1. On-Line SPE with the Flexcube/6460 QQQ
Dr. Sheher Bano Mohsin Mass Spectrometry Applications June 11, 2013
This overview of on-line Solid Phase Extraction (SPE) coupled with LC/MS/MS was created by Joe Weitzel in November This presentation provides 4 applications sections with data from Peter Abrahamsson (LC/MS PS EMEA), Thomas Glauner (EMEA Food team LC/MS chemist), Paul Stephens (EMEA Environmental marketing manager), Sheher Mohsin (AFO LC/MS AE) and customers: Tarun Anumol –University of Arizona, Sarah Gledhill – South East Water (UK).
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2. Introduction – Why Online SPE? Introducing the Flexcube for Online SPE
Sensitivity of the Mass Spectrometers
Application Examples
- Water Samples
- Samples with dirty Matrices
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3. Solid Phase Extraction (SPE)
Wouldn’t it be great if you could automate SPE while reducing sample and solvent volumes?
Most environmental customers find that manual SPE is a bottleneck in the lab. It is time consuming and lab intensive! Volume of water sample required can vary from 200ml – 1,000 ml. This also requires solvent for rinsing, eluting, and creating final volumes.
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4. Automated on-line SPE
Load 500ul-1800 µl water sample on the enrichment cartridge.
Valve switch, ‘backflush‘ desorb using HPLC gradient.
Separate on the analytical column and collect MRM tranistions on the QQQ.
Recondition enrichment cartridge during analysis.
Ready for next sample after ~ 20 minutes.
Basics of the Agilent on-line SPE LC/MS.
Everything controlled by one software package –MassHunter!
Customers have found that they can get analyses per SPE cartridge depending upon the cleanliness of the water sample. SPE system can be used with LC/QQQ or LC/TOF, or LC/QTOF.
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5. High sensitivity 6460 Triple Quadrupole
Sub-femtogram, on-column sensitivity (1pg reserpine; s/n 2000:1)
Five Orders of Linearity
Fast Pos/Neg switching, 30 millisec
“Dynamic Multiple Reaction Monitoring” for MRMs scheduled by peak retention times instead of time segments
Triggered MRM for confirmation
Agilent Jet Stream
Online SPE or enrichment can be used with either the LC/QQQ , LC/TOF, or LC/QTOF.
MassHunter acquisition controls entire system.
Most online SPE users utilize the 6460 LC/QQQ as the detector. Agilent Jet Stream thermal gradient
focusing technology was developed
to signifi cantly enhance sensitivity in
ESI-MS by improving the desolvation
and spatial focusing of ions. Superheated
nitrogen sheath gas confi nes the
nebulizer spray to more effectively dry
ions and concentrate them in a thermal
confi nement zone (Figures 1-3).
Desolvation reduces noise. Full
confi nement of the spray by the superheated
nitrogen gas eliminates sample
recirculation and reduces peak tailing
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6. Agilent MassHunter Software
Instrument Control
Real-time monitoring
Method set-up
Autotune
Qualitative Analysis
Chromatographic results
Spectral results
Find compounds
Quantitative Analysis
User filters
Compound results
Calibration curve
Reporting
Easily Customizable
Based on Excel and XML technology
MassHunter Software is available for LC/QQQ, LC/TOF, LC,QTOF, GC/MSD (data analysis only), GC/QQQ, GC/QTOF, and ICP-MS systems
Entire on-line SPE LC/QQQ controlled by MassHunter SW
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7. Agilent Online SPE System
Analytical column for separation
Switching valve with TEC
MS/MS
Quaternary pump
Autosampler
Binary pump for gradient elution
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8. What’s new with Online SPE?
Flexcube with Built in Valves and a Pump - all Controlled through Mass Hunter
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9. Agilent Online SPE System
Switching valve with TEC
Analytical column for separation
Replace with Flexcube
MS/MS
Quaternary pump
Autosampler
Binary pump for gradient elution
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10. Agilent 1200 Infinity Series Online SPE solution
Valves mounted in 1290 Infinity Flexible Cube
Solvent selection valve for up to three solvents
Low level detection
3 position Solvent Selection Valve helps with conditioning and loading SPE cartriges.
Condition with up to 3 solvents in paralell condition with the other cartrige in analytical flow path
Re-useable SPE cartriges.
Flexible Advanced Valve Solutions. Valve heads easy to mount
SPE Starter set
Direct injection
High Volume injection
Flask Sampling
Multi SPE Cartrges 2 position 10 port valve in the flex cube with 2 SPE cartrige, 1.8 ml of sample,
Software: MassHunter MH B.06.00, driver release A or higher to controll all features of 1290 Infinity Flexible Cube (G4227A)
List price of 1290 Infinity Flexible Cube including 2 valve drives ca. 9 kUSD
Reciprocating single-piston pump for flows up to 4 ml/min (60 bar)
up to two Quick-Change valves, according to application

