (+24 °C), +4, and -20 °C. For all five enzymes, the activity measured at day 0 (26 h after the drawing of the blood, considering 2 h for the drying of DBS and 24 h for the enzymatic reaction) has been considered 100%. The DBSs were stored in aluminum bags with desiccant packs. Results suggested that lysosomal enzymes are stable at 4 °C, and no loss activity was noted except for GLA (minus 29.5% if compared to day 0 levels) in agreement with published data47 (Figures S-2, S-3, and S-4). For the screening purpose, in our center, the median storage time between the drawing of the blood sample and the analysis was 32 h (minimum 20 h, maximum 58 h). The activities of GAA, ABG, GLA, ASM, and GALC enzymes in healthy newborn (48-72 h of life) and adult noncarriers, heterozygous carriers, and affected patients are reported in Table 2. Figure 4 shows the extracted ion chromatograms of a blank, a healthy control, and an affected patient for all five enzymes. It demonstrates the unambiguous distinguish of the activity of a healthy control from an affected patient. Since the assay is pursuing the detection of a lack of the enzymatic activity, a “positive” sample is qualified by the absence of any reaction product which means that its tracing resembles the blank one as generated by a mixture of all the reagents but without any enzyme. In order to avoid that an assay failed for any experimental reason

Figure 4. DBS samples analyzed by online trapping-and-cleanup/liquid chromatography coupled to mass spectrometry detection in multiple reaction monitoring (MRM) mode. The panels are organized in three columns, ordered from left to right: the typical signals from blank samples, from healthy controls, and from affected patients are shown. Each row represents a specific LSD. The chromatograms show excellent baseline resolution and no interfering peak. Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

6119

Figure 5. Extracted ion chromatograms of GLA enzyme activity in a healthy control, heterozygous carrier and affected patient.

(the enzymatic reaction did not take place because the adverse conditions) is misinterpreted as a “positive” sample (the enzymatic reaction did not take place because the targeted enzyme was missing in that patient), it is mandatory that each batch includes some control samples (e.g., samples coming from healthy indi6120

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

viduals where an enzymatic activity is expected) in order to dissipate any doubt on those samples not showing any enzymatic activity. Figure 5 shows the extracted ion chromatograms of GLA enzyme activity in a healthy control, in a heterozygous carrier,

and in a Fabry disease affected patient. In the latter heterozygous carrier the activities were lower than in control subjects but still well distinguished from the affected patient ones. The high activity level of heterozygous carriers was between 27.2% (GLA) and 73.9% (GAA) the mean activity in healthy adults, but the range of activities partially overlapped. Lack of GALC and ASM heterozygous carriers DBS did not allow related data. For GAA and ASM, the median enzyme activity in healthy adults was lower than in healthy newborns, but the higher end of the activity range is quite similar except for GALC. The lower end of the activity range is similar except for GALC and GLA. In both cases, activities were higher (5 times for GLA; 2 times for GALC) than in healthy newborns. The activities of all five enzymes in all samples were measured from a single 3.2 mm DBS per enzymatic reaction. We tried to use the universal extraction solution as proposed by Li et al.40 for measuring the five activities from one single DBS. Our results were absolutely not good and therefore able to confirm the published data (data not shown). A novel approach in determining GAA, GLA, GBA, and ASM activities from one extracted DBS and GALC from another DBS has been recently published.46 Online purification based on trapping on column and following chromatography step coupled to tandem mass spectrometry is a relatively new approach used to date, principally for drug assays. To the best of our knowledge, it is the first time it is implemented in the LSDs analysis. There are several factors required for making an analytical protocol suitable for large-scale screening studies. Those are not limited just to the short measurement time, but they concern as well the sample manipulation complexity and the sustainable running costs. The hereby-presented protocol follows the enzymatic reaction steps as described in ref 46 but skips any further sample treatment before the analytical measurement. After incubation, the resulting mixture is directly injected in the mass spectrometer without any preventive extraction and/or purification of the targeted analytes. The cleaning-up of the injected mixture, containing significant amounts of buffers as demanded for the enzymes activation, is performed through a fast online trapping-and-cleanup/liquid chromatography preceding the mass-spectrometer measurement. It implements an online solid phase extraction (SPE) in a fast and cheap way. The steps chained as described in the Experimental

Section enable the characterizing within one single measurement of the panel of five enzymes pertaining to the five lysosomal disorders. The aim of the present study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation step and with reduced running costs for the characterization of the five enzymes implicated in some lysosomal disorders. The entire assay involves a 2-day process. On the first day, the samples are punched and the enzymatic reaction is started. The time required for punching a batch of cards varies from lab to lab depending upon the device each lab uses. It is conceivable that it takes 20 min to have the plate ready for overnight incubation after the reaction cocktail addition. On the second day, the bundling of the 5 reaction mixtures and the centrifugation require 15 min before having all the samples ready for the mass spectrometry measurement. The cost for reagents, disposables, instrumentation, and personnel has been roughly estimated in 5-6 Euro per test (40000 test/year). Indeed, the cost per sample decreases to less than 2 Euro (including the recruitment of a new medical technologist) for the 5 assays in newborn screening laboratories where tandem mass spectrometry is already used for other tests. All enzyme activities from this new method showed an unambiguous difference between DBS from healthy adults and newborns and LDS affected patients. CONCLUSIONS The methodology hereby described enables the monitoring and the quantifying of 5 enzymes activity without any purification steps and in one single ‘4-min’ analytical run. The achievable repeatability and robustness of the methodology make it a prime candidate for implementation in routine clinical screening and quantification environments. SUPPORTING INFORMATION AVAILABLE Figures S-1-S-4. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 9, 2009. Accepted June 9, 2009. AC900504S

Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

6121