PAL System in Automated Sample

Preparation
For GC and LC

1 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Chiew Mei Chong
CCHONG@ctc.ch
+65 8711 9007

2 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Automation systems
An open, modular based concept

• Multiple techniques in one system

• Fully customizable
• Modular based
• Time saving with overlapping and prep ahead mode

3 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Instrument Lineup
PAL LSI, RSI and RTC
Liquid injection Multiple injection Fully automated sample prep. Ultimate Productivity
techniques Robotic Tool Change Dual Head Rail
Liquid injection, static
headspace, ITEX Dynamic
Headspace, MHE, SPME
Sample prep capability

PAL LSI PAL RSI PAL RTC PAL DHR

Liquid Injector with high High capacity, high Robotic Tool Change allows full To maximize productivity the PAL
capacity: up to 800 x 2ml productivity for LCMS & access to the modular toolbox for DHR performs tasks with two heads
vials GCMS frontend workflow automation in parallel

Injection mode flexibility

4 PAL is a registered trade mark of CTC Analytics AG, Switzerland
 Solutions we provide

Pharma/Life Science Food & Food Safety

USP-467 Flavor and Fragrance
Nitrosamines Pesticides
Leachables & Extractables Authenticity
Clinicals FAMEs
Proteomics
Metabolomics

Petrol chemicals
Off-Odor in drinking water Dye
POPs in soil
SVOCs in water
Purge and Trap

Environmental Chemicals

5 PAL is a registered trade mark of CTC Analytics AG, Switzerland

And more…
Principle of Solid Phase
Extraction and QuEChERS

6 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Principle of SPE
1) Removal/Trapping Strategy

Source: Solid-Phase Extraction - Chemistry LibreTexts

7 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Principle of SPE
2) Bind and Elute Strategy

Source: Solid-Phase Extraction - Chemistry LibreTexts

8 PAL is a registered trade mark of CTC Analytics AG, Switzerland

What is QuEChERS?
Sample preparation for pesticides, and more
• The acronym stands for
Quick Easy Cheap Efficient Rugged and Safe
• First published in 2003
by M. Anastassiades, S.J. Lehotay, and team1
• Originated from and is mainly used for pesticides
analysis
But: since 2003:
>1800 non-pesticide QuEChERS applications published e.g.
mycotoxins, antibiotics, vet drugs …
• Uses a 2-steps procedure
1. Extraction of a small sample portion
2. Clean-up with sorbent materials
Then GC-MS or LC-MS analysis

[1] Anastassiades, M.; Lehotay, S. J.; Štajnbaher, D.; Schenck, F. J. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-
phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412−431.

9 PAL is a registered trade mark of CTC Analytics AG, Switzerland

What is QuEChERS?
Which test portion size is acceptable?
• With QuEChERS sample sizes decreased significantly.
• A typical test portion size today is 10 g or less.
Bulk sample homogenization is mandatory
• Comminution must ensure that the test portion is
representative of the original bulk sample.
Liquid N2 comminution Source: fritsch-international.com
Cryogenic milling is recommended using dry ice or liqu. N2

• Lehotay, Michlig et al.2 showed recently

that even 1 g test portions did not generate measurable bias on precision.
Using a 2 g test portion for QuEChERS extraction
is a good practical choice for routine analysis without compromising accuracy
• Conclusion: Small sample sizes allow automated workflows and high sample throughput

[2] Lehotay, S. J., Michlig, N. and Lightfield, A. R. (2019) ‘Assessment of Test Portion Sizes after Sample Comminution with Liquid Nitrogen in the High-Throughput Analysis
of Pesticide’, Journal of Agricultural and Food Chemistry, 68(5), pp. 1468–1479. doi: 10.1021/acs.jafc.9b07685.

10 PAL is a registered trade mark of CTC Analytics AG, Switzerland

What is QuEChERS?
Step 1: Extraction and partitioning
• Principle Extraction
Liquid/liquid portioning using acetonitrile (MeCN)
High salt concentrations lead to 2-phases,
also modify the extraction selectivity vs. co-extractives.
After shaking and centrifugation
the MeCN phase taken for clean-up step
• Harmonization and Regulations
Lehotay et al. modified the method using acetate buffer3
Anastassiades et al. chose a citrate buffer4
• Both methods were accepted from independent standard organizations
The acetate-buffered version became AOAC Official Method 2007.01
The citrate-buffered version was named the CEN Standard Method EN 15662

[3] S.J. Lehotay, K. Maštovská, A.R. Lightfield, J. AOAC Int. 88 (2005), 615–629 & 60A.
[4] M. Anastassiades, E. Scherbaum, B. Tas¸ delen, D. Stajnbaher, in: H. Ohkawa, H. Miyagawa, P.W. Lee (Eds.), Crop Protection, Public Health, Environmental Safety,
Wiley-VCH, Weinheim, Germany, 2007, p. 439.