11. Online SPE with the Flex Cube
Column Compartment
Flexcube
1290 Autosampler
Autosampler
6460 QQQ
Autosampler with 900uL loop and extension seat capillary
1290
Binary Pump

12. Flex Cube and Autosampler
6 port valve
10 port valve
Single Piston Pump
Autosampler

13. Mass Hunter B 06.00 with Flexcube Control

14. System with alternating SPE loading: Load sample to SPE1 waste
SPE starter set
Flex cube pump
Column
Std. ALS 900 µL head
SPE2
1260 Bin Pump
SPE1
Load
System with alternating SPE loading:
Load sample to SPE1
waste

15. System with alternating SPE loading:
Flex cube
Column
Mass Spec
Load
Std. ALS 900 µL head
SPE2
1260 Bin Pump
SPE1
Elute
waste
System with alternating SPE loading:
Elute sample from SPE1 to column / Load sample 2 on SPE2

16. Application:
Quantitative LC/MS/MS analysis of 24 trace pharmaceuticals and consumer products in water using on-line SPE enrichment.
Tarun Anumol and Shane Snyder University of Arizona
Sheher Mohsin – Agilent Applications Scientist
Tarun Anumol and Shane Snyder are collaborators at the Un. of Arizona in Tucson. Sheher is located in Chicago, Il. This data will be published as an Agilent application note by February 2013.
11/15/2018

17. Compounds Analyzed Compound Class Atrazine Herbicide PFBS
Fluoro-surfactant
Bisphenol A
Plasticizer
PFOA
Caffeine
Stimulant
PFOS
Carbamazepine
Anti-seizure
Primidone
Anticovulsant
DEET
Insect-repellant
Simazine
Estrone
Hormone
Sulfamethoxazole
Antibiotic
Fluoxetine
Anti-depressant
Atenolol
Β-blocker
Gemfibrozil
Anti-cholesterol
TCPP
Flame-retardant
Ibuprofen
Analgesic
Testosterone
Meprobamate
Anti-anxiety
Triclocarban
Anti-microbial
Naproxen
Pain-reliever
Triclosan
Diltiazem
Anti-histamine
Trimethoprim
24 compounds consisting of common Pharmaceutical and Consumer Products found in Western US wastewaters, perfluorinated compounds, herbicides, and a flame retardant.
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18. Summary Experimental Methods
Collect 5 mL sample and store at 4C for preservation.
Filter sample through 0.2 µm filters.
Combine 1683 µL of sample (weight using analytical balance for accuracy) with 17 µL of a 10 ppb surrogate standard mix. Vortex for 30 sec.
Analyze by UHPLC/MS/MS with the Agilent 1290 LC coupled to an Agilent 6460 Mass Spectrometer LC/MS/MS.
Large volumes of sample are not required for this method. A 1.5 ml aliquot is taking by the autosampler from the 2 ml water sample in the autosampler vial.
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19. Chromatography
Simultaneous analysis of 24 PPCPs in both ESI+ & ESI- mode.
Injection Volume: 1.5 mL
Cycle time (Extraction + Analysis): 18.5 min
Trace Enrichment Cartridge: PLRP-S, 15 µm
Analytical Column: Poroshell 120 EC, 2.1x50 mm
The 18.5 minute cycle time include the on-line SPE and chromatography! PLRP-s polymeric reverse phase/polystyrene divinyl benzene/EC is endcapped
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20. Method Reporting Limits (ng/l)
Analyte
MRL
Atenolol 15
Meprobamate
0.5
Atrazine 5
Naproxen 10
Bisphenol A
PFBS
Caffeine
PFOA
Carbamazepine
2.5
PFOS
DEET
0.1
Primidone
Estrone 20
Simazine
1.5
Fluoxetine
Sulfamethoxazole
Gemfibrozil
TCPP
Ibuprofen
Triclocarban 1
Trimethoprim
Triclosan
S/N> 10 (by height) for 3 successive injections
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21. Evaluation of On-line Solid Phase Extraction and Direct Injection-LC/MS/MS for the Analysis of Chemicals on U.S. EPA’s Contaminant Candidate List 3
Sheher Bano Mohsin
Confidential
April 2010

22. Chemicals for LC/MS/MS Evaluation*
4,4’-methylenedianiline
3-hydroxycarbofuran
bensulide
clethodim
fenamiphos
fenamiphos sulfone
fenamiphos sulfoxide
methomyl
quinoline
tebuconazole
tebufenozide
thiodicarb
9 CCL3 chemicals CCL3 degradates
*Slide Taken From: Evaluation of On-line Solid Phase Extraction-LC/MS/MS for the Analysis of Chemicals on U.S. EPA’s Contaminant Candidate List 3 - Jody A. Shoemaker and Daniel R. Tettenhorst

