Deconjugation
of Steroid conjugates in urine
Lenny Lin
2022-09-14
+ H2O
Enzyme
Enzymatic Hydrolysis Mechanism
+ MeOH
Conc. H2SO4
Methanolysis Mechanism
+ MeOH
Solvolysis Mechanism
Enzymatic
hydrolysis SPE Dryness Derivatization GC-MS/MS
GC-MS/MS
LC-MS/MS
Dilution LC-MS/MS
96-well SPE plate 96-well SPE plate
THE SCOPES OF THIS PROJECT
Shortage of enzyme supply due to COVID
pandemic.
Some steroid conjugates were not cleaved
completely with enzyme.
Investigation of other hydrolysis methods
Labor-intensive SPE
A 96-well SPE plate
Automated SPE
THE ISSUES
Project plan
Enzymatic
Hydrolysis 1st SPE Dryness Solvolysis 2nd SPE Dryness LC-MS/MS
Break-down and Workflow
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
Synergy
Scientist team: * Planning
[x 4 scientists] * Expertise in separation science.
* Expertise in LC-MS/MS
Technologist team: * Experiments
[x 3 technologists]
Vendors: * Expertise in SPE application
* Expertise in Enzyme
Lenny: * Design of experiments
* Data analysis
* Reporting
* SPE programming
01. Enzymatic hydrolysis
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
Experimental scheme
Enzymatic
hydrolysis SPE Dryness Derivatization GC-MS/MS Modeling with
R
1. Optimized incubation time and dose of enzyme.
2. Some steroid conjugates were cleaved to free steroids completely, others were not.
The results
02. Solid phase extraction (SPE)
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
Cause and effect diagram
SPE RECOVERY
SPE SORBENT
SORBENT TYPE
SPE CAPACITY
WASHING SOLVENTELUTING SOLVENT
SPE PROCESS
PROGRAMMING
PROFILE
SPE Variables
4 Types
SPE
Sorbents
2 Types
with
pH-adjustment
Washing
Solvents
2 Types
Eluting
Solvents
Sampling
Sampling Sampling Sampling Sampling
Experimental scheme
Replicate I
SPE
Sorbents
Washing
Solvent
Eluting
Solvent
I I I
III II
I I I
III II
II …
III …
IV …
x 16 SPE cartridge
Experimental scheme
Replicate II
SPE
Sorbents
Washing
Solvent
Eluting
Solvent
I I I
III II
I I I
III II
II …
III …
IV …
x 16 SPE cartridge
Replicate III
SPE
Sorbents
Washing
Solvent
Eluting
Solvent
I I I
III II
I I I
III II
II …
III …
IV …
x 16 SPE cartridge
Replicate IV
SPE
Sorbents
Washing
Solvent
Eluting
Solvent
I I I
III II
I I I
III II
II …
III …
IV …
x 16 SPE cartridge
Data interpretation and reporting
Design of
Experiments
•Repeatability
•Randomization
•Blocking
Data
processing
•VBA
PivotTable
Two-way
ANOVA
1. Fine-tuned SPE automation program.
2. Recovery > 90%.
3. Selected one SPE sorbent, one washing solvent, and one eluting solvent.
The results
03. Combined Hydrolysis process
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
Process OF ENZYMATIC HYDROLYSIS AND SOLVOLYSIS
Enzymatic
Hydrolysis 1st SPE Dryness Solvolysis 2nd SPE Dryness LC-MS/MS
Experimental scheme
Dup. samples
Dup. controls
Dup. samples
Dup. controls
Dup. samples
Dup. controls
Dup. samples
Dup. controls
Dup. samples
Dup. controls
Enzymatic
Hydrolysis 1st SPE Dryness Solvolysis 2nd SPE Dryness LC-MS/MS
Data interpretation and reporting
PivotTable
Data
Visualization
1. A promising process. More yield of free steroids than that of enzymatic hydrolysis
alone.
2. Too many steps.
The results
04. methanolysis
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
+ MeOH
Conc. H2SO4
Methanolysis Process
Methanolysis Mechanism
Methanolysis SPE Dryness LC-MS/MS
Data interpretation and reporting
Data
processing
•VBA
PivotTable
•Data
Visualization
Although steroid conjugates were cleaved with methanolysis, the yields of free
steroids were not increased.
The result
outcome
01. Enzymatic Hydrolysis Optimization
02. Automated SPE fine-tuning
03. Promising Enzymatic Hydrolysis + Solvolysis
04. Adventure to Methanolysis
Outcome
1. Better understanding of deconjugation - combination of enzymatic hydrolysis and
solvolysis.
•The combination of enzymatic hydrolysis and solvolysis produced more free
steroids than that enzymatic hydrolysis did alone.
•Although methanolysis cleaved more steroid conjugates, the yields of free
steroids were not increased.
2. Sample extraction can be automated.
The outcome of
this project
Wrap-up
Synergy
•Plan
•Brain-storming
•Teamwork
Project
•Break-down
•Design of
Experiments
•Take risk for gains
•Adjustments
Data
Processing
•VBA
•PivotTable
•R
Data
Interpretation
•PivotTable
•Visualization
•Modeling
•ANOVA
1) Separation science: sample extraction (SPE, SLE, LLE), liquid chromatography
2) Statistical analysis, design of Experiments, ANOVA, metabolomics, …
3) Informatics: data processing, QC dashboard, electronic troubleshooting logbook
Something
even better to come …
Q & A
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