This document discusses automated sample preparation techniques for LC or LC/MS/MS using GERSTEL Solutions. It describes the Multi Purpose Sampler (MPS) which can perform various sample preparation steps automatically including liquid-liquid extraction, solid phase extraction, evaporation, and more. It also discusses the MAESTRO software which allows control and scheduling of complete automated sample preparation and analysis workflows. Several application examples are provided demonstrating automated sample preparation techniques coupled to LC/MS/MS for analysis of drugs of abuse in oral fluid and dried blood spots. Accuracy and precision results for the methods show most analytes within 15% of the target value.

1. GERSTEL Solutions
Automating Sample Preparation Techniques
For LC or LC/MS/MS
Fred Foster
Applications Scientist
GERSTEL, Inc.
Baltimore, MD, USA

2. “Ensuring precision throughout sample preparation is
critical for accurate analytical results.”
“Good sample preparation unlocks the potential for even
better analytical detection.”

3. Multi Purpose Sampler (MPS)
Integrated with LC/MS/MS
MPS

4. Multi Purpose Sampler (MPS)
Benchtop Workstation Integrated with GC
MPS

5. MPS Overview
• Standard autosampler for GC & LC
• XL version for extra capacity
• Dual rail PrepStation for extra capabilities
• Sample preparation & introduction
• Sample preparation WorkStation for off-line work
• Unique sample preparation technologies
• Integrated Software Control
MPS

6. MAESTRO
Software control designed to work in concert:
• ChemStation, MassHunter, AB SCIEX Analyst®
• Integrated sequence table
• Intuitive control of complete process
• Sample prep steps
• Evaporative parameters and solvent exchange
• Functional method switching with real time scheduler
• Barcode Reading & Tracking
• Method recording
• Ease of method transfer

7. MAESTRO with LC MassHunter software
MAESTRO

8. MAESTRO
PrepBuilder
• Significant time savings through simple editing
• Self explanatory sample prep steps, no macros
• Unlimited number of lines available within a PrepSequence

9. MAESTRO

10. MAESTRO
Prep Ahead Function
• Automated multi-sample
overlapping of sample prep
and GC or LC analysis
• Best possible productivity
• Optimal system utilization
Integrated Scheduler for planning and method optimization
E-Mail notification
Log file

11. Sample Preparation
Typical Manual Sample Preparation Techniques
• Pipetting
• Dilution
• Vortexing/Mixing
• Heating
• Sonication
• Evaporation
• Calibration Standard Preparation
• Liquid-Liquid Extraction
• Solid Phase Extraction

12. SPE
Automated Standard SPE, ITSP, DPX
DPX Pipette Tip
(patented)
ITSP Solutions, Inc.
Cartridge
(patented)
Conventional SPE
Cartridge
Packed Bed SPE Dispersive SPE

13. Original 3 mL 500 mg cartridge
GERSTEL style 3 mL 500 mg cartridge
Automated Solid Phase Extraction
SPE

14. SPE

15. SPE

17. DPX steps

18. ITSP
ITSP Solutions, Inc.

19. Automated SPE-LC/MS/MS for drugs of abuse in oral fluid

20. 1- “Drugs of abuse measured in urine or oral fluid for forensic and medical purposes.” ITSP Solutions, Inc. Application Note, accessed April 2017.
Automated SPE Prep Sequence1:
 The user transfers 1.5mL of Oral Fluid sample into a 2mL vial.
 Working internal standard (15uL) is added to the sample and the sample is mixed.
 Conditioning buffer (150uL) (20% ammonium acetate in water) is added to the sample and the sample is
mixed.
 The ITSP SPE cartridge (UCT C18 endcapped, 10mg) is washed using 100uL of wash solvent (0.2%
ammonium acetate in water) followed by 100uL of the conditioning buffer.
 The oral fluid sample (1mL) is added to the ITSP SPE cartridge at a flow rate of 5uL/sec.
 The ITSP SPE cartridge is washed using 100uL of the conditioning buffer.
 The analytes are then eluted from the ITSP SPE cartridge using 2 x 75uL aliquots of the buffered elution
solvent (0.2% ammonium acetate in methanol) and then diluted (1:2) to ensure good chromatography
and separation of isobaric compounds.

