Development of analytical methods for Catecholamines ... · Free catecholamines in plasma Free...
Transcript of Development of analytical methods for Catecholamines ... · Free catecholamines in plasma Free...
Development of analytical methods for Catecholamines, Metanephrines and related metabolites in urine and plasma matrices From sample preparation to LC/MS/MS
Linda Côté
Christophe Deckers
Agilent Technologies
Outline
• Introduction
• Why a unified approach?
• Description of each method • Sample preparation
• Chromatography
• Figures of merits
• Sample preparation • Why
• Which sample preparation technique to choose
• Conclusions
• Questions and Answers
2 For Research Use Only. Not for use in diagnostic procedures.
Free catecholamines in urine
Total metanephrines in urine
Free catecholamines in plasma
Free metanephrines in plasma
VMA in urine
HVA in urine
5-HIAA in urine
Which ones and in which matrix urine or plasma? 7 different methods
3 For Research Use Only. Not for use in diagnostic procedures.
Why a unified approach?
Challenge:
Many analysis to perform
with
One LC/MS/MS system
4 For Research Use Only. Not for use in diagnostic procedures.
Free catecholamines and metanephrines in urine
Total catecholamines and metanephrines in urine
Free catecholamines in plasma (in progress)
Free metanephrines in plasma (in progress)
VMA in urine (in progress)
HVA in urine (in progress)
5-HIAA in urine (in progress)
Which ones and in which matrix urine or plasma? Webinar April 2013
5 For Research Use Only. Not for use in diagnostic procedures.
Free catecholamines and metanephrines in urine
Total catecholamines and metanephrines in urine
Free catecholamines in plasma
Free metanephrines in plasma
VMA in urine
HVA in urine
5-HIAA in urine
Catecholamines and related metabolites in urine or plasma 4 methods with a unified approach
6
1
3
4
2
For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix
Linear range
Sample prep
Chromatography
Mobile phase
LC/MS/MS
7 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep
Chromatography
Mobile phase
LC/MS/MS
8 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
9 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
10 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
11 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
12 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
13 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
14 For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
15 For Research Use Only. Not for use in diagnostic procedures.
Catecholamines and metanephrines in a single run
16 For Research Use Only. Not for use in diagnostic procedures.
Catecholamines and metanephrines in a single run
17
• One SPE cartridge is used to recover both catecholamines and metanephrines
• SPE Bond Elut Plexa was chosen for best recoveries
• Simple acid elution for direct injection into LC-MS/MS
• pH control for stabilization of catecholamines
• Metanephrines are also retained under the same conditions, even though they
are methylated and do not contain the cis-diol moiety for the covalent linkage
binding mechanism
• Studies have shown that metanephrines do have some affinity for this sorbent
[1] Ann Clin Biochem 2009; 46: 129–136. DOI: 10.1258/acb.2008.008180
For Research Use Only. Not for use in diagnostic procedures.
Sample preparation
• Calibrators are prepared with clean urine matrix from Golden West
Biologicals
• Isotopically labelled Internal standards
• 24 hours collection of urine
• Native urine for free catecholamines and metabolites (typically for
catecholamines)
• Acid-hydrolysed urine for total (typically for metanephrines)
• Solid phase extraction (SPE) is used to cleanup urine
18 For Research Use Only. Not for use in diagnostic procedures.
Compound structures
19
Dopamine (D)
C8H11NO2
Neutral Mass: 153.08
Norepinephrine (NE)
C8H11NO3
Neutral Mass: 169.07
Epinephrine (E)
C9H13NO3
Neutral Mass: 183.09
3-Methoxytyramine (3-MT)
C9H13NO2
Neutral Mass: 167.09
Normetanephrine (NMN)
C9H13NO3
Neutral Mass: 183.09
Metanephrine (MN)
C10H15NO3
Neutral Mass: 197.11
O
OHNH
CH3 OH
CH3
OH
OHNH2
OH
OH
OH
NH2
OH
OH
OH
NHCH3
O
OHNH2
CH3
O
OHNH2
CH3 OH
For Research Use Only. Not for use in diagnostic procedures.
