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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


Why a unified approach?

Challenge:

Many analysis to perform

with

One LC/MS/MS system


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


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



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



1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



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



1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



ng/mL

0.08-100

mg/L

15.6-10000

pg/mL

5-2500

pg/mL


complex/ Bond

Elut Plexa SPE


BondElut PBA

SPE



- 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


Catecholamines and metanephrines in a single run


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


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


Compound structures

19

Dopamine (D)

C8H11NO2

Neutral Mass: 153.08

Norepinephrine (NE)

C8H11NO3


Epinephrine (E)

C9H13NO3


3-Methoxytyramine (3-MT)

C9H13NO2


Normetanephrine (NMN)

C9H13NO3


Metanephrine (MN)

C10H15NO3


O

OHNH

CH3 OH

CH3

OH

OHNH2

OH

OH

OH

NH2

OH

OH

OH

NHCH3

O

OHNH2

CH3

O

OHNH2

CH3 OH


Chromatography challenge

Need to keep resolution between E and NMN

Also between MN and 3-MT


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)


Calibration curves


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



24


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



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



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


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


Results Matrix Effects Observed Using SPE Procedure

28

Compound

Matrix

effects %*

(n = 9)

Accuracies %


(n = 9)


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



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



1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



ng/mL

0.08-100

mg/L

15.6-10000

pg/mL

5-2500

pg/mL


complex/ Bond

Elut Plexa SPE


BondElut PBA

SPE



- 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


Compound structures

30

5-Hydroxyindole-3-acetic acid (5-HIAA)

C10H9NO3


Homovanillic acid (HVA)

C9H10O4


Vanillyl mandelic acid (VMA)

C9H10O5



//upload.wikimedia.org/wikipedia/commons/a/aa/Homovanillic_acid.png

http://en.wikipedia.org/wiki/File:5-Hydroxyindolessigs%C3%A4ure_(5-HIAA).svg

http://en.wikipedia.org/wiki/File:Vanilmandelic_acid.svg

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


Example chromatogram

32

1. VMA

2. 5-HIAA

3. HVA

1

2

3


Calibration curves



34


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




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


Results Matrix Effects Observed

36

Compound

Matrix

effects %*

(n = 10)

Accuracies %


(n = 10)


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



1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



ng/mL

0.08-100

mg/L

15.6-10000

pg/mL

5-2500

pg/mL


complex/ Bond

Elut Plexa SPE


BondElut PBA

SPE



- 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


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


Plasma calibrator chromatogram with extra wash

39

1. Normetanephrine

2. Metanephrine

3. 3-Methoxytyramine

1

2

3


Calibration curves


Results Summary of analyte performance

41


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


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


Results SPE extraction procedure recoveries with spiked formic acid solutions with and without SPE

43

Compound

Absolute Recoveries %*

(n = 9)

Relative recoveries %


(n = 9)




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


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



1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



ng/mL

0.08-100

mg/L

15.6-10000

pg/mL

5-2500

pg/mL


complex/ Bond

Elut Plexa SPE


BondElut PBA

SPE



- 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


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


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


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


Plasma Catecholamines calibrator chromatogram

49

1. Norepinephrine

2. Epinephrine

3. Dopamine

1

2

3


Calibration curves


Results Summary of analyte performance

51


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


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


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


Sample preparation

54

• Why

• Which sample preparation technique to choose


Objectives of Sample Preparation

• Removal of interferences that affect detection and accuracy

• Reach lower detection limits

• Increase method robustness

• Decrease overall operating costs


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


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


Why did we use different sample preparation techniques?

1. Free and Total

Cats/Mets

2. VMA, HVA, 5-

HIAA

3. Mets 4. Cats



ng/mL

0.08-100

mg/L

15.6-10000

pg/mL

5-2500

pg/mL


complex/ Bond

Elut Plexa SPE


BondElut PBA

SPE

58

Sample Dependant

Analyte Dependant OH

OHNH2

O

OHNH

CH3 OH

CH3


http://en.wikipedia.org/wiki/File:5-Hydroxyindolessigs%C3%A4ure_(5-HIAA).svg

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


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


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


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


Metanephrines in plasma

65


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


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


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


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


Catecholamines in plasma Step 2 – Solid Phase Extraction (SPE)

68


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


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


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


More information

70

5991-6194EN 5991-6053EN 5991-6531EN 5991-6530EN


Questions

71

Thank You!


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