Catecholamine Analysis: Evaluation of Method Optimization to Improve Sensitivity and Reduce Limits of Quantitation using LC-MS/MS Adam Senior1, Alan Edgington1, Lee Williams1, Rhys Jones1, Helen Lodder1, Geoff Davies1, Steve Jordan1, Claire Desbrow1, Victor Vandell2 & Elena Gairloch2 1Biotage GB Limited, Distribution Way, Dyffryn Business Park, Ystrad Mynach, Cardiff, CF82 7TS, UK 2Biotage, 10430 Harris Oaks Blvd., Suite C, Charlotte North Carolina 28269, USA

© 2015 Biotage. All rights reserved. All trademarks are the property of their respective companies. MSACL 2015, San Diego www.biotage.com Part Number: P121

OH

OH

OH

NH

OH

OH

OH

NH2

NH2OH

OH dopamine

epinephrine

norepinephrine

a) b)

Introduction Catecholamines are biomarkers for the detection of diseases such as: hypertension, pheochromocytoma and neuroblastoma. The main target analytes are epinephrine, norepinephrine and dopamine (see Figure 1 for details) which are traditionally analyzed using liquid chromatography with electrochemical detection. This poster investigates various parts of the method development process to evaluate the sensitivity of LC MS/MS analysis. A highly sensitive LC-MS/MS system, a Shimadzu Nexera UHPLC coupled to an AB SCIEX Triple Quad 5500 MS was used for analysis. Method sensitivity in terms of precursor ion selection and MRM transitions, chromatography and solid phase extraction protocols were all evaluated for increased sensitivity.

Experimental Reagents Standards were obtained from LGC (Teddington, UK). Acetic acid (HAC), formic acid, ammonium hydroxide, hydrochloric acid, ammonium acetate, ammonium formate, ethylene glycol and LC MS grade chromatographic solvents were obtained from Sigma-Aldrich Chemical Co. (Poole, UK). 18.2 MΩ.cm water was drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK). Pooled human plasma was obtained from The Welsh Blood Service (Pontyclun, UK).

Sample preparation All extractions were performed using polymer-based SPE. EVOLUTE® EXPRESS WCX was evaluated in the 30 mg 96 fixed well plate format.

Sample pre-treatment: 300 μL of plasma was pre-treated by diluting 1:1 with various aqueous buffers and mixed thoroughly. Sample extraction: EVOLUTE® EXPRESS WCX 30 mg 96 well plate; 602-0030-PX01. 600 μL of pre-treated sample was applied per well of the FWP, extraction strategies are shown below in Table 1. Table 1. Extraction Strategies

Step Volume Strategy 1 Strategy 2 Condition 1 mL MeOH MeOH Equilibration 1 mL 50 mM NH4OAC 50 mM NH4OAC Sample load 600 μL plasma 1:1 50 mM NH4OAC plasma 1:1 50 mM NH4OAC Wash 1 1 mL 10% MeOH (aq) 50 mM NH4OAC Wash 2 1 mL propan-2-ol MeOH Elution 1 mL 5% formic acid in MeOH 5% formic acid in MeOH

Post extraction: 4 μL of ethylene glycol was added to each appropriate well of the collection plate prior to analyte elution. The eluates were evaporated to dryness at 40 °C under a stream of air and reconstituted in 200 μL 0.1% HCOOH in 2% MeOH .

UHPLC Conditions Instrument: Shimadzu Nexera UHPLC (Shimadzu Europa GmbH, Duisburg, Germany). Column: ACE Excel 2 C18-PFP 100 x 2.1 mm, 2μ (Hichrom ltd., Theale, UK). Mobile phase: A, 2 mM ammonium formate pH 3.2; B, MeOH; 0.4 mL min-1. Gradient: 2% B isocratic hold for 2.2 min, step to 100% B, hold 0.5 min, step to initial conditions, equilibrate 1.0 min. Column temperature: 40 °C Injection volume: 20 μL

Mass Spectrometry Instrument: Triple Quad 5500 mass spectrometer (AB Sciex, Framingham, US). Ions were acquired in the positive mode using a Turbo V ESI interface and either MRM or Scheduled MRM transitions.

