simultaneous analysis of catecholamines and metanephrines ......xavier rodriguez piró ©2015 waters...

Simultaneous Analysis of Catecholamines and Metanephrines by Mixed-mode SPE and and Metanephrines by Mixed mode SPE and

HILIC UPLC/MS/MS

Xavier Rodriguez Piróg

©2015 Waters Corporation 1

Outline

Background & ChallengesM th d lMethodology

Results Conclusions Conclusions


Backgroundg

Indicators of pheochromocytomas, neuroblastomas, ganglioblastomas, ganglioneuromasg g , g g

Norepinephrine Epinephrine DopaminelogP =-0.68 logP =-0.43 logP =-0.03


NormetanephrinelogP =-0.39

MetanephrinelogP = 0.00

3-methoxytyraminelogP = 0.68

Typical catecholamine and metanephrine analysis methodsp y

Reversed-phase HPLC with ion-pairing Electrochemical Detection Electrochemical Detection Separate methods for catecholamines and metanephrines Sample prep often including SPE with Alumina Long LC methods


Challengesg

Very polar compounds– Elute very early under reversed-phase conditionsy y p– Subject to Matrix Effects in urine (lots of early eluting salts and low

MW components) Basic Compoundsp

– Unstable at high pH Endogenous compounds Must separate EP from NMT Must separate EP from NMT

– Isobaric molecules which share MRM transitions Urine is acidified at collection

Needs to be buffered for extraction– Needs to be buffered for extraction– Capacity issues for IEX sorbent

Plasma: very low concentrations/ L


– pg/mL

Outline




Chromatography - HILICg p y

HILIC - Hydrophilic Interaction Chromatography— Term coined in 1990 by Alpert to distinguish from normal-phase*

HILIC is a variation of normal-phase chromatography without the disadvantages of using solvents that are not miscible in water— “Reverse reversed-phase” or “aqueous normal-phase” Reverse reversed phase or aqueous normal phase

chromatography

Designed to retain extremely polar analytes

Stationary phase is a POLAR material— Silica, hybrid, cyano, amino, diol, amide


Multi-modal Retention Mechanisms in HILIC

Combination of partitioning, ion‐exchange and hydrogen bondingand hydrogen bonding

• Polar analyte partitions between bulk mobile phase and partially immobilized polar layer on material surface

• Secondary interactions b t f il lbetween surface silanols and/or functional groups with the charged analyte leading to ion‐exchange

• Hydrogen bonding between positively charged analyte and negatively charged surface silanols


gD.V. McCalley, U. D. Neue, J. Chromatogr. A 1192 (2008) 225‐229

E.S. Grumbach, D.M. Diehl, U.D. Neue, J. Sep. Sci. 31 (2008), 1511‐1518

A. Méndez, E. Bosch, M. Rosés, U. D. Neue, J. Chromatogr. A 986 (2003), 33‐44

ACQUITY BEH AmideQ


Instrument Parameters in urine

ACQUITY I-Class MPA: 5:95 ACN:H2O with 50 mM NH4COOH (pH 3.0)

Time Flow %A %BMPB: 85:15 ACN:H2O with 30 mM NH4COOH (pH 3.0)Column: ACQUITY UPLC BEH Amide 1.7 μm; 2.1 x

100 mmSNW: MPB

Time Flow %A %B0.0 0.6 0.0 100.0

1.0 0.6 0.0 100.0

2.0 0.6 10.0 90.0WNW: MPB

Instrument: Xevo® TQDIonization: ESI+

2.1 1.0 10.0 90.0

2.5 1.0 30.0 70.0

2.6 1.0 0.0 100.0Ionization: ESI+

Capillary voltage: 0.5 kVDesolvation Gas: 900 L/hrCone Gas: 0 L/hr

3.9 1.0 0.0 100.0

4.0 0.6 0.0 100.0

Desolvation Temp: 550 ˚CSource Temp: 150 ˚C


High concentration of NH4COOH in MPB helps with peak shapeNeeds to be balanced with 15% H2O to maintain solubility

100

MPB Salt Concentration

%

100

5-HT

DA

EP NE

100

0.50 1.00 1.50 2.00 2.500

10 mMDA% 20 mM

100

0.50 1.00 1.50 2.00 2.500

%

30 mM


Time0.50 1.00 1.50 2.00 2.500

Danaceau et al., Bionanalysis 4(7) 783-794 (2012)

Column Temperature

%

10020 oCDA

EP

NE5-HT

%

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.400

30 oC

%

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.400

40 oC

%

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.400

50 oC

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

60 oC


Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

Danaceau et al., Bionanalysis 4(7) 783-794 (2012)

TQD Conditions Q

Analyte RT (min)MRM

transitionsm/z

Cone Voltage

(V)

CollisionEnergy

(eV)

3-methoxytyramine 0.83168.1>91.0168 1>119 0

2222

2418168.1>119.0 22 18

Metanephrine 0.89198.1>180.0198.1>165.

