Successful implementation of online SPE LC-MS/MS in the ... · Successful implementation of online...

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


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


Overview of applications


21

-Hyd

roxylase

Aro

matase


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


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)


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



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


289.2 > 97.05 (Testosterone-Quan)

3.27e73.23


331.2 > 97.1 (17-OH-Progesterone-Quan)

6.87e63.31

3.11


291.25 > 255.25 (Dihydrotestosterone-Quan)

3.06e53.71


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



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


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


females, 20-80 yrs, 20 per age decade


females using contraception (20-40yrs old)

*https://lifelines.nl/lifelines-research/general


Negative ion mode

Difficult to measure by LC-MS/MS

Low endogenous concentrations (low pmol/L)

Ion ratios not stable


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


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)


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


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


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 (pmol/L)

Low (109)

Medium (288)

High (1259)

Intra-assay %

4.0 3.4 2.0

Inter-assay %

6.0 5.7 5.3


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


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

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