IS: 5500 V Curtain: 35 psi Temperature: 700 °C Gas 1: 50 psi Gas 2: 50 psi

Table 2. MRM Parameters

Analyte Transition DP, V EP, V CE, V CXP, V epinephrine 166.1>107.1 148 8 24 16 norepinephrine 152.1>107.1 25 2 22 25 dopamine 154.1>91.1 50 9 29 13

Results Mass Spectrometry Optimization Source parameters were optimized to enhance the production of dehydrated

precursor ions to increase signal sensitivity of epinephrine and norepinephrine. Figure 2a demonstrates at least a two-fold increase in signal when using a suitably optimized MRM transition for epinephrine and norepinephrine. A concomitant increase in the

dehydrated dopamine precursor was not observed suggesting only the alkyl OH group is susceptible to dehydration. The accompanying blank chromatograms in Figure 2b demonstrate the transitions are free of interferences.

Chromatographic Optimization Chromatographic separation was optimized by comparison of 4 eluent systems with low concentrations of either MeOH or MeCN. Target analyte peak shape and height were maximized using 2 mM formate buffer pH 3.2; increasing the concentration of formic acid to 0.1% was detrimental to chromatography, as was use of acetate as a counter-ion. Increasing concentration of counter-ion resulted in an increase in background noise (data not shown). Use of MeOH as an organic modifier gave higher peaks and greater separation than with MeCN; a reduction in peak width was observed using MeCN

ACE Excel 2 C18-NH2 100 x 2.1 mm 2 µm

Restek Pinnacle BiPh 100 x 2.1 mm 1.9 µm

ACE Excel 2 C18-PFP 100 x 2.1 mm 2 µm

Restek Raptor BiPh 50 x 2.1 mm 2.7 µm

(data not shown). The optimized chromatographic separation was applied to 4 HPLC or UHPLC columns as demonstrated in Figure 3. The C18-PFP column was chosen for further work on the basis of its superior retention, separation, peak shape and height.

Extraction Optimization Sample pre-treatment optimization focused on control of pH below 8 to ensure the amine functional group remained charged whilst maintaining the neutrality of the hydroxyl groups. Additionally, pre-treatment should be at least 1 pH unit away from the approximate pKa of the WCX polymer in order to maximize binding.

The effect of variation in pre-treatment pH on recovery is demonstrated in Figure 4. Recoveries are dependent on extraction strategy (see table 2). Where the protocol is based on strategy 2, 0.1%

HCOOH is detrimental to recovery; for strategy 1, 0.1% HCOOH pre-treatment demonstrates improved recoveries. Sample pre-treatment with 50 mM ammonium formate gave unreliable data with poor repeatability (data not shown).

Several interference wash strategies were investigated, modifying aqueous interference washes with either buffer of organic modifier and modifying the nature and eluotropic strength of the organic interference wash. Figure 5 demonstrates the effect of variation in interference wash steps.

The effect on recovery by modification of the strength and choice of elution solvent acid modifier was investigated. The effect on analyte recovery is shown in Figure 6. The

chart demonstrates 5% formic acid in MeOH to be the most efficient recovery solvent. There may be the potential to reduce this to 2% by modification of the interference wash regime to replace 10% MeOH as an interference wash solvent with 10 mM to 50 mM ammonium acetate (data not shown).

Of the two principal extraction strategies investigated, a protocol based on strategy 1 demonstrated the greatest peak areas. In comparison, a protocol based on strategy 2 gave rise to lower analyte peak areas, see Figure 7 for details. Matrix interference is

lower using strategy 1 and higher using strategy 2. However, as demonstrated elsewhere in the poster relative recoveries were lower even if absolute recoveries were higher.