Chromatography challenge
Need to keep resolution between E and NMN
Also between MN and 3-MT
20 For Research Use Only. Not for use in diagnostic procedures.
Catecholamines and metanephrines in a single run Chromatography E/NMN and MN/3-MT Resolution is Critical
21
NE
MN
D
NMN
E
3-MT
1. Norepinephrine (NE)
2. Epinephrine (E)
3. Normetanephrine (NMN)
4. Dopamine (D)
5. Metanephrine (MN)
6. 3-Methoxytyramine (3-MT)
For Research Use Only. Not for use in diagnostic procedures.
Calibration curves
22 For Research Use Only. Not for use in diagnostic procedures.
Results Summary of Analyte Performance
23
Compound R2 Concentration Concentration
Accuracy
(%)
Intraday
CV (%)
Interday
CV (%)
(ng/mL) (nmol/L) n = 3 n = 3 n = 5
Dopamine 0.9997
1.56 10.2 107.5 1.0 2.7
62.5 408.0 99.1 1.7 2.0
1000 6528.3 101.3 0.1 0.3
Norepinephrine 0.9999
1.56 9.2 102.9 0.9 5.4
62.5 369.4 101.1 3.5 4.0
1000 5910.9 101.1 0.6 0.6
Epinephrine 0.9998
1.56 8.5 101.6 4.3 2.7
62.5 341.2 100.9 2.5 2.0
1000 5458.4 100.3 0.4 0.3
Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes
For Research Use Only. Not for use in diagnostic procedures.
Results Summary of Analyte Performance
24
Compound R2 Concentration Concentration
Accuracy
(%)
Intraday
CV (%)
Interday
CV (%)
(ng/mL) (nmol/L) n = 3 n = 3 n = 5
3-Methoxytyramine 0.9999
4.69 28 95.7 1.1 3.6
187.5 1121.4 102.9 0.9 2.0
3000 17942.1 100.0 0.2 0.3
Normetanephrine 0.9999
4.69 25.6 100.1 1.5 3.2
187.5 1023.45 102.0 1.1 2.5
3000 16375.2 100.7 0.2 0.2
Metanephrine 0.9999
4.69 23.8 100.5 0.3 2.8
187.5 950.7 102.0 0.5 2.2
3000 15210.6 100.8 0.1 0.2
Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes
For Research Use Only. Not for use in diagnostic procedures.
Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck)
25
Level 1 Level 2
Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%)
Dopamine Free 44.4 -75.0 61.4 3.4 377 – 629 509 2.8
Norepinephrine Free 31.3 – 51.6 38.4 5.8 156 – 239 192 4.8
Epinephrine Free 9.62 – 19.1 14.3 5.3 67.8 – 104 86.7 2
3-Methoxytyramine Total 28.6 – 48.7 44.7 3.8 381 – 572 557.7 2.2
Normetanephrine Total 220 - 366 300.7 2.4 1084 – 1630 1379.2 2.8
Metanephrine Total 69.0 - 116 91.2 2 434 - 655 612 2.5
• All measurements are in ng/mL
• Bio-Rad QC material was used. Ranges provided were for free
catecholamines and total metanephrines
For Research Use Only. Not for use in diagnostic procedures.
Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck)
26
Level 1 Level 2
Compound Free/Total Range (HPLC) Measured CV (%) Range (HPLC) Measured CV (%)
Dopamine Free 290-490 401 3.4 2465-4105 3323 2.8
Norepinephrine Free 185-305 227 5.8 920-1410 1135 4.8
Epinephrine Free 52.5-104 78 5.3 370-570 473 2
3-Methoxytyramine Total 171-291 267 3.8 2280-3420 3335 2.2
Normetanephrine Total 1200-2000 1641 2.4 5920-8900 7528 2.8
Metanephrine Total 350-590 462 2 2200-3320 3103 2.5
• All measurements are in nmol/L
• Bio-Rad QC material was used. Ranges provided were for free
catecholamines and total metanephrines
For Research Use Only. Not for use in diagnostic procedures.
Results Recoveries Observed Using SPE Procedure
27
Compound
Absolute
Recoveries %*
(n = 9)
Relative recoveries %
With ISTDs corrections**
(n = 9)
Average SD Range Average SD
Dopamine 73.5 2.4 95.0-103.4 100.0 2.5
Norepinephrine 112.5 4.9 99.0-102.4 100.0 1.0
Epinephrine 90.3 3.6 94.5-104.4 100.0 2.9
3-Methoxytyramine 53.2 3.6 94.4-102.5 100.0 3.0
Normetanephrine 88.7 7.5 97.3-102.0 100.0 2.0
Metanephrine 93.9 3.6 97.2-103.9 100.0 2.2
* ISTDs peak areas spiked in formic acid subjected to SPE compared with spiked formic acid without SPE
** Calculated concentrations with ISTDs peak area ratios corrections (with SPE) versus theoretical concentrations
For Research Use Only. Not for use in diagnostic procedures.
