Page 46 - Clinical-Medical Solution for Clinical Chemistry

LAAN-A-LM-E110

Application Liquid Chromatography Mass Spectrometry

News Measurement of Enzymatic Activities in Dried

Blood Spots with On-Line Solid Phase
No.C139 Extraction-LC/MS/MS System

Lysosomes are a type of intracellular organelle that uses a the ODS column and both are flowed by 300 μL/min of
variety of hydrolytic enzymes to digest waste matter. To organic solvent (solvent B). With the switching-back of
measure the enzymatic activity of lysosomes, methods the valve, occurring at 3 min, the Perfusion column is
using artificial fluorescent dyes and tandem mass re-equilibrated with a solvent A and delivered by pump
spectrometry are used. Of these methods, tandem mass A at 1.2 mL/min for 2.2 min. Using this system
spectrometry offers the advantage of being able to eliminates the need for desalting and purification
measure multiple enzymatic activities at the same time. processes.
In this example, a protocol developed at the Meyer DBS
Children's Hospital, Mass Spectrometry, Clinical
Chemistry and Pharmacology Laboratory (Florence, Italy) 30 μL Reaction Solution
was used to measure the enzymatic activity in dried Stand for 22 hrs, 37 ˚C (humid chamber)
blood spots (DBS) using an online solid phase extraction 150 μL 0.1 %HCOOH - CH3OH
(SPE) - liquid chromatograph - tandem mass spectrometer Transfer solution to the new plate
(LCMS-8050) system. Because using this system results in
samples being cleaned up during SPE, samples can be 150 μL 0.1 %HCOOH - CH3OH
inserted directly for measurement after enzymatic LC-MS/MS Analysis (2 μL Inject)
reaction, without any pretreatment processes.
Fig. 1 Preparation Process Flowchart
Q Sample Preparation and Analytical Conditions
GAA-P : C28H39N3O5 IDUA-P : C20H26N2O6
Three enzymes were targeted, alpha-iduronidase GAA-IS : C28H34N3O5D5 O O
(IDUA), acid alpha-glucosidase (GAA), and alpha- CH3 HO CH3
O O NH NH O
galactosidase A (GLA). DBS was used as sample. NH CH3 N NH O O H3C CH3
3.2 mm diameter disks were punched from the DBSs HO O O IDUA-IS : C19H24N2O6
and placed in a 96-well plate. Then a reaction solution GLA-P : C27H37N3O5 HO O O
containing respective enzyme substrates and internal GLA-IS : C27H32N3O5D5 NH NH O CH3
CH3 CH3
standard substance (Genzyme) was added to each well O O O O H3C
CH3 N NH
NH
and incubated for 22 hours at 37 ˚C. A flowchart of the
HO O O
preparation process is shown in Fig. 1.
Samples were analyzed using online SPE-LC and LCMS- Fig. 2 Structures of Enzymatic Reaction Products and
8050 system. Respective reaction products were Internal Standards
measured as the target compounds based on multiple Table 1 MRM Transitions
reaction monitoring (MRM) using an internal standard
substance. The structures of the target enzymatic Compounds Polarity Precursor ion Product ion
m/z
m/z
reaction products and the internal standards are shown
in Fig. 2. MRM transitions are listed in Table 1 and the IDUA-P + 391.2 291.2
LC and MS conditions in Table 2. IDUA-IS + 377.3 277.2
GAA-P + 498.4 398.3
Q Online Solid Phase Extraction-Tandem Mass GAA-IS + 503.4 403.3
Spectrometer System GLA-P + 484.3 384.3
The online SPE-LC/MS/MS system configuration is GLA-IS + 489.3 389.3
shown in Fig. 3. When the enzymatic reaction was •Note: P: Product, IS: Internal Standard
complete, the sample was injected directly and
measured. The trapping and cleanup procedure was
centered on a Perfusion column POROSš R1 and SPE column SPE column
separation chromatography was performed through a Autosampler Autosampler
Shim-pack XR-ODS. The two operations are articulated Analytical column LC/MS Analytical column LC/MS
through the following steps. Upon the injection, the
A B A B
sample is cleaned through the Perfusion column with Solvent delivery unit waist Solvent delivery unit waist
CTO-RVL CTO-RVL
an aqueous solution (solvent A) and delivered by pump Trapping and Cleanup Chromatographic Separation
A at 1.2 mL/min for 1 min. With the activation of the
valve, the Perfusion column is connected in line with Fig. 3 MRM Chromatograms of Each Target Compound
Table 2 Analytical Conditions
<LC> <MS>
Analytical Column : Shim-pack XR-ODS (75 mm L × 2.0 mm I.D., 2.2 μm) Instrument : LCMS-8050
Trapping Column : POROSš R1 (30 mm L × 2.1 mm I.D., 20 μm) Ionization Mode : ESI (+)
Solvent A : 0.05 % HCOOH-5 mM HCOONH 4 -H 2 O Interface Temperature : 100 ˚C
Solvent B : 0.1 % HCOOH-CH 3 OH DL Temperature : 100 ˚C
Ratio : 50 %B Heat Block Temperature : 100 ˚C
Flowrate : 0.6 mL/min Nebulizing Gas Flow : 3 L/min.
Oven Temperature : 30 ˚C Heating gas Flow : 5 L/min.
Injection Volume : 2 μL Drying Gas Flow : 15 L/min.
Analysis Time : 5.5 min