Due to the structure of the analytes shown in Figure 1 there was concern the extracted analytes may demonstrate volatility under certain conditions. Two strategies to reduce this effect were investigated: addition of HCl to the collection plate to promote formation of hydrochloride salts or addition of small volumes of ethylene glycol to act

as a ‘keeper solvent’. The results are shown in Figure 9. The chart demonstrates that there is analyte loss during extract blowdown and reconstitution. Addition of HCl has a detrimental effect on evaporative losses. The use of glycol as a keeper solvent gives the most beneficial effect to analyte recoveries.

The procedure gives linear recoveries from 100 to 2000 pg mL-1 when spiked into pooled human plasma as demonstrated in Figure 10. Coefficients of determination were greater than 0.99 for all analytes. S/N ratios at 100 pg mL-1 were demonstrated >10:1 (data not shown).

Conclusions » We demonstrate that the use of EVOLUTE EXPRESS WCX 96 fixed well plates to

extract polar catecholamines from pooled human plasma can be used to perform a sensitive, linear assay.

» LOQs were demonstrated to be 100 pg mL-1 for each analyte (per mL of sample loaded) based on an observable peak at 10:1 S/N.

» An extraction protocol based on strategy 2 demonstrates reduced signal suppression when compared to a protocol based on strategy 1. Work is ongoing to fully optimize the extraction protocol.

» Reducing elution volume to 0.5 mL for a protocol based on strategy 1 gave rise to increased recoveries compared to strategy 2. This could be due to increased co-extractives with increasing elution volume.

» Work is ongoing to increase assay recoveries, reduce matrix interference and improve assay reliability, e.g. with the use of high pH extraction, neutralized on collection.

Figure 3. Chromatographic Separation With 2% MeOH in 2 mM formate pH 3.2 (cyan: norepinephrine / blue: epinephrine / red: dopamine)

Figure 1. Catecholamine Structures

Figure 2. XICs Showing Dopamine MRMs (blue: dehydrated precursor / magenta: non-dehydrated precursor)

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epinephrine norepinephrine dopamine

Catecholamine Recovery with Differing Pre-treatment (0.5 mL elute)

50 mM NH4OAC strat1 50 mM NH4OAC strat2

0.1% HCOOH strat1 0.1% HCOOH strat2

Figure 4. Catecholamine Recovery Comparing Sample Pre-treatment (100 pg mL-1 spike plus IS)

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Catecholamine Recovery Comparing Elution Solvent and Modifier

5% HCOOH in MeOH 2% HCOOH in MeOH

2% HCOOH in MeCN 0.5% HCl in MeOHFigure 6. Recovery Comparing Elution Solvent / pH Modifier (100 pg mL-1)

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epinephrine norepinephrine dopamine

Normalized Catecholamine Area Response with Differing Pre-treatment (0.5 mL elute)

50 mM NH4OAC strat1 50 mM NH4OAC strat2

0.1% HCOOH strat1 0.1% HCOOH strat2Figure 7. Relative Catecholmine Area Responses

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epinephrine norepinephrine dopamine

Catecholamine Recovery Comparing Interference Washes with Other Conditions Fixed

10% MeOH (aq) 50 mM NH4OAC 100% IPA 1:1 IPA:MeCN 100% MeOH

Figure 5. Catecholamine Recovery Comparing Interference Washes (100 pg mL-1)

epinephrine

dopamine

norepinephrine

Figure 10. Calibration Curves, Plasma Extraction 10 to 2000 pg mL-1

0.020.040.060.080.0

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epinephrine norepinephrine dopamine

Blow-down / Recovery of Catecholamines With Differing Collection Plate Eluate Modifiers

5% FA / 2µL Et glycol 2% FA / 2µL Et glycol

5% FA / no treatment 2% FA / no treatment

5% FA / 50µL 50mM HCl in MeOH 2% FA / 50µL50 mM HCl in MeOH

Figure 9. Recovery Comparing Analyte Blow-down/Recovery (50 ng epinephrine/dopamine, 250 ng epinephrine)