1818

818

Normetanephrine 1.16184.1>166.0184 1>134 1

2012

818184.1>134.1 12 18

Dopamine 1.24154.0>91.0154.0>119.0

2222

2018

Epinephrine 1.38184.1>166.0184.1>107.0

2020

820

Norepinephrine 1.93170.0>152.0170.0>107.0

1414

620

Compounds with matched colors share common precursors


Compounds with matched colors share common precursorsSome share common fragments as well

Cats/Mets in urine ChromatographyChromatography

Cal Std 50 ng/mLCal_50_131216_003 Sm (SG, 2x2) 2: MRM of 5 Channels ES+

168 068 > 90 985 (3 MT)0 83

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100 168.068 > 90.985 (3-MT)1.17e60.83

Cal_50_131216_003 Sm (SG, 2x2) 2: MRM of 5 Channels ES+ 198.068 > 180 (Metanephrine)0.89

3-MT

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100 8.62e6

Cal_50_131216_003 Sm (SG, 2x2) 1: MRM of 8 Channels ES+ 154.004 > 91.044 (Dopamine)

9.49e41.22

MTN

DA

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

Cal_50_131216_003 Sm (SG, 2x2) 1: MRM of 8 Channels ES+ 184.068 > 166.04 (Epi/Normet)

8.34e51.16

1.38 EpiNMT

DA

%

100

0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

Cal_50_131216_003 Sm (SG, 2x2) 3: MRM of 3 Channels ES+ 170.004 > 152.042 (Norepinephrine)

7.00e41.92

NE


Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

02.04

Column: BEH Amide, 2.1 x 100 mm; 1.7 μm

Instrument Parameters in plasmap

ACQUITY UPLC MPA: 5:95 ACN:H2O with 30 mM NH4COOH (pH 3.0)

Time Flow %A %BMPB: 85:15 ACN:H2O with 30 mM NH4COOH (pH 3.0)Column: ACQUITY UPLC BEH Amide 1.7 μm; 2.1 x

100 mmSNW: MPB

Time Flow %A %B0.0 0.6 0.0 100.0

1.0 0.6 0.0 100.0

2.0 0.6 10.0 90.0WNW: MPB

Instrument: Xevo® TQ-SIonization: ESI+

2.1 1.0 10.0 90.0

2.5 1.0 30.0 70.0

2.6 1.0 0.0 100.0Ionization: ESI+

Capillary voltage: 0.5 kVDesolvation Gas: 900 L/hrCone Gas: 150 L/hr

3.9 1.0 0.0 100.0

4.0 0.6 0.0 100.0

Desolvation Temp: 550 ˚CSource Temp: 150 ˚C


High concentration of NH4COOH in MPB helps with peak shapeNeeds to be balanced with 15% H2O to maintain solubility

MS transitions and compound specific parameters - plasmap p

Analyte RT (min)MRM

transitionsCone

Voltage CollisionEnergy

m/z (V) (eV)

3-Methoxytyramine 0.84151.1>91.16151.1>119.2

1717

2218

Metanephrine 0.91180>165.1180>148.1

3535

1620

Normetanephrine 1.17166.1>134.1166.1>149.1

5050

1610

Dopamine 1.25137.1>91.1154.1>137.2

5029

1810

Epinephrine 1 40184.1>166.1 15 8

Epinephrine 1.40166.1>107 15 18

Norepinephrine 1.98152>135.2152>79.2

4620

1420


ChromatographyEndogenous plasma Cats/Metsg p

3 MT DA3-MT DA

MTN EP

NMT NE


Compound 3-MT MTN NMT DA EP NE

Conc. (pg/mL) 7 32 71 0 29 361

Oasis Family of Sorbents:Reversed-Phase and Mixed Modes


Oasis® 2x4 MethodSelect Sorbents

Characterize your Analyte and Select the Sorbent

For Strong AcidspKa 10S l

For AcidspKa 2-8

For BasespKa 2-10

SelectOasis® WAX

SelectOasis® WCX

SelectOasis® MAX

Select Oasis® MCX

*also quatsalso quats

Neutralsare Retained on

all Sorbents


all Sorbents

Oasis® 2x4 Method:

For Bases:pKa 2-10Use Oasis® MCX

For Strong AcidspKa 10Use Oasis® WCX

For AcidspKa 2-8Use Oasis® MAXUse Oasis MCX Use Oasis WAX Use Oasis WCX Use Oasis MAX

P S l

Protocol 2P S l

Protocol 1Prepare Sample

Condition/EquilibrateLoad Sample

W h

Prepare Sample

Condition/EquilibrateLoad Sample

W h

Neutrals

Wash:5% NH4OH

Elute 1:100% MeOH

Wash:2% Formic acid

Elute 1:100% MeOH 100% MeOH

Elute 2:2% Formic Acid in MeOH

100% MeOH

Elute 2:5% NH4OH in MeOH


Bases Strong AcidsStrong Bases Acids

Oasis® WCX Ion Exchange Retention Map for Weak Basesp

maxOasis® WCX SORBENT CHARGE

(N) ( )

n

max (N) (-)Sorbent (N)

entio

n Weak Base (N)Unonized

Weak Base (+)Ionized

Sorbent (N)Unionized Sorbent (-)

Ionized

Ret

e IonizedWeak Base (+)Ionized

0 2 4 6 8 10 12 14

minpKa of analytepKa of sorbent


Maximum Retention When Sorbent and analyte are IonizedpH0 2 4 6 8 10 12 14

WCX Extraction Procedurefor Urinary Cats and Mets

Condition WCX 30 mg Plate1 mL MeOH then 1 mL Water

y

Sample Pretreatment400 µL urine

Add 1 mL of 500 mM NH4CH3COO + 40 µL IS (1000 ng/mL)(1000 ng/mL)

Load Entire pretreated sample

Wash1 mL of 20 mM NH4CH3COOfollowed by 1 mL of MeOH

Elute2 x 250 µL

(85:15 ACN:H2O+ 2% formic acid)


Inject 10 µL

WCX Extraction Procedurefor Urinary Cats and Mets

Condition WCX 30 mg Plate1 mL MeOH then 1 mL Water

y

Sample Pretreatment400 µL urine

Add 1 mL of 500 mM NH4CH3COO + 40 µL IS (1000 ng/mL)(1000 ng/mL)


WCX enables loading, retention and elution at pH

Sample PreparationOasis µElution Plate Technologyµ gy

Patented plate design Ideal for SPE cleanup and analyte enrichment of Ideal for SPE cleanup and analyte enrichment of

small sample volumes (10 µL to 375 µL) Elute in as little as 25µL; up to 15X concentration No evaporation and reconstitution required

–Eluates can be directly injected –Saves time–No evaporative loss

Speed 96 well plate in

WCX μElution Extraction Procedurefor Plasma Cats and Mets

Condition WCX μElution Plate200 μL MeOH then 200 μL Water

Sample Pretreatment250 µL plasma

Add 250 µL of 50 mM NH4CH3COO + 50 µL IS (2 5 ng/mL)(2.5 ng/mL)


Wash200 μL of 20 mM NH4CH3COO

followed by 200 μL 50:50 ACN:IPA

Elute2 x 25 µL

(85:15 ACN:H2O+ 2% formic acid)


Inject 15 µL

WCX μElution Extraction Procedurefor Plasma Cats and Mets

Condition WCX μElution Plate200 μL MeOH then 200 μL Water

Sample Pretreatment250 µL plasma

Add 250 µL of 50 mM NH4CH3COO + 50 µL IS (2 5 ng/mL)(2.5 ng/mL)


WCX enables loading andelution at pH

Outline




Cats & Mets in urineRecovery and Matrix Effectsy

100%

40%

60%

80%

0%

20%

40%

-40%

-20%

-80%

-60% Recovery

Matrix Effects


Matrix Effect improvement vs.Reversed-Phase Chromatographyg p y

100%

40%

60%

80%

0%

20%

40%

-40%

-20%

Recovery

-80%

-60% Matrix Effects

Matrix Effects (RP)


