ASMS 2011 Sensitive & Robust Accurate Mass Analysis of Small Molecules Using a Dual-Spray Thermal Gradient Focusing ElectrosprayIonization Source

Sh E Ti hShane E. TichyMP 091Agilent Technologies, Inc.  Santa Clara, CA June 2011June 2011

Introduction

It has been demonstrated that TOF instruments canreliably yield low part-per-million (ppm) mass errorsthrough the use of real time reference mass correction.Although reference mass ions can be produced by

Experimental

Analytical nebulizer with active cooling Referenceg p y

“teeing in” suitable reference compounds to the mainanalyte flow, this approach often suffers from ionizationsuppression or solvent gradient effects. Each of thesefactors can affect either the analyte or reference massion response. Loss of analyte ion response will affectlimits of detection and quantitation (LOD/LOQ) andoverall linearity, while loss of reference mass ionresponse typically leads to increased mass errors.

with active cooling Reference nebulizer with thermal shielding

In this study, we utilize a high resolution Q-TOFinstrument equipped with a novel dual-spray thermalgradient focusing electrospray ionization (ESI)interface (Dual Agilent Jet Stream) in which apneumatically nebulized reference mass solution issprayed into the thermal confinement region ofelectrosprayed droplets. The resulting reference massions are largely unaffected by LC gradient conditions

Figure 1: Dual Agilent Jet Stream source provides enhanced sensitivity and robustreference mass ionization.

Gradient Study:ions are largely unaffected by LC gradient conditionsand are highly tolerant of matrix effects. Furthermore,the reference spray did not affect the analyte responsefor the compounds studied.

Experimental

Gradient Study:Alprazolam standards from 5 to 5000 pg/µL concentrations wereseparated at 0.75 mL/min on a 2.1 x 50 mm C18 column using a rapidgradient from 5-95% acetonitrile with 0.1% formic acid (FA). A 5ppmextraction window was used to generate quantitative data. Referencemasses at 121.050873 and 922.009798. were used to provide internalmass correction throughout the gradient study.

Sample Preparation:

Agilent 1290 Infinity UHPLC:An Agilent 1290 Infinity UHPLC system comprising abinary pump, thermostatted autosampler and columncompartment was used to develop high matrix and widegradient narrow-bore analytical methods to test therobustness of the new source design.

p pA commercially available alprazolam standard (Cerilliant) was used toprepare neat calibration standards from 5 to 5000 pg/µL inwater/acetonitrile. Reference ion standards (Agilent ) were prepared in95% acetonitrile at 2µM for the purine standard and 1µM for the HP-0921standard.UHPLC Conditions:

Mobile Phase A: 0.1% FA in WaterAgilent 6540 Ultra High Definition (UHD) Q-TOF:An Agilent 6540 Q-TOF was used to evaluate theperformance of a novel dual-spray thermal gradientfocusing ESI interface. The instrument was operated inExtended Dynamic Range mode for the gradient studyand High Resolution mode for the matrix study.

Dual-Spray Agilent Jet Stream Source:An Agilent Jet Stream interface was modified to add a

B: 0.1% FA in ACNInjection Volume 1 µL + needle wash, 10 sec. Column Eclipse Plus, 1.8 µM, 2.1 x 50

mmColumn Temp. 50 °C

An Agilent Jet Stream interface was modified to add athermally shielded reference nebulizer in addition to theanalytical nebulizer with active cooling. (Figure 1). A1200 series Isocratic pump and 1:100 splitter was usedto provide variable flow control of the reference masssolution for the reference sprayer. A precision sapphiresplitter was used to provide a 1:3 split of the analyticalsprayer gas supply to control gas pressure for thereference sprayer.

Binary Flow 0.75 mL/minBinary Gradient 0.0 min 5% B

0.25 5% B 1.75 95% B2.17 5% B3 5% B

Title ASMS 20xxPage 2

p y 3 5% BStop Time 3.0 minPost Time Off

Results and DiscussionIonization mode Positive ion

Dual-Spray AJSOperational mode Extended dynamic range

Matrix Study:A red wine sample was spiked with reserpine at 150 pg/µL andseparated at 0.6 mL/min on a 2.1 x 150 mm C18 column usinga 40 minute gradient from 1-99% acetonitrile with 0.1% formicacid Empirical formula confirmation was applied to 30

Drying gas temp. 300 °CDrying gas flow 7 L/minSheath gas temp. 350 °CSheath gas flow 10 L/minNebulizer pressure 40 psi

acid. Empirical formula confirmation was applied to 30replicate runs spanning 24 hours of continuous operation tocalculate the mass accuracy of the reserpine standard.Reference masses at 121.050873 and 922.009798. were usedto provide internal mass correction throughout the gradientstudy.

Sample Preparation:

Nozzle voltage 0 VCapillary voltage 3250 VFragmentor 130 VSkimmer 47 V

Mass Range m/z 50 - 1000

A red wine sample was spiked with reserpine at 150 pg/µL andvortexed for 5 seconds. No filtration or centrifugation wasapplied to the sample prior to analysis. Reference ion standards(Agilent) were prepared in 95% acetonitrile at 2µM for thepurine standard and 1µM for the HP-0921 standard.

