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Successful implementation of online SPE LC-MS/MS in the clinical research laboratory
Martijn van Faassen, PhD student Prof. Dr. Ido Kema
University Medical Center Groningen Department of Laboratory Medicine, Special Chemistry Groningen, The Netherlands
©Wonderlab Comic
Sample prep – LC-MS
“Dilute-and-shoot”
Liquid-Liquid Extraction (LLE)
Supported Liquid Extraction (SLE)
Solid Phase Extraction (SPE) – offline/online
Department of Laboratory Medicine Subdepartment: Special Chemistry
Focus on routine patient care (20 FTE) Low volume, specialized tests Specialized in chromatography, mass spectrometry Several techniques: - HPLC-UV/Fluorometry/ECD - GC-FID, GC-MS and GC-MS/MS - LC-MS/MS - Ion chromatography - Atomic absorption spectroscopy - Infrared spectroscopy
Mass spectrometry since 1975, LC-MS since 2003
Decreased total analysis time – SPE and LC in parallel
No manual sample pretreatment / Reduction human
errors
“Optimal” SPE (superior clean-up)
Increased accuracy and precision
Easy upscaling of production (e.g. research projects)
Method development
Minimal sample volume needed
Pros
Online SPE
Skilled personnel for development (operation)
and trouble shooting
Robustness system important
Medium sized throughput
Carry-over
Points of consideration
Online SPE
Our “toys”
Systems:
3 x Quattro Premier XE – Symbiosis Pharma
System (Spark Holland)
1 x Xevo TQ-MS - Symbiosis Pharma System
(Spark Holland)
2 x Xevo TQ-s – Online SPE Manager
(Waters) & ACQUITY UPLC I-Class
1 x 5500 – Shimadzu HPLC
Current LC-MS/MS assays in routine patient care:
Catecholamines and metanephrines in plasma
Catecholamines, metanephrines, serotonin and 5-hydroxyindoleacetic acid (5HIAA)
in urine
25-OH-Vitamin-D3 in plasma
Cortisol in urine, saliva and hair
Methylmalonic acid in plasma
Tryptophan, 5-hydroxytryptophan, serotonin and 5-HIAA in platelet-rich-plasma
Methylhistamine and imidazoleacetic acid in urine
Vitamin B3 metabolites in urine
Testosterone in plasma
Androgen profile: androstenedione, 17-OH-progesterone, testosterone,
dihydrotestosterone, progesterone in plasma
Aldosterone in plasma
For research purposes: Testosterone in saliva (male, female and paediatric)
Cortisol and melatonin in plasma and saliva
6-Sulfatoxymelatonin in urine
Tryptophan, kynurenine, 3-OH-kynurenine in urine/plasma
Thyroid hormone profile in plasma (T2, rT2, T3, rT3, T4)
Vitamin K in plasma (K1, MK-4)
Vitamin D profiling in mother’s milk and fat tissue
1,25-diOH Vitamin D3 in plasma (+25-OH Vit D3)
IGF-I in plasma
Fulvestrant in plasma
C4 in plasma (intermediate bile acid)
Cortisol and metabolites in urine (THE, THF, allo-THF, cortisone, cortisol)
ACQUITY Online SPE Manager
Online SPE coupled the UPLC
Precipitation is necessary
Several SPE chemistries available:
- Xbridge C8
- Xbridge C18
- Oasis WCX
- Oasis HLB
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
ACQUITY Online SPE Manager
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Inline elution to column
SPE cartridges 1x10mm 2x96 cartrdiges
High pressure dispenser For extraction and wash solvents
SPE extraction and wash
Valves for extraction and wash solvents (up to 23)
Online SPE
Short movie – OSM in action
Illustrations
Androgen profile in plasma
Aldosterone in plasma
Overview of applications
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
21
-Hyd
roxylase
Aro
matase
Androgen profile in plasma
Androgen profile
Instrumentation
ACQUITY Online SPE Manager (OSM)