21. Drugs of Abuse Method LC Method Parameters
Mobile Phase: A - 5mM ammonium formate with 0.05% formic acid
B –0.05% formic acid in methanol
LC Gradient:
Analytical Column: Agilent Poroshell 120, C18 column (3.0 x 50mm, 2.7 um)
Run time: 6.5 minutes.
Injection volume: 2.0uL (loop over-fill technique)
Column Temperature: 55 oC
Drugs of Abuse Method Mass Spectrometer Parameters:
Operation: Electrospray positive mode
Gas Temperature: 350oC
Gas Flow (N2): 5 L/min
Nebulizer pressure: 35 psi
Sheath Gas Heater: 250 oC
Sheath Gas Flow (N2): 11 L/min
Capillary voltage: 4000 V
Delta EMV: +500 V
deltaRT (min): 0.5 min
Time
(min)
Flow
(mL/min)
Pressure
(bar)
% B
0 0.5 600 5
0.5 0.5 600 5
1.5 0.5 600 30
3.5 0.5 600 70
4.5 0.5 600 95
6.49 0.5 600 95
6.5 0.5 600 5

22. 6x10
0
0.025
0.05
0.075
0.1
0.125
0.15
0.175
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
1
1.025
1.05
1.075
1.1
1.125
1.15
1.175
1.2
1.225
Cpd 9: D5-Amphetamine: +ESI MRM Frag=70.0V CF=0.000 DF=0.000 CID@5.0 (141.1000 -> 124.1000) 1011.d
11289
1 1
Counts vs. Acquisition Time (min)
0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3
Automated SPE-LC/MS/MS results from extracted low QC

23. LCMS Tool
• Near-Zero carryover
• Low detection limits
• Fast
• Reproducible
• Integrated MAESTRO software control

24. 6x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
1.75
1.8
1.85
1.9
1.95
2
+ESI TIC MRM CF=0.000 DF=0.000 (** -> **) 1007.d
1 1
Counts vs. Acquisition Time (min)
0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3
Results of Carryover experiment:
blue = extracted low standard
orange = injected blank sample immediately following injection of extracted high standard

25. RelativeResponsesRelativeResponses
Morphine
Ketamine

26. RelativeResponses
RelativeResponses
Nordiazepam
Cocaine

27. 0.00
20.00
40.00
60.00
80.00
100.00
120.00
Morphine
Oxymorphone
Hydromorphone
Oxycodone
Codeine
Hydrocodone
6-monoacetylmorphine
d-Amphetamine
MDA
O-Desmethyl-cis-Tramadol
MDMA
methamphetamine
MDEA
Benzoylecgonine
Ketamine
Norketamine
7-Aminoclonazepam
cis-Tramadol
Cocaine
Methylphenidate
nor-Fentanyl
Meperidine
Normeperedine
PCP
Norbuprenorphine
Meprobamate
Fentanyl
EDDP
Flurazepam
Buprenorphine
Norpropoxyphene
Bromazepam
Midazolam
Nitrazepam
Clonazepam
Propoxyphene
Flunitrazepam
Methadone
alpha-Hydroxyalprazolam
Amitriptyline
Estazolam
Clobazam
Carisoprodol
Lorazepam
Oxazepam
Triazolam
Alprazolam
Temazepam
Nordiazepam
Diazepam
Accuracy and Precision
Ave. % Precision Ave. % Accuracy
Accuracy data averaged 101% and precision data averaged 5.97% for all 50 drugs of abuse analyzed.

