O-CON1718E Comparison of FIA and on-column LC-M/M method to measure simultaneously ABG, AM, GAA, GALC, GLA and IDUA activities AM 2017 Th 091 Misa Tanaka, Jun Watanabe, Toshikazu Minohata, Junichi Masuda, Eishi Imoto himadzu Corporation. 1, Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604-8511, JAAN
Introduction Lysosome is a membrane-enclosed organelle and contains hydrolytic enzymes. They can digest virtually all kinds of biomolecules. ome techniques such as fluorometric and spectrophotometric assays have been developed to measure these enzyme activities. Multiplex flow injection analysis using tandem mass spectrometry (FIA-M/M) method has been developed that simultaneously measures the activities of the enzymes, alphaglucocerebrosidase (ABG), sphingomyelinase (AM), alpha-glucosidase (GAA), galactocerebrosidase (GALC), alpha-galactosidase A (GLA) and alpha-iduronidase (IDUA). The use of mass spectrometric techniques has exhibited advantages over the other techniques in the simultaneous quantification of several targets. However, FIA-M/M method can exhibit background noise levels that lead to inaccurate data interpretation. Here we compared and evaluated FIA LC-M/M method and on-column LC-M/M method to simultaneously measure these enzyme activities. Methods and Materials 6-plex NeoLD reagent (erkinelmer Inc.) was used for this study. This kit contains assay buffer, substrates for 6 enzymes and internal standards. 3.2 mm diameter disks were punched from the DBs and placed in a 96-well plate. Assays were run according to the procedure of the kit with overnight incubation (16-20 hours). ABG products AM ABG ABG substrate (ABG-) AM substrate (AM-) GAA GALC GAA substrate (GAA-) GALC substrate (AM-) GLA IDUA GLA substrate (GAA-) IDUA substrate (GAA-) Figure 1 6 substrates for 6 enzymes; I are deuterated forms of enzyme products 2
Figure 2 6-plex NeoLD reagent workflow Flow injection analysis (FIA) and on-column analysis were achieved using LC-M/M system; UHLC was coupled to triple quadrupole mass spectrometer (Nexera with LCM-8050, himadzu Corporation, Kyoto, Japan). The mobile phase consisted of (A) water + 0.1 % formic acid and (B) Acetonitrile + 0.1 % formic acid. LC-M/M with electrospray ionization was operated in multiple-reaction-monitoring (MRM) mode. Analytical Conditions HLC conditions (Nexera M system) Flow Injection Analysis Mobile phase : A: water + 0.1 % formic acid B: Acetonitrile + 0.1 % formic acid Isocratic Flow : B 84 % Flow rate : 0.3 ml/min Injection volume : 10 μl On Column Analysis Mobile phase : A: water + 0.1 % formic acid B: Acetonitrile + 0.1 % formic acid Flow rate : 0.2 ml/min Injection volume : 1 μl Column : GL ciences MonoTower C18 100 mm (3.0 um x 50 mm x 2 pieces) Time program : 0 min. B 75 % > 2 min. B 75% > 3 min. B 90 % > 6 min. B 100 % 10 min. B 100 % > 10.1 min. B 75 % M conditions (LCM-8050) Ionization EI, ositive MRM mode 3
Table 3 MRM Transition Compound recursor ion ion Compound recursor ion ion ABG-I 391.4 271.3 GALC-I 417.4 ABG-* 384.3 GALC- 412.4 ABG- 546.4 264.2 GALC- 574.5 264.25 AM-I 405.4 GLA-I 489.3 389.3 AM- 398.4 GLA- 489.3 384.2 AM- 563.4 184.1 GLA- 646.4 546.25 GAA-I 503.3 403.3 IDUA-I 431.3 322.2 GAA- 498.3 398.2 IDUA- 426.2 317.2 GAA- 660.4 560.25 IDUA- 602.3 317.2 * means product Result In Japan, when newborn screening of lysosomal storage disease using LC-M/M is implemented, it is necessary to perform validation of the analysis method. The FIA method has been developed as the analysis method of 6-plex using tandem mass spectrometer. We evaluated both analysis method of FIA and on-column. 4