Results Matrix Effects Observed Using SPE Procedure
28
Compound
Matrix
effects %*
(n = 9)
Accuracies %
With ISTDs corrections**
(n = 9)
Average SD Range Average SD
Dopamine 102.1 3.9 95.4-102.5 100.0 2.3
Norepinephrine 30.1 5.2 94.7-104.4 100.0 3.6
Epinephrine 107.1 3.2 95.2-104.6 100.0 3.7
3-Methoxytyramine 88.2 8.5 95.4-104.0 100.0 3.1
Normetanephrine 93.8 3.1 96.4-101.8 100.0 2.8
Metanephrine 103.0 1.3 95.3-101.3 100.0 2.8
* ISTDs peak areas spiked in urine subjected to SPE compared with spiked formic acid subjected to SPE
** Calculated concentrations with ISTD corrections (urine with SPE) versus theoretical concentrations
For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
29 For Research Use Only. Not for use in diagnostic procedures.
Compound structures
30
5-Hydroxyindole-3-acetic acid (5-HIAA)
C10H9NO3
Neutral Mass: 191.06
Homovanillic acid (HVA)
C9H10O4
Neutral Mass: 182.06
Vanillyl mandelic acid (VMA)
C9H10O5
Neutral Mass: 198.05
For Research Use Only. Not for use in diagnostic procedures.
Sample preparation
• Calibrators are prepared with clean urine matrix from
Golden West Biologicals
• Isotopically labelled Internal standards
• 24 hours collection of urine
• Dilution of urine (vortex and centrifuge) 1 in 10 with
0.2% formic acid in water
31 For Research Use Only. Not for use in diagnostic procedures.
Example chromatogram
32
1. VMA
2. 5-HIAA
3. HVA
1
2
3
For Research Use Only. Not for use in diagnostic procedures.
Calibration curves
33 For Research Use Only. Not for use in diagnostic procedures.
Results Summary of Analyte Performance
34
Compound R2 Concentration Concentration
Accuracy
(%)
Intraday
CV (%)
Interday
CV (%)
(mg/L) (µmol/L) n = 3 n = 3 n = 5
VMA 0.9999
0.078 0.39 106.8 3.9 3.9
12.5 63.1 100.1 1.3 1.7
100 504.6 100.5 0.6 0.5
5-HIAA 0.9998
0.078 0.41 109.7 3.4 3.4
12.5 65.4 99.1 2.2 1.7
100 523.1 100.8 0.1 0.1
HVA 0.9999
0.078 0.43 102.2 4.2 3.2
12.5 68.6 99.9 1.7 1.3
100 548.9 100.5 0.4 0.3
Note: Signal to noise ratios and CVs indicate that LLOQs are lower than measured here for all analytes
For Research Use Only. Not for use in diagnostic procedures.
Results Inter-run Over 3 Days for Commercial QC (BioRad Lyphocheck)
35
Level 1 Level 2
Compound Range (HPLC)
Measured CV (%) Range (HPLC)
Measured CV (%)
VMA 2.1-3.1 2.5 2.3 11.2-16.8 14.6 2.4
5-HIAA 2.2-3.4 2.8 2.0 20.8-31.2 27.6 2.8
HVA 1.00-1.40 1.3 5.8 13.0-19.6 15.8 3.9
• All measurements are in mg/L (n=5)
• Bio-Rad QC material was used. Ranges provided were for HPLC method
For Research Use Only. Not for use in diagnostic procedures.
