esi and apci

Introduction

The invention and commercialization of electro-spray ionization (ESI) was revolutionary whenapplied to the field of organic analytical chem-istry. For the first time, chemically and thermallyfragile molecules (proteins, drugs, environmentalcompounds, to name a few classes) could easilyand reliably be analyzed in high sensitivity byLC/MS techniques. ESI is used today in thousandsof laboratories around the world for routine highsensitivity analysis of these and other analytes. As a testimony to the utility of this technique, itsinventor, Professor John Fenn, was one of therecipients of the Nobel Prize in chemistry in 2002.

As remarkable as the technique of ESI is, it doesnot ionize all organic compounds of interest thatmay be eluted from an HPLC. It has been estimatedthat about 80% of all analytes respond by ESI,leaving a significant fraction as undetectable. Thislimitation was noticed early in the development ofESI, and other ionization techniques were devel-oped almost simultaneously to fill this need. The technique of atmospheric pressure chemicalionization (APCI) and the related atmospheric

Simultaneous Electrospray andAtmospheric Pressure Chemical Ionization: The Science Behind theAgilent Multimode Ion Source

Technical Overview

Steven M. Fischer and Patrick D. PerkinsAgilent Technologies

pressure photoionization (APPI) are the best-known alternatives.

Further study of ESI and APCI pointed up funda-mental differences between them.[13] The sprayconditions and voltage conditions for optimal ESIoperation are very different than for optimal APCIoperation. In optimized ESI, ion formation occursin the following sequence:

1. A surface charge is induced on the emergingliquid by imposition of an external electricalfield

2. The charged liquid is converted to a spray ofdroplets

3. The droplets shrink due to solvent evapora-tion, which increases the surface charge density

4. Droplets undergo fission as a consequence ofthe high surface field strength

5. When a droplet is small enough, ions are ejectedfrom the droplet due to the high field strengthat the surface of the droplet

Simultaneous Electrospray and Atmospheric Pressure ChemicalIonization: The Science Behind the Agilent Multimode Ion Source

2

Agilent Technologies

Under optimal APCI conditions, ion formationoccurs as follows:

1. A spray of droplets is formed

2. The spray is dried completely creating a mix-ture of solvent molecules and analyte mole-cules in the gas phase

3. The solvent molecules and analyte moleculespass through a corona discharge

4. The corona discharge ionizes the solvent mole-cules, creating solvent ions

5. The solvent ions transfer charge to the analytemolecules, creating analyte ions

From the above lists, it should be apparent thatthe spray formation and timing of ion generationare different in ESI and APCI. As a result of thesedifferences, all MS manufacturers have developeddedicated optimized ion sources for each tech-nique. Users who want to analyze a sample usingboth techniques generally must run the analysisusing one source first, and if there is no response,run the sample on the instrument using the othersource. This significantly decreases the number ofsamples per day that may be analyzed on a giveninstrument.

In response to this diminished throughput causedby the fundamental differences between ESI andAPCI, novel ion sources have been developed tocombine the two ionization techniques into onesource.[4,5] The differences in performance of thesecombination (also known as dual-mode or multimode) sources may be traced to how theyaddress the incompatibilities between ESI andAPCI. Do they perform the ionization in the sameregion of space, or do they separate the ionizationregions and attempt to optimize the performanceof each? And if the ionization regions are sepa-rate, how is the sample delivered to the tworegions and how is the separation maintained?This document describes the features of theAgilent multimode ionization source and how itaddresses these incompatibilities, permitting high-flow, high-sensitivity ESI and APCI to be per-formed independently or simultaneously on asingle injection of a sample.

Experimental

All experiments were performed using an Agilent1100 Series LC system comprised of a binarypump, autosampler, thermostatted column com-partment; and an Agilent 1100 Series LC/MSD SL(G1956D) quadrupole mass spectrometer. TheLC/MSD was equipped with a multimode source,dedicated ESI source, or dedicated APCI source asneeded. The sources for the LC/MSD are all inter-changeable. The multimode source is currentlysupported on G1946 LC/MSD instruments, modelsB, C, and D, and on all G1956 LC/MSD instru-ments, models A and B. However, some of thesemodels need both hardware and software upgradesto be able to control the multimode source; seeyour Agilent representative for details.