28. LLE
Automated LLE-LC/MS/MS for Vitamin B compounds in sports drinks

29. mVORX
• Vortex and Orbital shaker for
the MultiPurpose Sampler
(MPS)
• Planar, programmable orbital
agitation with movements
from 200 to 3000 rpm
• 8 x 2 mL; 2 x 4 mL; or 1 x 10
mL vials

30. CF-200 Compact
Centrifuge
Up to 6 vials
2mL/10mL vials
4500 RPM/2000g
Centrifuge

31. Filtration

32. Vitamin B Liquid-Liquid Extraction Procedure:
1. 1g of food sample is placed into a 10mL vial and capped with a magnetically transportable cap
and placed onto the autosampler.
2. Add 9mL of 50% acetonitrile in acidified water (containing an antioxidant) to the food sample.
3. Add 10uL of the internal standard solution to the food sample.
4. Vortex for 5minutes at 3000rpm.
5. Centrifuge samples for 5minutes at 2000g.
6. Filter 1mL of the resulting supernatant through a 0.45um, regenerated cellulose syringe filter
(Agilent p/n: 5042-1392).
7. Transfer 75uL of the filtrate into a 2mL vial.
8. Add 675uL of 0.1% formic acid in water (containing an antioxidant).
9. Inject 2uL into the LC/MS/MS system.
LLE

33. LLE

34. LLE

35. Infant Formula
Recovery Drink
Breakfast Shake
LLE

36. Dried Blood Spot (DBS) Analysis

37. DBSA Compatible Blood Spot Cards
Perkin Elmer 226 Whatman FTA DMPK-C
Whatman FTA DMPK-BWhatman FTA DMPK-A
Card format 84 x 53 mm

38. Manual DBS analysis workflow

39. Custom Workstations

40. Automated DBS analysis

41. Automated DBS analysis

42. SPExos
xos
Online SPE with
Cartridge Exchange

43. SPExos
• New cartridge can be used for each
sample
• Eliminates cross contamination associated with
multiple use of on-line columns
• Small particle sizes (<10 µm)
• “LC-like” separation
• All standard SPE materials available
• 100% analyte transfer to LC
• Low detection limits without
need for evaporation steps
• Can be combined with other
sample preparation techniques
using the MPS

44. Automated DBS analysis

45. Automated DBS analysis

46. Mass Transitions and MS Parameters for Compounds tested

48. Optimization of DBS Desorption Parameters
Volume of Desorption Solution
Spot DBS Desorb Hot Cap
1 1mL H2O 40oC
2 2mL H2O 40oC
3 1mL 10%MeOH in H2O 40oC
4 1mL H2O 40oC
Temperature of Desorption Solution
Spot DBS Desorb Hot Cap
1 2mL H2O 40oC
2 2mL H2O 60oC
3 2mL H2O 80oC
4 2mL H2O 100oC
pH of Desorption Solution
Spot DBS Desorb Hot Cap
1 2mL H2O 80oC
2 2mL 0.1% FA 80oC
3 2mL 0.1% acetic acid 80oC
4 2mL 0.1% NH4OH 80oC