11 PAL is a registered trade mark of CTC Analytics AG, Switzerland

What is QuEChERS?
Step 2: Clean-up of the extract – The most suitable sorbent mix?
• dSPE is a manual step (= dispersive SPE)
Mixing the extract with various sorbent materials
Matrix specific mixes used

• Challenges and Limitations

Low recovery for fatty samples
Matrix effects with complicated matrices
[5]
e.g. spices, tea, and oils, or a high chlorophyl contents

• “Modified QuEChERS“ search in Google Scholar:

5700 publications since 2003 deal with modified clean-up of the QuEChERS extract6
Customized dSPE clean-ups are used matrix dependent with mixes of many different sorbent
materials:
Like PSA, C18, GCB, CarbonX®, MgSO4, Clorofiltr®, Z-Sep®, Z-Sep+®, ZrO2, EMR®, …
or freezing the extract
[5] From: Lehotay, S.J. 2013. Revisiting the Advantages of the QuEChERS Approach to Sample Preparation. Separation Science Webinar.
[6] Google Scholar search result: “modified QuEChERS”, 2003 to today.
12 PAL is a registered trade mark of CTC Analytics AG, Switzerland
QuEChERS Methods – AOAC vs. CEN

S T AN DA RDIZED
E c l e a n-up NOT
dS P
r e c e i pe s in use
ma n y

[5] From: Lehotay, S.J. 2013. Revisiting the Advantages of the QuEChERS Approach to Sample Preparation. Separation Science Webinar.

13 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Automated µSPE Extract Clean-up
using the PAL System

14 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Why use µSPE?
Compare to the classical cartridge SPE
Works like
Classical SPE µSPE a
mini LC co
lumn !
• Limited selectivity • High selectivity
High sample and solvent volumes compares to LC separation
Requires evaporation with N2 Sharp elution peak profile, no concentration
End volume >>100 µL in vial Final volume < 100 µL (or online)
• Vacuum operated • Positive pressure w liquid syringe
• Drying before elution • No drying step
• Manual operation • Walk away automation
The h
Time consuming Fast with < 10 min a pate
Low sample throughput High productivity conta
filter a
Batch processing Prep on chromatographic timescale
• No QA/QC • Traceable
As of manual operation Processing well documented
21CFR11 compatible
15 PAL is a registered trade mark of CTC Analytics AG, Switzerland
 Qu E Ch E R S
Quick Easy Cheap Effective Rugged Safe
Crude Extract Preparation Manual dSPE clean-up Automate

Centrifu Centrifu
Extract Salt out Dispersi Shake
ge ge Injection
sample sample sample ve SPE sample sample
Add MgSO4 MgSO4 + Variations
Add Acetonitrile Phase separation Phase separation To LC or GC
+ Na-based salt PSA / C18 / GCB

16 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Automated
Qu E Ch E R S
Quick Easy Cheap Effective Rugged Safe
Crude Extract Preparation Automate clean-up Automate

Centrifu Centrifu
Extract Salt out Dispersi Shake
ge ge Injection
sample sample sample ve SPE sample sample
Add MgSO4 MgSO4 + Variations
Add Acetonitrile Phase separation Phase separation To LC or GC
+ Na-based salt PSA / C18 / GCB

17 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Automated
Qu E Ch E R S
Quick Easy Cheap Effective Rugged Safe
Crude Extract Preparation Automate clean-up

Centrifu
Extract Salt out
ge µSPE Injection
sample sample sample
Add MgSO4 MgSO4 + PSA
Add Acetonitrile Phase separation To LC or GC
+ Na-based salt + C18 + CarbonX

18 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Standard QuEChERS Protocol*
Add per 1 g of sample:
Typ. 10 g of sample 1 mL acetonitrile with 1% HOAc
ISTD (e.g. triphenylphosphate) Extraction
Shake 1 min
0.4 g anh. magnesium sulphate,
0.1 g sodium chloride
Salt out For CEN 15662:
0.1 g trisodium citrate dihydrate
Shake 1 min 0.05 g disodium hydrogencitrate
For AOAC 2007.01:
0.1 g anh. sodium acetate
Centrifuge