The LC/MS conditions for the analysis of the sam-ples in this note vary with sample type; therefore,they are stated in the sidebars in the appropriatesection.

Results and Discussion

The Agilent multimode source is a compact ionsource that fits on the left side of the LC/MSDquadrupole mass spectrometer. Some distinguish-ing features of the source are highlighted (Figure 1).

Concepts behind simultaneous (mixed mode) ESIand APCI operation

A functional view of this source is given in Fig-ure 2. The top part of the source is the ESI zonewhere the conditions exist for optimal electro-spray ion formation. The bottom part of thesource contains the APCI zone where optimalconditions exist for this ionization process. Someof the functional elements of this source are alsoshown and are discussed in detail below.

3Simultaneous Electrospray and Atmospheric Pressure ChemicalIonization: The Science Behind the Agilent Multimode Ion Source


ESI zone with nebulizer, charging electrode,and reversing electrodeThis source uses the same patented orthogonalESI geometry with a grounded nebulizer as isfound on Agilents dedicated ESI source. Thisdesign has proven to be extremely robust, direct-ing the vast majority of solvent and spent dropletsto waste while selectively diverting the ions to thecapillary entrance of the mass spectrometer. Thegrounded nebulizer permits the mass spectrome-ter to be interfaced directly to other devices, e.g.,capillary electrophoresis instruments, withoutfear of electrical interactions or special isolationprecautions. The elements in the nebulizer arecarefully optimized to provide the proper dropletsize and size distribution for maximum produc-tion of ions. The charging electrode in the ESIzone (Figure 3) induces the surface charge on theliquid to start the ion formation, and ESI ions aregenerated here. The nebulizing gas flow pushesthe droplets and ESI ions past the reversing elec-trode towards the APCI zone below.

APCI corona needle

Nebulizer

Sensor for vapor temperature Field-shaping electrodes

IRemitters

APCI counterelectrode

Figure 1. (left) The Agilent multimode ion source mounted on the MSD quadrupole mass spectrometer. (right) Source opened toshow a few of the inner parts.

Capillary

HPLC Nebulizer

Drying gas

Corona needle

Thermalcontainer

Chargingelectrode

Reversing electrode

Sensor for vapor temperature

APCI counterelectrode

inlet

ESI Zone

APCI Zone

IR emitters

Figure 2. An overview diagram of the multimode source

4Agilent Technologies

Infrared emittersA major innovation in this ion source is a set ofinfrared (IR) emitters (Figure 4) to aid in dryingthe aerosol. These emitters give off light in abroadband emission whose maximum is at 3 m,the absorption wavelength of the hydrogen-bondedhydroxyl group of water. About 40% of the emit-ters power occurs in this absorption band, andthe emitters maximum power output is 270 W,enough to completely and instantly vaporize 2 mL/min of 100% water and maintain the vaportemperature at 250 C. For production of ESI ions,the emitters act in a similar role as heated dryinggas, causing the droplets to shrink, undergo fis-sion (Coulombic explosion) and eject ions. Thus,the nitrogen drying gas consumption of an instru-ment using a multimode source is reduced consid-erably compared to that of an instrument with adedicated ESI source using only heated dryinggas. Typical nitrogen consumption is 57 L/minusing the multimode source, whereas 1214 L/minis usual for a standard ESI source. The power tothe IR emitters is controlled by feedback from avapor temperature sensor located in the exit gasflow stream of the multimode source. As the flowand/or mobile phase composition changes during

an LC/MS analysis or during restoration of theinitial gradient conditions, the emitter powerchanges to maintain the same vapor temperature(the vaporizer temperature as set in the soft-ware). This feedback control maintains ESI andAPCI at the conditions for optimal performanceand minimizes decomposition of thermally sensi-tive analytes that may be present.