Results Matrix Effects Observed
36
Compound
Matrix
effects %*
(n = 10)
Accuracies %
With ISTDs corrections**
(n = 10)
Average SD Range Average SD
VMA 91.5 6.4 95.6-108.3 100.0 3.9
5-HIAA 93.2 3.3 94.6-115.2 100.0 5.8
HVA 91.3 1.5 92.9-103.9 100.0 3.1
Measurements done at 10 different concentrations ranging from 0.078 to 100 mg/L
* Peak areas from urine spiked compared with 0.2% formic acid-H2O solutions spiked
** Calculated concentrations of urine spiked with ISTD corrections versus theoretical concentrations
For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
37 For Research Use Only. Not for use in diagnostic procedures.
Sample preparation
• Calibrators are prepared with clean plasma matrix from
Golden West Biologicals
• Isotopically labelled Internal Standards
• Solid phase extraction (SPE) is used to cleanup plasma
38 For Research Use Only. Not for use in diagnostic procedures.
Plasma calibrator chromatogram with extra wash
39
1. Normetanephrine
2. Metanephrine
3. 3-Methoxytyramine
1
2
3
For Research Use Only. Not for use in diagnostic procedures.
Calibration curves
40 For Research Use Only. Not for use in diagnostic procedures.
Results Summary of analyte performance
41
Compound R2 Concentration Concentration
Accuracy
(%)
Intraday CV
(%)
Interday CV
(%)
(pg/ml) (nmol/L) n = 3 n = 3 n = 5
3-Methoxytyramine 0.9997
15.63 0.09 115.4 2.4 1.9
78.13 0.47 97.6 1.8 1.6
1250 7.5 100.4 1.1 1.8
10000 59.8 100.0 0.4 1.0
Normetanephrine 0.9996
15.63 0.09 117.3 2.7 4.9
78.13 0.43 100.0 2.4 2.5
1250 6.8 97.1 2.5 1.8
10000 54.6 100.4 0.4 0.8
Metanephrine 0.9998
15.63 0.08 116.6 1.7 1.9
78.13 0.40 96.5 2.5 2.2
1250 6.3 100.2 1.1 0.9
10000 50.7 100.3 0.6 0.6
For Research Use Only. Not for use in diagnostic procedures.
Results Inter-run over 3 days for commercial QC materials (ChromSystems)
42
Level 1 (n=3)
Level 2 (n=3)
Compound Measured (pg/mL)
CV (%)
Measured (pg/mL)
CV (%)
3-Methoxytyramine -- -- 1768.9 0.8
Normetanephrine 95.9 5.1 7369.9 3.0
Metanephrine 85.0 2.2 1620.8 3.2
For Research Use Only. Not for use in diagnostic procedures.
Results SPE extraction procedure recoveries with spiked formic acid solutions with and without SPE
43
Compound
Absolute Recoveries %*
(n = 9)
Relative recoveries %
With ISTDs corrections**
(n = 9)
Average SD Range Average SD
3-Methoxytyramine 75.5 19.4 94.4-115.4 100.0 5.7
Normetanephrine 28.0 8.2 95.4-117.3 100.0 5.1
Metanephrine 74.5 14.7 96.4-116.6 100.0 2.9
*: Peak areas spiked in formic acid subjected to SPE compared with spiked formic acid without SPE
**: Calculated concentrations with ISTDs peak area ratios corrections (with SPE) versus theoretical concentrations
For Research Use Only. Not for use in diagnostic procedures.
Results Matrix effect and Recovery efficiency
44
Compound
Matrix
effects %
(n = 9)
Recovery
efficiency %
(n = 9)
Average SD Average SD
3-Methoxytyramine 78.3 7.2 99.1 16.9
Normetanephrine 36.0 13.7 87.7 27.0
Metanephrine 73.0 9.0 104.4 17.3
A: neat standard solutions
B: plasma extracted then spiked (post-ext)
C: spiked plasma then extracted (pre-ext)
Matrix effect % = B/A * 100
Recovery efficiency % = C/B * 100
For Research Use Only. Not for use in diagnostic procedures.
Unified approach in method development
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
Chromatography Pursuit-PFP Pursuit-PFP Pursuit-PFP Pursuit-PFP
Mobile phase 0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
0.2% formic acid
- Methanol
1 mM
ammonium
fluoride -
Methanol
LC/MS/MS 1290/6460 1290/6460 1290/6460 1290/6460
45 For Research Use Only. Not for use in diagnostic procedures.
Sample preparation
Challenge: Need to measure low pg/mL in plasma, therefore must cleanup and concentrate
• Calibrators are prepared with clean plasma matrix from Golden West Biologicals
• Isotopically labelled Internal Standards
• Catecholamines are readily oxidized and unstable in whole blood and plasma. Stabilizers should be added to plasma.
