Arjen Gerssen - RIKILT [Alleen

From HPLC to UHPLC and how to continue?
Van HPLC naar UHPLC en hoe nu verder?
Arjen Gerssen
Outline
RIKILT?
Marine biotoxins
● HPLC
● UHPLC
NanoUHPLC
microUHPLC
RIKILT – Institute of Food Safety –
Wageningen UR
RIKILT contributes to European food and feed safety and
health with high-quality, independent research and
consultancy
Results of RIKILT are more often found in the media
Marine biotoxins
Natural toxins produced by algae which can end-up in
filter feeders
Marine biotoxins
In Europe three different intoxication syndromes
● ASP (Amnesic Shellfish Poisoning)
● PSP (Paralytic Shellfish Poisoning)
● DSP (Diarrhoea Shellfish Poisoning)
Goal of this research
2.0
1.0
Conventional LC-MS/MS
Traditional: LC-MS/MS C18 and acidic chromatographic
conditions
● Problems; Overlap ESI-/+, Peak shape YTX
ESIESI+
AZA1
PTX2
OA
GYM
0
YTX
SPX1
5
10
Retention time (min)
15
Like always with multi methods
Okadaic acid
Yessotoxins
Azaspiracids
Pectenotoxins
Cyclic imines
Conventional LC-MS/MS
More polymeric based columns and alkaline conditions
Separation in ESI+ and ESISomewhat poor peak shape
AZAs
negatief
Excellent sensitivity for acid
containing compounds (OA,
YTX)
positief
AZA1
PTX2
OA
YTX
0
5
GYM
10
Retention time (min)
Gerssen et al. 2009, J Chrom. A 1216 1421-1430
SPX1
15
Finished!... Or not?
Problem with reference methods in legislation
● Improvements are not formally allowed and
changing legislation is a tedious procedure
UHPLC instead of UPLC
Increase the throughput of the method (LC ~ 20 min)
Upgrade the conventional method towards latest
generation equipment
● Improve in speed of analysis
● Include more toxins in same procedure
Pin-F
Pin-E
SPX1
Pin-G
GYM
UHPLC method developed
Analytical column: BEH C18 100mm x 2.1mm, 1.7µm
Flow of 0.6 mL/min
Time
A (%)
B (%)
Analytical column temp 40C
0
70
30
0.5
70
30
3.5
10
90
4
10
90
4.1
70
30
5
70
30
Detection with Xevo TQ-S
ESI+
and
ESI-
27 toxins (50 transitions)
well time 3msec
New faster method
5 min per analysis
Extreme valuable additional information i.e. EFSA
But more data analysis
Application of improved method
Rapid method allowed us to do additional research on
these samples
Analysis of 100 samples
● HPLC = 33h
● UPLC = 8h
Conclusion for HPLC and UHPLC
Both conventional LC and UHPLC can be used for routine
monitoring
With UHPLC method possible to incorporate more toxins
UHPLC is incorporated in the Dutch routine program
Needs
Cheaper solutions saving valuable standards and
solutions
REDUCTION OF COSTS
Moving to µLC and nLC devices
Cons
● Sample-size is usually not limited
● Nano equipment does not comply with the
requirements we want to have in small molecule
world
Pros
● Increase costs of high-grade solvents
● Less contamination of source etc.
● Sample size limited i.e. forensic applications or
bioactivity-driven hazard identification
Our definition (UPLC - µUPLC – nUPLC)
UPLC
2.1mm
µ
n
0.15mm
0.085mm
1-10µl/min
<1µl/min
UPLC
1mm
High flow
>600µl/min
Conventional flow
100-600µl/min
Low flow
10-100µl/min
Gerssen et al. LC-GC North America, Special Issue 2014, 32-5, 18-23
Start with nanoUHPLC (2009)
Application to > 100 veterinary drugs in porcine,
succesfull (column 75µm i.d. flow 600nl/min)
● Improved sensitivity (180 fold increase peak area)
● Drift sensitivity low
● Green chemistry
But
● Analysis time doubled
(16 to 33 min)
● And..
Drawback of the NanoUPLC
Manual connections introduces void volume
Difficult to rapidly change columns
Plug-and- play µUPLC (Ikey)
Ceramic device
50mm x 150µm, 1.7µm particles (BEH C18)
Max pressure 10000 psi
Integrated ESI emitter
No manual connections when changing a column!!
UHPLC compared to µUHPLC (steroids)
UPLC (urine spiked at 2ppb)
8.0× 10
5µl inject ion
6.0× 10 5
Intensity
100
80
4.0× 10
β-T
α-T
tr 7.16 min
Area: 35290
5
t r 7.95 min
Area: 27557
2.0× 10 5
60
0
6.5
7.0
20
8.0
8.5
T rizaic (urine spiked at 2ppb)
8.0×105
0
9.8
7.5
t r (min)
40
9.9
10.0
t r (min)
10.1
6.0×105
Intensity
Relative intensity (%)
5
β-T
tr 8.44 min
Area: 78638
4.0×105
α -T
tr 9.18 min
Area: 44537
2.0×105
0
8.0
0.5µl injection
8.5
9.0
t r (min)
9.5
10.0
UHPLC compared to µUHPLC (steroids)
Asymmetry factor
UPLC sym = 1.37±0.34
µUPLC sym = 1.39±0.11
Relative intensity (%)
UPLC
UPLC
100
tr 7.16 min
80
W 1/ 2
60
40
W 10%
20
b a
0
6.8
7.0
7.2
7.4
7.6
t r (min)
Effective plate number
UPLC Neff = 48711
µUPLC Neff = 46829
Relative intensity (%)
µUPLC
Trizaic
100
tr 8.44 min
80
60
W 1/ 2
40
20
0
8.0
W 10 %
b a
8.2
8.4
8.6
t r (min)
8.8
9.0
Differences for marine biotoxins methods
Mobile
phase (µL)
Injection
volume (µL)
Analysis
time (min)
HPLC
8000
10
20
UHPLC
3000
5
5
µUHPLC
45
0.2
15
AZA3
AZA1
AZA2
Conclusions
Currently majority of the methods changed from
HPLC to UHPLC
New possibilities of µUHPLC more and more applicable
for routine use
Chemisch Weekblad (C2W) 26 September 2014
UHPLC
µUHPLC
µUHPLC
UHPLC
[email protected]

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