APCI zone with IR emitters, corona needle, andAPCI counter-electrodeThe droplets continue down into the thermal con-tainer that surrounds the APCI zone of the source.Here, radiation from the IR emitters completelyconverts the droplets to solvent and analyte vaporto create the conditions for optimal APCI. A coronadischarge is struck between the APCI needle anda counter-electrode (Figure 5). This discharge ion-izes the solvent molecules which subsequentlytransfer their charges to the analyte molecules,


IR emitters

Nebulizer

Corona

sensor

HPLC inlet

needle

Chargingelectrode

Reversing electrode

Thermal container

Separator

Vaportemperature

Figure 4. Diagram of the multimode source showing the rela-tionship of the IR emitters and vapor temperature sensor withrespect to other parts of the source

Charging electrode

Reversing electrode

Nebulizer

Figure 3. A close-up diagram of the ESI zone of the multimodesource

5Agilent Technologies Simultaneous Electrospray and Atmospheric Pressure ChemicalIonization: The Science Behind the Agilent Multimode Ion Source

producing ions. A separator in the middle of thesource screens most of the ESI ions from reactionwith the corona discharge present in the APCIzone. The ESI ion stream on one side of the sepa-rator and the APCI ion stream from the other sidecombine below the separator and are guided simul-taneously into the capillary entrance of the MSD.

Simultaneous (mixed mode) ESI and APCI

From the above discussion, it may be seen that thedesign of the multimode source solves the prob-lems of maintaining separate (but connected) ESIand APCI regions in a simple yet elegant fashion.The liquid from a single nebulizer is first chargedand converted to an aerosol in the presence of anelectrical field, the conditions for optimal ESI ionformation. Then, the residual droplets and ESIions are pushed past the reversing electrode intothe heated region on their way to the APCI zone.

The reversing electrode is the electrical boundarybetween the ESI and APCI zones, and without itions would tend to migrate back toward the charg-ing electrode instead of forward toward the massspectrometer entrance. In the thermal containerspace between the ESI and APCI zones, the IRemitters convert the residual droplets to vaporwhile having no effect on any ESI ions that may be present. Once the vapor reaches the APCI zone,the conditions exist for optimal APCI ion forma-tion. Thus, simultaneous efficient production ofESI and APCI ions is a function of the overalldesign of the source and the interaction of itsunique parts.

If desired, the multimode source also functionswell in ESI-only or APCI-only modes. To performonly ESI, the APCI corona needle voltage is turnedoff while leaving the ESI charging electrode volt-age on, and only ESI ions are produced. To per-form only APCI, the ESI charging electrode isturned off while leaving the APCI corona needlevoltage on, and only APCI ions are produced. Thesevoltages are under data system control, and thusthe instrument may be switched between ESI,APCI, and mixed mode operation manually with aclick of the mouse, automatically during an analy-sis, or automatically during a sequence.

Coronaneedle

Counterelectrode pin

Figure 5. A close-up diagram of the APCI zone of the multi-mode source


Proof of concept: ESI-only, APCI-only and simulta-neous (mixed mode) ESI and APCI

The proof of concept for simultaneous ESI andAPCI operation is shown in Figure 6. A mixture of bovine insulin and indole was infused into themultimode source mounted on the LC/MSDquadrupole system. Bovine insulin, like all pro-teins possessing multiple basic sites, yields a char-acteristic multiple-ion spectrum in positive ESImode; it has no response in APCI mode. Indole,being a small molecule with a weakly basic site,protonates poorly under ESI conditions butresponds well in positive APCI mode. When a mix-ture of the two was analyzed using the multimodesource, bovine insulin had a strong response inESI-only mode, whereas indole had a very weakresponse. The opposite was true in APCI-onlymode, with indole having a strong response. Bothcompounds responded well when the simultane-ous ionization mode was enabled. This and othertests indicate that the multimode source producesESI and APCI spectra similar to those of the dedi-cated sources and that the two ionization modesmay be switched off, on, or combined for simulta-neous ionization.