• Step 1: Protein precipitation and lipids removal with “Captiva NDLipids” filter cartridges
• Step 2: Solid phase extraction (SPE) is used to further cleanup plasma
46 For Research Use Only. Not for use in diagnostic procedures.
Sample Preparation – Plasma pretreatment
47
Pretreatment of samples:
• Plasma should be kept frozen at -80 °C until sample analysis
• Stabilizer solutions:
• Sodium metabisulfite: 317 mg/mL of sodium metabisulfite in water
• EDTA 0.5M
• For sample plasma, calibrators and QCs
• Add 2% v/v of each stabilizer solution
For Research Use Only. Not for use in diagnostic procedures.
Column: Analytical: Pursuit 3 PFP, 2 x 150 mm, 3 µm Guard: Pursuit 3 PFP MetaGuard 2 mm Column Temp: 30 °C
Injection volume: 20 µL
Needle Wash: 1:1:1:1 MeOH:ACN:IPA:H2O + 0.1% formic acid 20 seconds Injector Temp: 4 °C Mobile Phase: A: 1 mM Ammonium Fluoride in Water B: Methanol Flow rate: 0.3 mL/min.
1290 Pump Gradient:
Stop time: 10 min.
Requilibration time: 3 min.
Method LC Conditions (1290)
48
Time (min) %B
0.0 0
3.8 0
4.0 95
10 95
For Research Use Only. Not for use in diagnostic procedures.
Plasma Catecholamines calibrator chromatogram
49
1. Norepinephrine
2. Epinephrine
3. Dopamine
1
2
3
For Research Use Only. Not for use in diagnostic procedures.
Calibration curves
50 For Research Use Only. Not for use in diagnostic procedures.
Results Summary of analyte performance
51
Compound R2 Concentration Concentration
Accuracy
(%)
Inter-day CV
(%)
n = 3 (pg/mL) (nmol/L) n = 3 n = 3
Norepinephrine 0.9999
5 0.03 107.6 4.7
20 0.12 95.4 1.2
250 1.5 98.6 1.4
2500 14.8 100.2 0.3
Epinephrine 0.9998
5 0.03 108.4 2.1
20 0.11 96.5 1.1
250 1.4 97.5 1.6
2500 13.6 100.6 0.3
Dopamine 0.9997
5 0.03 108.7 3.2
20 0.13 98.8 2.6
250 1.6 98.1 0.9
2500 16.3 99.6 1.1
For Research Use Only. Not for use in diagnostic procedures.
Results Intra and Inter-day over 3 days for commercial QC materials (ChromSystems)
52
Compound QC Level Measured Value Intra-day (n=3)
pg/mL - nmol/L
Intra-day CV% (n=3)
Measured Value Inter-day (n=3)
pg/mL - nmol/L
Inter-day CV% (n=3)
Norepinephrine 0010 0020
240 1756
1.42 10.4
3.6 0.7
242 1767
1.43 10.4
3.0 0.8
Epinephrine 0010 0020
93.4 451
0.51 2.46
1.6 0.7
92.3 449
0.50 2.45
1.2 0.4
Dopamine 0010 0020
164 595
1.07 3.88
0.9 0.5
162 597
1.06 3.90
0.8 0.9
For Research Use Only. Not for use in diagnostic procedures.
Results Matrix effect and Recovery efficiency
53
Compound
Matrix
effects %
(n = 3)
Recovery
efficiency %
(n = 3)
Average SD Average SD
Norepinephrine 42.3 1.9 56.3 6.3
Epinephrine 70.1 6.6 56.5 2.4
Dopamine 118.5 21.5 58.7 4.3
A: neat standard solutions
B: plasma extracted then spiked (post-ext)
C: spiked plasma then extracted (pre-ext)
Matrix effect % = B/A * 100
Recovery efficiency % = C/B * 100
For Research Use Only. Not for use in diagnostic procedures.
Sample preparation
54
• Why
• Which sample preparation technique to choose
For Research Use Only. Not for use in diagnostic procedures.