Cats & Mets in urineLinearityy

Res

pons

e

100

200 NormetanephrineR2 = 0.999

ng/mL-0 50 100 150 200 250 300 350 400 450 500

-0

nse 300

400

EpinephrineR2 = 0.999

ng/mL

Res

pon

-0

100

200

R 0.999


ng/mL-0 50 100 150 200 250 300 350 400 450 500

-0

Cats & Mets in urineQC ResultsQ

3‐MT Metanephrine Normetanephrine

QC Spike Conc. Acc Bias %CV Acc Bias %CV Acc Bias %CV

1.6 94.4% ‐5.6% ‐2.6% 98.9% ‐1.1% ‐2.9% 98.8% ‐1.2% ‐3.9%

8 95.9% ‐4.1% ‐3.0% 100.5% 0.5% ‐1.7% 101.0% 1.0% ‐4.2%

80 101.8% 1.8% ‐4.3% 104.0% 4.0% ‐0.9% 103.4% 3.4% ‐1.6%

400 109.8% 9.8% ‐5.0% 101.9% 1.9% ‐2.1% 100.8% 0.8% ‐0.5%

Mean 100.5% 101.3% 101.0%

Dopamine Epinephrine Norepinephrine

QC Spike Conc. Acc Bias %CV Acc Bias %CV Acc Bias %CV

1.6 92.1% ‐7.9% ‐9.5% 95.6% ‐4.4% ‐6.2% 94.0% ‐6.0% ‐7.2%

8 96.5% ‐3.5% ‐11.0% 95.4% ‐4.6% ‐3.2% 98.7% ‐1.3% ‐3.3%

80 102.8% 2.8% ‐2.0% 103.4% 3.4% ‐1.5% 99.2% ‐0.8% ‐14.8%

400 103.4% 3.4% ‐2.4% 103.0% 3.0% ‐1.9% 98.3% ‐1.7% ‐12.9%

Mean 98.7% 99.4% 97.5%


Mean 98.7% 99.4% 97.5%

Cats & Mets in plasmaRecovery and Matrix Effectsy

Recovery and Matrix Effects

80%

100%

120% RecME

40%

60%

80%

0%

20%

-40%

-20%


Cats & Mets in plasmaCalibration summaryy

R2 Mean % Dev. Max % Dev. Endogenous(pg/mL)

Corrected Calibration Range

3‐MT 0.9993 0.25% 2.89% 7 17‐2007

Metanephrine 0.9997 0.00% 2.50% 32 42‐2042

Normetanephrine 0 9998 0 00% 1 72% 71 81 2081Normetanephrine 0.9998 0.00% 1.72% 71 81‐2081

Dopamine 0.9994 ‐0.33% 4.57% 0 10‐2000

Epinephrine 0.9990 0.84% 11.83% 29 79‐10079

Norepinephrine 0.9995 0.00% 2.59% 361 411‐10411


Calibration Curves in plasmaDopamine and Metanephrinep p

DopamineR2 = 0.9994

MetanephrineR2 = 0.9997


Cats & Mets in plasmaQuality Control ResultsQ y

QC spike concentration

40 pg/mL 100 pg/mL 400 pg/mL 800 pg/mL

Mean S.D. %CV Mean S.D. %CV Mean S.D. %CV Mean S.D. %CV

3‐MT 99.9% 7.4% 7.4% 99.2% 3.0% 3.1% 105.9% 1.8% 1.7% 93.9% 2.6% 2.8%

Metanephrine 99.9% 2.0% 2.0% 97.6% 0.8% 0.8% 107.3% 1.2% 1.1% 94.6% 1.7% 1.7%

Normetanephrine 99.8% 1.6% 1.6% 96.8% 1.7% 1.8% 104.6% 0.4% 0.4% 93.4% 1.0% 1.1%

Dopamine 97.0% 7.2% 7.4% 91.2% 3.4% 3.7% 103.7% 3.1% 3.0% 95.6% 2.7% 2.8%

200 pg/mL 500 pg/mL 2000 pg/mL 4000 pg/mL

Mean S.D. %CV Mean S.D. %CV Mean S.D. %CV Mean S.D. %CV

Epinephrine 97.3% 4.3% 4.4% 98.8% 2.2% 2.2% 100.8% 1.4% 1.4% 97.0% 2.6% 2.6%

Norepinephrine 105.1% 7.7% 7.4% 102.6% 8.2% 8.0% 96.7% 1.3% 1.3% 97.1% 4.2% 4.3%


All accuracies and all %CV’s

Conclusions

Rapid, robust chromatography with baseline separation of epinephrine and normetanephrine– BEH Amide UPLC– Reduction in matrix effects vs. RP chromatography for urine

Efficient extraction in 96-well formatWCX bl t ti t H 7 0– WCX enables extraction at pH

Questions ?Q


simultaneous analysis of catecholamines and metanephrines ......xavier rodriguez piró ©2015 waters...

Documents