118.1

m/z0 1000 2000

Max: 90624

Indole

1434.3 1912.0

m/z0 1000 2000

Max: 103701

118.1 2867.9

1434.3

m/z0 1000 2000

Max: 771491912.1

118.1

2867.8

InsulinIndole

Insulin

MM

MM-APCI

MM-Mixed

-ESI

Indole 1147.7

1147.6

Figure 6. Proof of concept of simultaneous (mixed mode) ESIand APCI using a mixture of indole (50 pmol/L) and bovineinsulin (9 pmol/L). (top) Source in ESI-only mode, exhibitinga response from insulin with a weak response from indole.(middle) Source in APCI-only mode, with only indole showinga response. (bottom) Source in mixed mode, with both indoleand insulin showing strong responses.

LC ConditionsColumn: None (infusion)

0.1% acetic acidFlow rate: 0.1 mL/min

MS ConditionsSource: Multimode, positive ESI-only,

APCI-only, or mixed modeDrying gas flow: 5 L/minNebulizer: 40 psigDrying gas temp: 350 CVaporizer temp: 200 CCapillary voltage: 2500 VCorona current: 2 APeakwidth: 0.2 minTime filter: OnScan: 1003000 m/zFragmentor: 150 VStepsize: 0.1 m/z


Another example of this performance is shown inFigure 7. This was a mixture of four compounds,each of which responded most strongly in a singleone of four possible modes: negative ESI, negativeAPCI, positive ESI, or positive APCI. By settingthe ionization parameters to one of these possibil-ities, e.g., positive ESI mode, only the response ofcrystal violet was seen. However, injection of thesame mixture using mixed mode operation andpositive/negative polarity switching allowed detec-tion of all four compounds using a single injectionand analysis. A broad range of response like thisis a welcome feature for analyses such as com-pound screening and high throughput analysis inearly drug discovery.

LC ConditionsColumn: 30 x 2.1 mm ZORBAX SB-C18,

3.5 m (Agilent part no. 873700-902)Mobile phase: 35:65 water:methanol with 0.2% acetic acidGradient: None (isocratic)Flow rate: 0.4 mL/minColumn temp: RoomInjection volume: 1 L

MS ConditionsSource: Multimode, mixed mode with positive/negative

switchingDrying gas flow: 5 L/minNebulizer: 60 psigDrying gas temp: 350 CVaporizer temp: 200 CCapillary voltage: 1000 V (positive and negative) Corona current: 2 APeakwidth: 0.2 minTime filter: OnSIM ions

(positive mode): 372.2 m/z (crystal violet), 168.1 m/z (carbazole)(negative mode): 165.1 m/z (hexanesulfonic acid), 193.1 m/z

(9-phenanthrol)Fragmentor:

(positive mode): 160 V at 168.1 m/z, 180 V at 372.2 m/z(negative mode): 140 V at 165.1 m/z, 160 V at 193.1 m/z

min0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

0.51

4

1.14

1

min0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

0.67

0

0.95

8

Negative Ion

Hexanesulfonicacid (ESI) m/z 165

9-Phenanthrol(APCI) m/z 193

SO

O OH

OH

Positive Ion

Crystal Violet(ESI) m/z 372

Carbazole(APCI) m/z 168

NH

N+

NN

Figure 7. Proof of concept ofmixed mode operation with positive/negative polarityswitching using the multimodesource demo sample. These fourcompounds each respond pri-marily in only one ionizationmode and only one polarity butmay be detected in a singleanalysis.