Dispersive SPE Dispersive

1 mL aliquot for clean-up,
add PSA, C18, GCB, MgSO4, ….
SPE
Shake 1 min Clean-up
Centrifuge

GC-MS, LC-MS Direct LCMS analysis of acidic pesticides

(phenoxyalkanoic group)

* QuEChERS - Mini-Multiresidue Method for the Analysis of Pesticides, M Anastassiades

19 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Standard QuEChERS Protocol
Add per 1 g of sample:
10 g of sample 1 mL acetonitrile with 1% HOAc
ISTD (e.g. triphenylphosphate) Extraction
Shake 1 min
0.4 g anh. magnesium sulphate,
0.1 g sodium chloride
Salt out For CEN 15662:
0.1 g trisodium citrate dihydrate
Shake 1 min 0.05 g disodium hydrogencitrate
For AOAC 2007.01:
0.1 g anh. sodium acetate
Centrifuge
The heart of the ITSP system is

X
Dispersive SPE Clean-up
a patented cartridge
1 mL aliquot for clean-up,
add PSA, C18, GCB, MgSO4, ….
replaced by
containing customer-defined
filter and/or SPE media.
Shake 1 min µSPE
Centrifuge One cartridge
for
GC-MS, LC-MS Direct LCMS analysis of acidic pesticides all matrices
(phenoxyalkanoic group)

20 PAL is a registered trade mark of CTC Analytics AG, Switzerland

µSPE Extract Clean-up
Fully automated QuEChERS clean-up workflow – The PAL µSPE Kit

Sample Rack Optional ISTD and

with raw extracts APs, Cleaning
Solvents

Elution Rack
with cleaned extracts
Conditioning and
Elution Solvents

Conditioning Rack
for 54 cartridges

21 PAL is a registered trade mark of CTC Analytics AG, Switzerland

How the PAL Runs µSPE Clean-up

22 PAL is a registered trade mark of CTC Analytics AG, Switzerland

µSPE Sample Clean-up
Workflow steps explained
1. The µSPE cartridge is activated with elution solvent (optional)
2. The QuEChERS extract is loaded into the syringe (150 to 300 µL only)
3. The syringe takes the cartridge on the needle and moves to the elution rack
4. Loading the raw extract onto the µSPE cartridge and elute the pesticides
5. Elute pesticides with elution solvent (optional)
6. The used µSPE cartridge is discarded, the syringe washed
7. The PAL takes the injection syringe
8. Take ISTD, calibrants, or analyte protectants for sandwich injection (optional)
9. Take and inject the cleaned pesticide fraction to GC-MS or LC-MS

Transfer
µSPE Sample Condition Sample to Wash µSPE Elute cleaned
Clean Up µSPE cartridge µSPE cartridge cartridge Sample to Vial

23 PAL is a registered trade mark of CTC Analytics AG, Switzerland

QuEChERS and µSPE Clean-up Workflow
Application Examples

24 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Hill Laboratories, Hamilton, NZ
Many Years Experience for > 900 Matrices and >1000 Samples/wk
• Reduced labor in sample prep
• Automated, compared to original Quechers
• Wider range of lipid content, e.g. incl. Avocados
• Wider range of matrix contaminants, e.g. dried spices
• Off-line workstations require only 1/5 of GC runtime
and can serve several GC-MS systems.

25 PAL is a registered trade mark of CTC Analytics AG, Switzerland

QuEChERS Clean-up Workflow
Automated µSPE clean-up of QuEChERS extracts for pesticide analysis7
NO clean-up

µSPE cleaned

High resolution LC-MS chromatograms for crude avocado extract

and the same extract diluted 2-fold then cleaned up by µSPE.
Crude ITSP clean- dSPE clean-up
Sample up

“The combination of automation on a PAL autosampler and use of the elution solution
developed, resulted in acceptable recoveries of 274 pesticides, with low relative
standard deviations and minimal LC-MS/MS matrix suppression”7
[7] Morris, B. D. and Schriner, R. B. , 2015. Development of an Automated Column Solid-Phase Extraction Cleanup of QuEChERS Extracts, Using a Zirconia-Based Sorbent,
for Pesticide Residue Analyses by LC-MS/MS’, J. Agric. Food Chem., 63, p. 5107−5119. doi: 10.1021/jf505539e.