Xevo TQ-S
Online SPE & LC
Xbridge C8, 10µm cartridges (1x10mm)
Waters Cortecs UPLC C18, 1.6µm, 2.1x100mm
-Mobile phase: A) 0.05%FA + 2mM NH4Ac in 10% MeOH
B) 0.05%FA + 2mM NH4Ac in MeOH
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
200µL plasma, 25µL internal standard mix, 25 µL protein disrupting
buffer
Incubation for 30minutes at room temperature
Fill up to 1000µL with water
40µL is injected onto OSM system
Xbridge C8 for online SPE extraction:
Protocol
• Conditioning: Methanol (0.50mL), Magic Mix (1.0mL), Water(1.0mL )
• Extraction: Water (1.0mL )
• Wash: 20% MeOH + 0.1% ammonia (0.5mL)
30% MeOH + 0.1% FA (0.5mL)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Elution time: 0:90 minute
Flow: 0.4mL/min, temperature column 40˚C
Total run-time per sample: 6:00 minutes
Time (min) Flow rate (mL/min)
% A % B
Initial 0.40 65 35
4:00 0.40 20 80
5:00 0.40 20 80
5:10 0.40 65 35
6:00 0.40 65 35
Androgen profile in plasma
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Time2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20
%
0
100
150513_125 2: MRM of 4 Channels ES+
287.25 > 97.1 (Androstenedione-Quan)
7.07e62.96
150513_125 1: MRM of 12 Channels ES+
289.2 > 97.05 (Testosterone-Quan)
3.27e73.23
150513_125 1: MRM of 12 Channels ES+
331.2 > 97.1 (17-OH-Progesterone-Quan)
6.87e63.31
3.11
150513_125 3: MRM of 4 Channels ES+
291.25 > 255.25 (Dihydrotestosterone-Quan)
3.06e53.71
150513_125 4: MRM of 4 Channels ES+
315.25 > 97.1 (Progesterone-Quan)
4.10e53.94
Androstenedione, 5.6 nmol/L
Testosterone, 18 nmol/L
17α-Hydroxyprogesterone, 4.4 nmol/L
Dihydrotestosterone, 1.1 nmol/L
Progesterone, 0.26nmol/L
Androgen profile in plasma
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
0
10
20
30
40
50
60
70
80
90
100
Androstenedione
Testosterone
17-OH-P
DHT
Progesterone
Increase in % organic in SPE wash steps
% R
eco
very
Chromatography on SPE
Validation results (Summary)
Precision
Intra-assay n=10 on one day
Inter-assay: n=2 replicates on 12 different days (over 5 weeks)
LLOQ
On six different days, samples with decreasing concentrations
analyzed. LLOQ: CV ≤ 20% and S/N >10
Recovery
Within 85 -115% for all compounds
Androgen profile in plasma
Analyte LLOQ (nmol/L)
Progesterone 0.03
17-OH-progesterone 0.05
Androstenedione 0.03
Testosterone 0.04
Dihydrotestosterone 0.09
Imprecision
Intraassay (n = 10) Interassay (n = 12 different days)
Mean (nmol/L) CV (%) Mean (nmol/L) CV (%)
Progesterone 0.27 4.6 0.27 3.7
7.6 1.2 7.6 2.6
46 2.1 45 2.2
17-OH-Progesterone 0.50 2.8 0.50 2.4
7.4 0.6 7.4 2.7
36 2.2 36 2.5
Androstenedione 0.36 3.6 0.35 2.2
2.6 1.6 2.6 2.7
24 1.9 23 2.3
Testosterone 0.37 3.1 0.37 2.7
3.9 1.4 3.8 2.8
51 1.2 50 2.9
Dihydrotestosterone 0.13 5.3 0.12 13
1.6 2.4 1.6 4.3
5.3 4 5.3 3.8 FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Outlook - Reference interval study
Samples from population study Lifelines*
120 plasma and urine samples from healthy
males, 20-80 yrs, 20 per age decade
120 plasma and urine samples from healthy
females, 20-80 yrs, 20 per age decade
40 plasma and urine samples from healthy
females using contraception (20-40yrs old)
*https://lifelines.nl/lifelines-research/general
Aldosterone in plasma
Negative ion mode
Difficult to measure by LC-MS/MS
Low endogenous concentrations (low pmol/L)
Ion ratios not stable
Aldosterone in plasma
Instrumentation
Waters Online SPE Manager (OSM)
Waters Xevo TQ-S
Online SPE & LC
XBridge C18, 10µm cartridges (1x10mm)
Waters ACQUITY CSH Phenyl-Hexyl, 1.7µm, 2.1x100mm