Results of FIA method sample 1 (Blank) sample 2 (Normal) 1:ABG_I 391.40>271.30(+) CE: -2 1:ABG_I 391.40>271.30(+) CE: -2 ABG 2:ABG_ 384.30>0(+) CE: -2 2:ABG_ 384.30>0(+) CE: -2 3:AM_I 405.40>0(+) CE: -3 3:AM_I 405.40>0(+) CE: -3 AM (x1,000) 7.5 4:AM_ 398.40>0(+) CE: -3 4:AM_ 398.40>0(+) CE: -3 GAA 11:GAA_I 503.30>403.30(+) CE: -1 1.5 (x1,000) 12:GAA_ 498.30>398.20(+) CE: -1 11:GAA_I 503.30>403.30(+) CE: -1 12:GAA_ 498.30>398.20(+) CE: -1 0.1 0.2 0.3 0.4 min 0.1 0.2 0.3 0.4 min 5:GALC_I 417.40>0(+) CE: -2 3.0 5:GALC_I 417.40>0(+) CE: -2 3.0 GALC 6:GALC_ 412.40>0(+) CE: -2 3.0 6:GALC_ 412.40>0(+) CE: -2 0.1 0.2 0.3 0.4 min 0.1 0.2 0.3 0.4 min GLA 1.5 9:GLA_I 489.30>389.30(+) CE: -1 10:GLA_ 484.30>384.20(+) CE: -1 9:GLA_I 489.30>389.30(+) CE: -1 1.5 10:GLA_ 484.30>384.20(+) CE: -1 7:IDUA_I 431.30>322.20(+) CE: -17.0 7:IDUA_I 431.30>322.20(+) CE: -17.0 IDUA (x1,000) 8:IDUA_ 426.20>317.20(+) CE: -17.0 8:IDUA_ 426.20>317.20(+) CE: -17.0 Figure 3 Results of FIA method; MRM chromatograms of each target compound ample #1 (Blank): sample containing no blood; ample #2 (Normal): ample containing enzymes with all activities All peaks were clearly detected for enzyme decomposition products in ample #1 and repeatability of measurement was good (data not shown). These results were indicated to be able to distinguish sample #1 and sample #2 in all enzyme activity data. However, all data in sample #1, especially ABG, GALC, GLA, showed significant peaks in their product chromatograms. 5
Result of on-column method Then, an analysis using on-column separation was used to confirm the reason of the peak in product MRMs of the blank sample. In this analysis, 3 MRMs,, and ubstrate, were monitored. The result are shown in Figure 4. ABG 1:ABG_I 391.40>271.30(+) CE: -2 AM 1.5 4:AM_I 405.40>0(+) CE: -3 I 2:ABG_ 384.30>0(+) CE: -2 5:AM_ 398.40>0(+) CE: -3 3:ABG_ 546.40>264.20(+) CE: -29.0 (x10,000,000) 6:AM_ 563.40>184.10(+) CE: -2 ubstrate 7.5 1 min GAA 16:GAA_I 503.30>403.30(+) CE: -1 I GALC 7:GALC_I 417.40>0(+) CE: -2 I 17:GAA_ 498.30>398.20(+) CE: -1 8:GALC_ 412.40>0(+) CE: -2 3.0 18:GAA_ 660.40>560.25(+) CE: -2 (x10,000,000) 9:GALC_ 574.50>264.25(+) CE: -3 7.5 1 min GLA 13:GLA_I 489.30>389.30(+) CE: -1 I IDUA 10:IDUA_I 431.30>322.20(+) CE: -17.0 I 14:GLA_ 484.30>384.20(+) CE: -1 3.0 11:IDUA_ 426.20>317.20(+) CE: -17.0 15:GLA_ 646.40>546.25(+) CE: -2 3.0 12:IDUA_ 602.30>317.20(+) CE: -2 7.5 1 min Figure 4 Results of on-column method; MRM chromatograms of each target compound ample #2 (Normal) was analyzed. In MRM chromatograms of ABG, GAA, GALC and GLA products, interfering peaks at the same retention time as those of substrates were found (red arrow in Figure 4). This peaks were probably produced by in-source fragmentation of the substrates. ince these peaks could not be chromatographically separated from enzymatic reaction products in FIA method, they were creating a peak detected even without enzyme activity. In the case of AM, the interfering peak observed (blue arrow) is probably due to in-source fragmentation of GALC I or GALC product. 6
Conclusions Measurement of six enzymatic activities in DB (6-plex assay) by FIA and on column method was conducted by LCM-8050 system. FIA method had higher throughput than with column separation, but it is expected to be less precise and accurate. Acknowledgement oster presenters thank Dr. Osamu Ohara and Dr. Kazuhiro ato (Kazusa DNA Research Institute) for measurements and data analysis. Disclaimer: himadzu LCM-8050 and Nexera M system are intended for Research Use Only (RUO). Not for use in diagnostic procedures. First Edition: June, 2017 himadzu Corporation, 2017