26 PAL is a registered trade mark of CTC Analytics AG, Switzerland

U.S. Department of Agriculture
Steve Lehotay application for critical food stuff fish, meat, spices
• Also for a wide range of lipid contents, e.g. salmon
• Critical range of matrices, e.g. dried spices
• Shorter clean-up time
• Resulted in a longer GC-MS uptime for increased
sample througput
• Less regular maintenance for higher productivity

“Injector liner after 230 matrix

injections, only little dirt found”

27 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Solid Peak Integration in Critical Matrices
Acephate and pyriproxyphen as sensitive compounds
• Recoveries 90 – 110% for wide
range of matrices
e.g. apple, kiwi, carrot, orange, olive,
wheat, basil, pork, salmon
• Symmetrical peak shapes
• High selectivity in LPGC-MS/MS
• Low background interference
• Solid low-level integration
Less manual rework required
Spiked here at 10 ng/mL
• Qualifier ions meet the 10% criteria

[8] Steven J. Lehotay, Lijun Han, Yelena Sapozhnikova, Automated Mini-Column Solid-Phase Extraction Cleanup for High-Throughput Analysis of Chemical
Contaminants in Foods by Low-Pressure Gas Chromatography-Tandem Mass Spectrometry, Chromatographia, 13.06.2016, DOI 10.1007/s10337-016-3116-y.

28 PAL is a registered trade mark of CTC Analytics AG, Switzerland

QuEChERS Extraction AND Clean-up
Complete workflow from extraction to injection on a PAL System9

[9] C.M. Chong, Lim, G., Lv, J., Zhu, B., Huebschmann, H.-J. . 2021. Fully Automated QuEChERS in Analyzing Organophosphate Pesticides in Orange Juice. Poster
at the LAPRW 2021.

29 PAL is a registered trade mark of CTC Analytics AG, Switzerland

 Extraction and Clean Up Workflow
User generated workflow using the PAL Method Composer10
❶ ❶
Add 400 µL sample
into a 2 mL vial
❷
❷
Add 3 x 200 µL acetonitrile
❸
Liquid-Liquid Extraction

Orange ❹
juice
Add 250 µL sat. NaCl test
portion Orange
❸ juice
Wait for phase separation + MeCN Orange
❹ juice
Clean-Up

Use 250µL extract + MeCN

µSPE

for µSPE clean up + NaCl Inject

after
Inject

µSPE
clean-up
[10] C.M. Chong and H.-J. Huebschmann. 2021. Fully Automated QuEChERS Extraction and Cleanup
of Organophosphate Pesticides in Orange Juice. LCGC North America 39(s1) 12-16.

30 PAL is a registered trade mark of CTC Analytics AG, Switzerland

31
Methacrifos

Sulfotep
Phorate

Terbufos
Fonofos

Disulfoton

Tolclofos-methyl
Fenchlorphos (Ronnel)

Malathion
Fenthion
Parathion
Bromophos methyl
Chromatogram of Orange Juice

Bromfenvinfos-methyl
Chlorfenvinphos

Bromophos-ethyl
Tetrachlorvinphos

Bromfenvinphos
Iodofenphos + Fenamiphos
Prothiofos
Profenofos
Total Ion Chromatogram from GC-MS/MS analysis

PAL is a registered trade mark of CTC Analytics AG, Switzerland

Ethion
Chlorthiophos
Triazophos + Sulprofos
TriazophosTris(1,3-dichloroisopropyl) phosphate (IS)
Carbophenothion
Edifenphos

Piperonyl butoxide

Leptophos
----- Unspike Orange Juice
----- Orange Juice with Post Spike 100ng/mL

Coumaphos
Comparing Spiked and Blank Runs
Level 10 ng/mL in orange juice and quantitative calibration

Methacrifos Sulfotep

Spiked at 10 ng/mL

Blank

Fonofos Tolclofos-methyl

Spiked at 10 ng/mL

Blank

32 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Conclusions
Using a PAL System for QuEChERS extraction and µSPE clean-up
• Fully automated PAL workflows are available for QuEChERS extraction and clean-up
• Only one clean-up cartridge is used for all food matrices
including ‘fatty’ and difficult matrices
No wrong clean-up any more
Greatly facilitates lab logistics
• Fast extraction and clean-up during the chromatographic runs
High sample throughput with “prep ahead” mode
Online injection to GC-MS and LC-MS, optional off-line processing
• Low test portion sizes are achieving LOQs < 5 ng/g
Recoveries were in the range of 70%-115%, precision 2 to 11% RSD
In a commercial orange juice sample we detected 1.8 ng/mL malathion
• Higher sample throughput
Significantly extended the instrument uptime
Less preventive maintenance shutdown.

33 PAL is a registered trade mark of CTC Analytics AG, Switzerland

Thank you