-Mobile phase: A) 10% MeOH
B) MeOH
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
300µL plasma + 100µL internal standard – mix for 10min
Biotage SLE extraction protocol (96-well 400µL plate)
After evaporation dissolve in 200µL 20% MeOH
50µL is injected onto OSM system
XBridge C18 for online SPE extraction:
Protocol
• Conditioning: Methanol (0.50mL), Magic Mix (0.5mL), Water(0.5mL )
• Extraction: Water (1.0mL )
• Wash: 10% MeOH + 0.1% ammonia (2.0mL)
15% MeOH (0.5mL)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Time (min) Flow rate (mL/min)
% A % B
Initial 0.40 100 0
3.50 0.40 15 85
4.50 0.40 15 85
4.60 0.40 100 0
5.50 0.40 100 0
Elution time: 1:00 minute
Flow: 0.4mL/min, temperature column 40˚C
Total run-time per sample: 6:30 minutes
Protocol
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
N8
Time2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
150316_045 Sm (Mn, 1x1) MRM of 4 Channels ES- 359.2 > 189.05 (Aldosteron 1)
6.97e4
S/N:PtP=40.12
3.052.85
150316_045 Sm (Mn, 1x1) MRM of 4 Channels ES- 366.2 > 194.1 (Aldosteron 1-d7)
2.25e5
3.27
Aldosterone in plasma: 105 pmol/L
7 fmol/2.5 picogram on column
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Validation results (summary)
Precision
Intra-assay n=20 on one day
Inter-assay: n=2 replicates on 20 different days (over 10 weeks)
LLOQ aldosterone: 20 pmol/L
Recovery
Within 85 -115%
Aldosterone in plasma
Aldosterone (pmol/L)
Low (109)
Medium (288)
High (1259)
Intra-assay %
4.0 3.4 2.0
Inter-assay %
6.0 5.7 5.3
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Method comparison
Overview current applications/projects
25-OH-vitamin D3/D2 in plasma
Cortisol/cortisone in saliva/urine
Catecholamines & metanephrines in plasma
Thyroid hormone profile in plasma (T2, rT2, T3,
rT3, T4)
IGF-I (conversion of offline method to online
method)
Time2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70
%
0
100
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70
%
0
100
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70
%
0
100
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70
%
0
100
140813_Corticosteroids_099 2: MRM of 12 Channels ES+ 363.3 > 121.1 (Cortisol Quan.)
3.73e5Area
3.1017755
140813_Corticosteroids_099 2: MRM of 12 Channels ES+ 367.3 > 121.1 (Cortisol-d4 Quan.)
1.33e7Area
3.08677678
140813_Corticosteroids_099 2: MRM of 12 Channels ES+ 361.3 > 163.15 (Cortison Quan.)
3.60e6Area
3.29183708
140813_Corticosteroids_099 2: MRM of 12 Channels ES+ 368.3 > 169.15 (Cortison-d7 Quan.)
2.68e7Area
3.261385502
Cortisol = 0.73 nmol/L
Cortisone = 5.6 nmol/L
Time1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
100
1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90
%
0
1002.79
3.11
2.92
3.32
2.65
2.98
2.84
2.54 2.622.67 2.69
2.30
2.65
2.83
L-DOPA
Dopamine
Norepinephrine
Epinephrine
3-Methoxytyramine
Normetanephrine
0.020 nmol/L
0.31 nmol/L
33 nmol/L
0.040 nmol/L
0.13 nmol/L
0.50 nmol/L
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
Summary
Online SPE-LC-MS/MS in our lab
Increase in assay portfolio, without extra hands
Increase in sample throughput and quality
6 technicians responsible for all routine LC-MS/MS
assays
Costs are mainly determined by depreciation,
maintenance and cartridges
Outlook: Consolidation and focus on other multi-analyte
assays
Acknowledgements
UMCG
Ido Kema Claude van der Ley Jasper Krijnen Anna van der Veen Hillie Adema Carlijn Boulet
Waters
Darwin Asa Kendon Graham
Thank you for your attention