23. Online SPE Instrumentation in Schaumburg
Agilent 1200 Infinity Series Online-SPE solution system comprising:
G1312B Binary Pump with modular degasser
G1329B Standard Autosampler
G4227A Flexible Cube with 2-position/10-port valves,
G1316C Thermostatted Column Compartment.
6460 Triple Quadrupole LC/MS with Jet Stream Technology
Online SPE
Instrumentation
in Schaumburg
Confidential
April 2010

24. Configuration showing the plumbing for direct on-column injection and on-line SPE.

25. LC/MS/MS Parameters Binary Pump:
• Solvent A: Water + 0.1% formic acid.
Solvent B: ACN + 0.1% formic acid.
Analysis Flow rate: 0.4 mL/min.
Column: ZORBAX EclipsePlus C18 3.0x50mm 1.8 μm, 50 °C.
Flexible Cube:
Valves: 2 of 2-position/10-port Quick-Change valves.
Flow rate: 1.0 mL/min.
Solvent:
A1: 3% MeOH/Water,
B1: ACN/IPA/MeOH/Water 1/1/1/1 v/v/v/v.
Standard Autosampler fitted with 900 μL metering head
Auto-SPE Injection volume: 900 μL using a draw and eject speed of 1,000 μL/min.
Direct Injection volume: 90 μL
Triple Quadruple MS method with Agilent Jet Stream thermal gradient focusing technology: • Gas temperature: 300 °C. Gas flow: 10 L/min. • Nebulizer: 45 psi • Sheath gas temperature: 350 °C. Sheath gas flow: 11 L • Capillary: 4,000 Volt. Nozzle: 0 Volt

26. Dynamic MRM Transitions
Cmpd Name
Prec Ion
Prod Ion
Frag (V)
CE (V)
Cell Acc (V)
Ret Time (min)
Ret Window
Polarity
4-4-diaminodiphenylmethane
199.1
106
120 28 3
6.74
1.81
Positive
Bensulide
398
356
100 1 2
11.06
1.11
314 6
158 26
Carbofuran-3-hydroxy
238.1
181
110 8
8.15
0.94
163 11
Clethodim
360.1
206 12 4
11.67
1.15
166
130 27
Fenamiphos
304.1
234
150 15
10.28
1.03
217 23
202 31
Fenamiphos-sulfone
336.1
308
9.13
0.91
266 18
188 24
Fenamiphos-sulfoxide
320.1
292 9
8.64
0.99
233
171
Methomyl
107
170 17
1.14 95
Quinoline
103 30
6.97 77
Tebuconazole
308.1
125 42
10.49
1.05 70
Tebufenozide
353.2
297 85
10.85
1.09
133
Thiodicarb
355 80
9.17
1.28
108 14 88
Dynamic MRM Transitions

27. Superimposed chromatograms Showing Reproducibility of Retention time as well as Area Counts for two runs Alternating Between two Different SPE Cartridges. The calibration standard used has a concentration of 10 ppt (ng/L) each for all 12 compounds with quantifier and qualifier ions. Each run alternates between two different SPE cartridges.

28. Mix at 1 ppt Tebufenozide Tebuconazole Carbofuran-3-Hydroxy Quinoline
Fenomiphos
Bensulide
Fenomiphos Sulfoxide
Fenomiphos Sulfone
4,4-diaminodiphenyl methane
Thiodicarb
Methomyl
Clethodim