Objectives of Sample Preparation
• Removal of interferences that affect detection and accuracy
• Reach lower detection limits
• Increase method robustness
• Decrease overall operating costs
55 For Research Use Only. Not for use in diagnostic procedures.
Types of Interferences in Biological Samples
Major causes of matrix effects:
• Salts – generally elute early in the run
• Abundant Proteins – most prominent interference
• Lipids, phospholipids, and lysophosphatidylcholines – difficult to
remove
• Surfactants, dosing agents, excipients
• Phthalates and plasticizers from plastic ware
56 For Research Use Only. Not for use in diagnostic procedures.
Instrumentation affects selection of Sample Preparation Techniques
57
More Specific ← Instrument Separation and Detection Specificity ← Less Specific
Less Specific → Sample Preparation Specificity → More Specific
Sample Prep Technique
Interference Removed
Dilute & Shoot Filtration Liquid/Liquid Extractions
Supported Liquid Extractions (SLE)
Dried Matrix Spotting
Precipitation QuEChERS Lipid Removal
‘Hybrid' Filtration
Solid Phase Extraction
Lipids No No No Some No No Yes Yes Yes
Oligomeric Surfactants No No No No No No No Yes Yes
Particulates No Yes No Some No Yes Yes Yes Yes
Pigments No No No Some No No Yes No Yes
Polar Organic Acids No No Yes Yes No No Yes No
Proteins No No Yes Yes Yes Yes Yes Yes Yes
Salts No No Yes Yes No No No No Yes
Suggested Agilent Product
Agilent Autosampler
Vials
Captiva Syringe Filters
Chem Elut Captiva ND Bond Elut QuEChERS
Captiva ND LIPIDS Bond Elut Silica and Polymeric
SPE
For Research Use Only. Not for use in diagnostic procedures.
Why did we use different sample preparation techniques?
1. Free and Total
Cats/Mets
2. VMA, HVA, 5-
HIAA
3. Mets 4. Cats
Matrix Urine Urine Plasma Plasma
Linear range 1.56-3000
ng/mL
0.08-100
mg/L
15.6-10000
pg/mL
5-2500
pg/mL
Sample prep Boronate
complex/ Bond
Elut Plexa SPE
Dilution WCX SPE Captiva NDLipids/
BondElut PBA
SPE
58
Sample Dependant
Analyte Dependant OH
OHNH2
O
OHNH
CH3 OH
CH3
For Research Use Only. Not for use in diagnostic procedures.
Why did we use different sample preparation techniques?
59
Consideration 1: Sample type (Urine vs Plasma)
• The dirtier the sample, the better sample cleanup you need to remove
interferences
• If the sample matrix is not removed you may have: carry-over, lipid build-up,
ion suppression, low signal to noise ratio
• A dirty sample increases operational costs, impacting instrument
maintenance, sample re-runs, column lifetime
Consideration 2: Analyte characteristics and LLOQs
• To reach pg/ml levels, you need a sample prep technique that concentrates
the analyte
• The analyte’s chemical properties are unique (Polarity, Log P, pKa, solubility)
• Some analytes have stability limitations
For Research Use Only. Not for use in diagnostic procedures.
What is Solid Phase Extraction (SPE) Four steps to Selective cleanup and Elution
60
Sample loading Retention Rinsing Elution
Green = Blue and Yellow
Blue is more non polar than yellow
Blue is retained
For Research Use Only. Not for use in diagnostic procedures.
Catecholamines and metanephrines in urine Solid Phase Extraction (SPE)
61
Prepare complexed samples:
0.5 mL urine*, calibrators, QCs*
Add 40 µL of internal standards mix
Add 0.8 mL of Diphenyl-Boronate complexing agent
Verify pH, must be between 7.5-9.5. If necessary adjust to pH 8.5 with NH4OH
Step 1: Condition SPE cartridge (Bond Elut Plexa, 30 mg, 3 mL) with:
1 mL of MeOH
1 mL of wash buffer 0.2 M NH4Cl-NH4OH
Step 2: Add complexed samples
Step 3: Wash with 1 mL of 5% MeOH wash buffer 0.2 M NH4Cl-NH4OH
Dry at full vacuum for 5 minutes
Step 4: Elute with 1 mL of 5% formic acid in water. Apply vacuum 5” Hg for 30 seconds
Transfer to autosampler vial
* Native for free catecholamines, hydrolyzed for total metanephrines
(add 25 µL HCl 6N, incubate at 90 deg. C for 25 min., cool at RT)
[1] Ann Clin Biochem 2009; 46: 129–136. DOI: 10.1258/acb.2008.008180
[2] Talwar et al., Journal of Chromatography B, 769 (2002) 341–349
For Research Use Only. Not for use in diagnostic procedures.