Infrared emitters provide conditions for optimalAPCI

APCI requires different spray and voltage condi-tions than ESI for maximum production of ions. Ithas been known for many years that the best APCIperformance is obtained when both the solventand the analyte are in the vapor state. All instru-ment manufacturers, including Agilent, have addi-tional heating on their dedicated APCI sources orAPCI probes to achieve these conditions. Theinfrared emitters of the multimode source accom-plish the same feat, completely vaporizing theHPLC effluent and any analytes it contains, toproduce this vapor. Figure 8 shows replicate injections of diphenhydramine, a compound thatresponds well by ESI and APCI, at an HPLC flowrate of 0.4 mL/min. As the vaporizer temperaturewas increased from 60 C to 250 C, the HPLCeffluent was converted essentially completely tothe vapor state, and the APCI response increasedapproximately five-fold.


3.5 m (Agilent part no. 873700-902)Mobile phase: 50:50 water:acetonitrileGradient: None (isocratic)Flow rate: 0.4 mL/minColumn temperature: RoomInjection volume: 1 L

MS ConditionsSource: Multimode, positive APCI-onlyDrying gas flow: 5 L/minNebulizer: 20 psigDrying gas temp: 350 CVaporizer temp: 60, 115, 250 CCapillary voltage: 2500 VCorona current: 6 APeakwidth: 0.05 minTime filter: OnSIM ions: 167.1, 256.2 m/zFragmentor: 120 V


Vaporizer = 250 C(500%)



min2 4 6 8 10 12 14

0

400000

800000

1200000

1600000

min2 4 6 8 10 12 14

0

400000

800000

1200000

1600000

min2 4 6 8 10 12 14

0

400000

800000

1200000

1600000

Figure 8. Improvement in diphenhydramine response with increasing IR emitter vaporizer temperature. Response increased by 5X over the temperature range of the experiment.


A more dramatic illustration of this effect isshown in Figure 9. The compound here is reser-pine, also a compound that responds well by bothESI and APCI and is generally used to test thesensitivity of LC/MS systems. Reserpine has alower vapor pressure than diphenhydramine whencompared at the same temperature, so greatervaporizer temperatures are needed to convert theanalyte into the gas phase. The experiment con-sisted of performing five consecutive injections of reserpine, 10 pg each, increasing the vaporizertemperature by 25 C, performing another fiveinjections, and so forth up to the temperaturelimit. Then the vaporizer was allowed to cool to its starting temperature while injections of reser-pine were continued. The flow rate and mobilephase during this experiment was 1.0 mL/min of75:25 methanol:water with 5 mM ammonium for-mate, typical for APCI, which performs better athigher flow rates.

The signal response for reserpine increasedalmost 17 times as the vaporizer temperature wasincreased. Furthermore, the response of the emit-ters was quite rapid. The command to raise thetemperature occurred simultaneously with aninjection. In the 20 seconds that elapsed while theanalyte traversed the tubing and column from theALS to the ion source, the gas temperature essen-tially reached and stabilized at the new value, 25 C higher. The source also cooled reasonablyrapidly, dropping from 250 C to 125 C in aboutthree minutes. A final point of this figure is thatthe increased signal also resulted in better repro-ducibility of the measured response. Data obtainedat the lowest vaporizer temperature, 125 C, had a higher %RSD value of 13% (four replicates) com-pared to the data taken at higher temperatures.


3.5 m (Agilent part no. 873700-902)Mobile phase: 25:75 water:methanol with 5 mM ammonium

formateGradient: None (isocratic)Flow rate: 1.0 mL/minColumn temperature: RoomInjection volume: 1 L, 5 injections at each vaporizer temperature

MS ConditionsSource: Multimode, APCI-onlyDrying gas flow: 5 L/minNebulizer: 20 psigDrying gas temp: 350 CVaporizer temp: 125250 C in 25 C stepsCapillary voltage: 3000 VCorona current: 6 APeakwidth: 0.1 minTime filter: OnSIM ion: 609.3 m/zFragmentor: 200 V