49. Ketamine: Rat Blood Amitriptyline: Rat Blood Ketoprofen: Rat Blood
QC 5ng/mL 1 5.55 111 QC 50ng/mL 1 43.0 85.9 QC 50ng/mL 1 50.7 101
QC 5ng/mL 2 4.71 94.1 QC 50ng/mL 2 45.6 91.3 QC 50ng/mL 2 52.3 105
QC 5ng/mL 3 4.26 85.1 QC 50ng/mL 3 40.7 81.3 QC 50ng/mL 3 49.5 99.1
QC 5ng/mL 4 4.21 84.3 QC 50ng/mL 4 43.1 86.3 QC 50ng/mL 4 54.5 109
mean 4.68 93.6 mean 43.1 86.2 mean 51.8 104
SD 0.539 SD 1.76 SD 1.87
%CV 11.5 %CV 4.08 %CV 3.61
QC 50ng/mL 1 41.2 82.3 QC 500ng/mL 1 417 83.5 QC 500ng/mL 1 435 87.0
QC 50ng/mL 2 43.0 85.9 QC 500ng/mL 2 468 93.6 QC 500ng/mL 2 535 107
QC 50ng/mL 3 40.3 80.6 QC 500ng/mL 3 420 83.9 QC 500ng/mL 3 543 109
QC 50ng/mL 4 40.7 81.4 QC 500ng/mL 4 458 91.7 QC 500ng/mL 4 541 108
mean 41.3 82.6 mean 441 88.2 mean 514 103
SD 1.02 SD 22.7 SD 45.5
%CV 2.47 %CV 5.15 %CV 8.86
QC 500ng/mL 1 403 80.7
QC 500ng/mL 2 445 88.9
QC 500ng/mL 3 401 80.3
QC 500ng/mL 4 437 87.4
mean 422 84.3
SD 19.4
%CV 4.61
Accuracy and Precision of Dried Rat Blood Spots

50. Ketamine: Bovine Blood Amitriptyline: Bovine Blood Ketoprofen: Bovine Blood
QC 5ng/mL 1 5.81 116 QC 50ng/mL 1 55.7 111 QC 50ng/mL 1 51.6 103
QC 5ng/mL 2 4.59 91.7 QC 50ng/mL 2 52.0 104 QC 50ng/mL 2 45.4 90.8
QC 5ng/mL 3 5.37 107 QC 50ng/mL 3 55.5 111 QC 50ng/mL 3 51.3 103
QC 5ng/mL 4 4.61 92.2 QC 50ng/mL 4 49.2 98.4 QC 50ng/mL 4 48.9 97.8
mean 5.09 102 mean 53.1 106 mean 49.3 98.6
SD 0.521 SD 2.68 SD 2.47
%CV 10.2 %CV 5.05 %CV 5.00
QC 50ng/mL 1 57.5 115 QC 500ng/mL 1 499 100 QC 500ng/mL 1 546 109
QC 50ng/mL 2 57.2 114 QC 500ng/mL 2 546 109 QC 500ng/mL 2 492 98.5
QC 50ng/mL 3 58.9 118 QC 500ng/mL 3 597 119 QC 500ng/mL 3 497 99.3
QC 50ng/mL 4 54.4 109 QC 500ng/mL 4 596 119 QC 500ng/mL 4 574 115
mean 57.0 114 mean 559 112 mean 527 105
SD 1.63 SD 40.7 SD 34.2
%CV 2.86 %CV 7.27 %CV 6.49
QC 500ng/mL 1 596 119
QC 500ng/mL 2 597 119
QC 500ng/mL 3 600 120
QC 500ng/mL 4 596 119
mean 597 119
SD 1.72
%CV 0.288
Accuracy and Precision of Dried Bovine Blood Spots

51. RelativeResponses
Ketamine Calibration Curve:
Rat blood
Ketamine Calibration Curve:
Bovine blood
RelativeResponses

52. Additional Application Notes

53. Cooling
Stirring
15 position
Agitators

54. Sonicator

55. mVAP
55
Multi-Position Evaporation Station mVAP

56. MPS robotic
Automated Dilutions with
Weight Verification

57. MPS robotic

58. MPS Workstation
Enhanced Sample Preparation
Your Application
Your Solution

59. MPS Workstation
Enhanced Sample Preparation
Liquid extraction of consumer product
Your Solution
Add extraction solvent to
vials.
Mix samples at user
defined speed and time.
Filter portion of extract
prior to injection.

60. MAESTRO allows the
autosampler to go from this…

61. …to this!

62. Thank you
for your
attention!

63. ASMS 2017 – Booth 524
Poster Tuesday – TP 463
“Drugs of Abuse in Oral Fluids: Automated SPE Extraction and
LC/MS/MS Analysis using a Robotic Autosampler.”