UHPLC Conditions:

Acquisition rate 4 spectra/secReference Mass Flow 20 µL/min

Quantitative Results from Gradient Study:The quantitation results for Alprazolam with therapid gradient method yielded an excellent fit to alinear calibration curve across three orders of

Mobile Phase A: 0.1% FA in WaterB: 0.1% FA in ACN

Injection Volume 1 µL + needle wash, 10 sec. Column Eclipse Plus, 1.8 µM, 2.1 x 150 mmColumn Temp. 60 °CBi Fl 0 6 L/ ilinear calibration curve across three orders of

magnitude with a correlation coefficient (R2) of0.99937.

Alprazolam - 8 Levels, 8 Levels Used, 33 Points, 33 Points Used, 0 QCs6x10

11.11.21.31.41.5

y = 297.099166 * x - 199.703628R^2 = 0.99891848

Res

pons

e

Binary Flow 0.6 mL/minBinary Gradient 0.0 min 1% B

40 min 99% BStop Time 40 minPost Time 5.0 min

-400 0 200 600 1000 1400 1800 2200 2600 3000 3400 3800 4200 4600 5000 5400-0.10

0.10.20.30.40.50.60.70.80.9

Ionization mode Positive ionDual-Spray AJS

Drying gas temp. 325 °CDrying gas flow 10 L/minSheath gas temp. 350 °CSheath gas flow 12 L/minConcentration (ng/ml)

Area %RSD for the lowest level (5 pg on-column)was 7.3% with five replicates. Calculatedaccuracies were within 15% across the range ofsample concentrations, demonstrating thatquantitation is not compromised by the secondsprayer or the use of reference mass ions.

Concentration

Sheath gas flow 12 L/minNebulizer pressure 35 psiNozzle voltage 0 VCapillary voltage 3250 VFragmentor 125 VSkimmer 47 V

Title ASMS 20xxPage 3

Concentration (pg/µL) 5 25 50 250 500 2500 5000

% Accuracy 112.0 87.8 100.4 91.6 104.0 99.9 100.1

Reproducibility (%RSD, n = 5) 7.3 4.5 3.3 1.9 1.4 1.6 1.8

Signal/Noise 12.4 53.3 145.1 560.2 871.1 1178.0 1165.1

MS Acquisition: Reserpine in Wine

Mass Range m/z 50 - 1000Acquisition rate 1 spectra/secReference Mass Flow 20 µL/min

Results and DiscussionMass Accuracy Results from Matrix Study:A scatter plot of the mass errors from the formulaconfirmation results exhibited mass errors of lessthan 1-ppm. Given the 24 hour duration of the

Stability of Reference Mass Ion Response:An overlay of the EIC traces from each of the 30 runs forthe 121.0509 and 922.0098 reference massesdemonstrates the stability of the reference mass ion signalacross a wide gradient with a complex matrix. Given the

study, these results would be impressive evenwithout the presence of a complex, heavy matrix.This illustrates the stability of the UHD Q-TOFsystem and the effectiveness of the real-time masscorrection.

3

g plow flow of reference mass solution (20 µL/min), it is likelythat the reference ions are desolvated prior to encounteringthe thermal confinement region of the analyte spray. Thiswould largely eliminate solvent and matrix effects andsubstantiates the utility of the dual sprayer approach forreference ion generation.

-1

0

1

2

0 10 20 30 40

Err

or

(pp

m)

EIC 121

-3

-2

Injections

Robustness & Reproducibility of Dual-Spray AJSSource:A l f th T t l I Ch t (TIC) lt

EIC 922

An overlay of the Total Ion Chromatogram (TIC) resultsfor each of the 30 runs shows the remarkableconsistency of both the 1290 Infinity UHPLC system andthe Dual Agilent Jet Stream source. Conclusions

• The Gradient study demonstrated that quantitativeresults could be obtained across a 5-95% acetonitrileresults could be obtained across a 5-95% acetonitrilegradient using a Dual-Spray Agilent Jet Stream (AJS)source with external reference mass correction.

• The Matrix study demonstrated that the referencemass ion response of the Dual-Spray AJS source waslargely unaffected by LC gradient conditions or matrix

effects.

The Extracted Ion Chromatogram (EIC) trace of thereserpine masses indicates the relative scale of thesample peak at 13.831 minutes. Elution times forthe reserpine peak varied by less than 0.05 minutesand the %RSD for the peak areas was 1.66%.

• The Matrix study demonstrated the remarkableconsistency of the Dual-Spray AJS source analyteresponse and the chromatographic performance of the1290 Infinity UHPLC system.

• When a Dual-Spray AJS source is used in conjunctionwith an Agilent Ultra High Definition (UHD) Q-TOF

Title ASMS 20xxPage 4

with an Agilent Ultra High Definition (UHD) Q-TOFsystem, it is possible to obtain sub-ppm massaccuracies throughout a 24 hour study.