Diphenyl boronate-catecholamine complex
62
“The diphenyl boronate forms a stable negatively charged complex (Fig. 1) with cis-
hydroxyl groups of catecholamines, which is strongly retained on a C18 extraction
sorbent when operating in alkali media. This allows for column washing with
methanol-buffer solutions to remove interfering compounds without the loss of the
catecholamines which are eluted by disrupting the complex under acid conditions.”
[2] Talwar et al., Journal of Chromatography B, 769 (2002) 341–349
For Research Use Only. Not for use in diagnostic procedures.
Metanephrines in plasma
65
Pretreatment of samples:
0.5 mL plasma, calibrators, QCs
Add 50 µL of internal standards mix
Add 0.5 mL of 10 mM NH4H2PO4 buffer pH 6.5
Step 1: Condition SPE cartridge (SampliQ WCX, 30 mg, 1 mL) with:
1 mL of MeOH
1 mL of 10 mM NH4H2PO4 buffer pH 6.5
Step 2: Add samples
Step 3: Wash with 1 mL H2O, 1 mL Methanol, 1 mL 0.2% formic acid in acetonitrile
Dry at full vacuum for 5 minutes
Step 4: Elute with 2 x 250 µL of 2% formic acid in acetonitrile.
Apply vacuum 5” Hg for 60 seconds
Evaporate under nitrogen flow at 40 deg. C
Reconstitute with 100 µL of 0.2% formic acid in water
For Research Use Only. Not for use in diagnostic procedures.
Catecholamines in plasma Step 1 – Protein precipitation and lipids removal
66
• Add 1.5 mL of 0.5% formic acid in acetonitrile to a Captiva NDLipids
cartridge (3 mL, PN: A5300635) or plate
• Add 50 µL of internal standard solution
• Add 750 µL of pretreated sample plasma or calibrators or QCs
• Mix 3-5 times with a 1.5 mL empty pipette tip
• Wait 5-10 minutes
• Place under vacuum at 7” Hg for 2 minutes, then at 15” Hg until dry
• Use filtrate for SPE
For Research Use Only. Not for use in diagnostic procedures.
Captiva NDLipids Lipid removal and filtration principles
Features:
- Captiva particulate filter
removes protein interferences
- Proprietary Lipid Stripping Media
removes lipids
- Non-Drip Membrane
ease of use
67 For Research Use Only. Not for use in diagnostic procedures.
Catecholamines in plasma Step 2 – Solid Phase Extraction (SPE)
68
Pretreatment of samples:
Use filtrate from step 1
Add 2 mL of 100 mM NH4H2PO4 buffer pH 10
1: Condition SPE cartridge (BondElut PBA, 100mg, 3 mL, PN: 12102127) with:
1 mL acetonitrile
1 mL 5% formic acid in methanol
1 mL 100 mM NH4H2PO4 buffer pH 10
2: Add pretreated samples
3: Wash with:
2 mL 1% NH4OH in 95% methanol
2 mL 1% NH4OH in 95% acetonitrile
2 mL 1% NH4OH in 30% acetonitrile
Dry at full vacuum for 5 minutes
4: Elute with:
3 x 500 µL of 5% formic acid in methanol
Apply vacuum 5” Hg for 60 seconds
Evaporate under nitrogen flow at 35 deg. C
Reconstitute with 100 µL of 0.1% formic acid in water
For Research Use Only. Not for use in diagnostic procedures.
Conclusions
69
• A unified approach where the same chromatography principles and
the same modern LC/MS/MS instrumentation were used to analyze
different compounds and matrices
• Novel and selective sample preparation techniques were adapted to
the matrix and to the desired LLOQ
• Four analytical methods that are robust, sensitive and precise were
developed
For Research Use Only. Not for use in diagnostic procedures.
More information
70
5991-6194EN 5991-6053EN 5991-6531EN 5991-6530EN
For Research Use Only. Not for use in diagnostic procedures.
Questions
71
Thank You!
For Research Use Only. Not for use in diagnostic procedures.
For more information on Agilent’s Clinical Research Solutions, visit www.agilent.com/chem/clinicalresearch