29. Mix at 0.1 ppt

30. 1 ppt and 0.1 ppt 1 ppt 1ppt 1 ppt 0.1 ppt 0.1 ppt 1 ppt 0.1 ppt 1 ppt

31. Direct Injection and online-SPE Same amount on column

32. Fenomiphos Sulfoxide
1.0 ppt
0.5 ppt
0.1 ppt

33. Fenomiphos Sulfoxide 0.1 ppt to 100 ppt
On-line SPE

34. Fenomiphos Sulfone
1 ppt
0.5 ppt
0.1 ppt

35. Fenomiphos Sulfone 0.1 ppt to 100 ppt
Online -SPE

36. Tebufenozide
1 ppt
0.5 ppt
0.1 ppt

37. Tebufenozide
Online SPE

38. Thiodicarb

39. Thiodicarb
Online SPE

40. Clethodim

41. Clethodim 0.1 ppt to 100 ppt
Online-SPE

42. Bensulide
1 ppt
0.5 ppt
0.1 ppt

43. Bensulide
Online SPE

44. Tebuconazole
1 ppt
0.5 ppt
0.1 ppt

45. Tebuconazole 0.1 ppt to 100 ppt
Online SPE

46. Fenomiphos
1 ppt
0.5 ppt
0.1 ppt

47. Fenomiphos
Online SPE

48. Carbofuran-3-Hydroxy
1 ppt
0.5 ppt
0.1 ppt

49. Carbofuran-3-Hydroxy
Online SPE

50. Quinoline
1 ppt
0.5 and 0.1 ppt

51. Quinoline 0.1 to 1000 ppt
Online SPE

52. Water Samples Analysis
Water samples from a lake-fed municipal water supply (tap), residential well (ground), a suburban collection basin (WestLake) and a flood control reservoir (Spring Creek) were analyzed. Notably, both WestLake and Spring Creek are in the drainage basin of nearby public and private golf courses. Samples were filtered with 0.45 um disk filters prior to analysis.

53. Tebuconazole in Water Spring Creek 50 ppt WestLake 37 ppt
Ground ppt
Tap ppt

54. Lake Water and Ground water
Results are based on the average of duplicate analyses, for the one quantifier transition (308.1 to 70.0) and one qualifier transition (308.1 to 125.0). Tebuconazole is used as a fungicide for turf control. Both WestLake and Spring Creek are in the drainage basin of nearby public and private golf courses.
Lake Water
Amount ~ 50 ppt
Ground Water

55. Performance data for all compounds present in the study based on the calculated signal to noise at 0.1 ppt and assuming an LOQ at approximately 10: 1 signal-to-noise. The signal to noise calculation is based on peak area and noise is RMS x 3.
Compound Name
Level (ppt)
Signal to Noise (S/N)
Nominal 10:1 S/N
4-4-diaminodiphenylmethane
0.1 25
0.04
Bensulide
10.3
Carbofuran-3-hydroxy
34.4
0.03
Clethodim 7
0.15
Fenamiphos
301.2
0.005
Fenamiphos-sulfone
7.9
Fenamiphos-sulfoxide
8.2
Methomyl
8.3
Quinoline*
500
0.002
Tebuconazole
427
0.0025
Tebufenozide
9.4
Thiodicarb 9
* uncertainty due to unresolved carryover

56. Trace level analysis of herbicides in drinking and surface water by online-SPE LC/triple quadruploe MS to the lower ppt range Edgar Naegele, Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany
Confidential
April 2010

57. MRM and dynamic MRM MS method
Confidential
April 2010

58. Chromatograms of a calibration standard with a concentration of 100 ppt (ng/L) each for all 28 pesticides measured by the final SPE-LC dynamic MRM method with quantifier and qualifier ion.
Confidential
April 2010

59. Water samples from a tap, Rhine river, and a spring spiked with 25 ppt of all 28 pesticides. The retention time window from 14.5 minutes to 19.1 minutes is shown. The table shows the average measured concentrations of all pesticides (n=12) within the retention time window dependent on the sources of water together with precision RSD and accuracy.
Confidential
April 2010

60. Conclusions
We have demonstrated an integrated, cost effective scheme to do on-line SPE coupled to LC/MS/MS. The technical barriers and potential technology transfer problems associated with traditional online SPE system have been avoided. The direct coupling of the SPE cartridges (PLRP-S, 2.1x12.5mm, 15-20um) to the LC/MS/MS system was done through the integrated valves. The use of Dynamic MRM enables complex mixtures to be efficiently analyzed. We have demonstrated excellent performance with challenging contaminants on the contaminant list. LOQ for all components was nominally 1 ppt or lower. Results for real samples, including ground water, lake and flood control reservoir, the latter two obviously containing visible swimming organisms, produced measurable tebuconazole results.

61. Expand Your Versatility
Agilent 1200 Infinity Series Online SPE solution
Expand Your Versatility
Boost Performance
enrichment of analytes and matrix removal for utmost sensitivity
Save Time
individual configurations for broad automation flexibility
Reduce Costs
1290 Infinity Flexible Cube contains a built-in pump
For online sample preparation, upgrade your system with Agilent 1290 Infinity Flexible Cube featuring
built-in pump to condition and flush re-usable cartridges
solvent selection valve
Reduce costs: the 1290 Infinity Flexible Cube comes with a built-in piston pump, so that for many applications no second LC pump is needed, i.e. save second LC pump in your set-up

62. Journal of Chromatography A, 1286 (2013) 166– 174 Quantitative analysis of naphthenic acids in water by liquid chromatography–accurate mass time-of- flight mass spectrometry
Ralph Hindle, Matthew Noesthedena, Kerry Perub, John Headley Vogon Laboratory Services Ltd., Unit 4, 34 Griffin Industrial Pt., Cochrane, Alberta T4C 0A3, Canada Water Science & Technology Directorate, Environment Canada, 11 Innovation Blvd., Saskatoon, SK S7N 3H5, Canada
Confidential
April 2010