10


Good sensitivity in ESI, APCI, and mixed mode

The multimode source can achieve detection limitsin the low picogram range in all modes of opera-tion. Shown in Figure 10 are replicate injections of the compound reserpine. As is noted on the leftside of the figure, the measured sensitivity was 36 in positive APCI mode (top), 32 in positive ESImode (middle), and 36 in positive mixed mode(bottom). The source produced comparable signal/noise values for this compound in all modes,suggesting that mixed mode operation achieved

essentially the same sensitivity as obtained in adedicated mode. Furthermore, these sensitivityvalues were similar to a dedicated ESI or dedi-cated APCI ion source on the same instrument(data not shown; current sensitivity specificationsfor this instrument are 20:1 signal/noise witheither a dedicated ESI or dedicated APCI source).There is typically no compromise in sensitivity forthe analysis of a single compound analyzed underthe same conditions.


min10 20 30 40 50

500

1000

1500

2000

2500

3000

3500

Vaporizer temperature

250 C

225 C

200 C

175 C

150 C

125 C

Relativepeak area

16.8

15.1

12.7

8.3

3.7

1.0

Rel. std dev.4 replicates

2.0%

1.7%

2.1%

2.9%

1.5%

13.4%

75:25 MeOH:water, 5 mM ammonium formate1 mL/min

Figure 9. Improvement in response of repetitive 10 pg reserpine injections with increasing IR emitter vaporizer temperature.Response increased by 16.8X over the temperature range of the experiment. The data also shows the rapid controlled response ofthe IR emitters to a 25 C step change in the setpoint.

11

While the instrument with a multimode sourcehas excellent response for a single compound, asshown above, this does not mean that all com-pounds in the same analysis will respond well.ESI and APCI are chemical ionization processes.They often have different optimum source con-ditions for different analytes in a mixture. Anexample of this would happen if two compoundscoeluted, but each needed a significantly differentvaporizer temperature for optimal response. Inthis case, one must select a compromise tempera-ture for the vaporizer. (Note that this situation isno different than exists for a dedicated ionsource.) With the greater number of compoundsthat may be ionized with a multimode source inmixed mode, it is more likely that one will have touse a compromise setting for a parameter; howev-er, the multimode source is designed to reduce theimpact of those compromises.

Agilent Technologies Simultaneous Electrospray and Atmospheric Pressure ChemicalIonization: The Science Behind the Agilent Multimode Ion Source

MM -ESI 1 pg Reserpine

S/N = 32

MM-APCI1 pg Reserpine

S/N = 36

MM-Mixed 1 pg Reserpine

S/N = 36

min16 18 20 22 24 26

400

800

1200

1600

6000

10000

14000

18000

24 26

3000

5000

7000

min16 18 20 22 24 26

min16 18 20 22

Figure 10. Typical sensitivity data for the multimode source in positive polarity operation, repetitive 1 pg reserpine injections. (top)APCI-only mode, (middle) ESI-only mode, (bottom) mixed mode operation.


3.5 m (Agilent part no. 873700-902)Mobile phase: 25:75 water:methanol with 5 mM

ammonium formateGradient: None (isocratic)Flow rate: 0.4 mL/min ESI and mixed mode, 1.0 mL/min APCIColumn temperature: RoomInjection volume: 1 L

MS ConditionsSource: Multimode, positive APCI-only, ESI-only,

or mixed modeDrying gas flow: 5 L/minNebulizer: 20 psig APCI, 60 psig ESI, 40 psig mixed-modeDrying gas temp: 350 CVaporizer temp: 250 CCapillary voltage: 3000 APCI, 2000 V ESI and mixed-modeCorona current: 6 A APCI, 2 A mixed modePeakwidth: 0.08 min ESI and mixed-mode, 0.04 min APCITime filter: OnSIM ion: 609.3 m/zFragmentor: 200 V

12


Analysis of thermally labile compounds: IR emittersand temperature feedback

ESI is renowned for its ability to ionize thermallylabile substances such as proteins and drugs,which in part gives rise to its great utility. As statedpreviously, the multimode source uses IR emittersto aid in drying the droplets during the ESIprocess. A natural concern is that the emittersmay introduce enough heat, or introduce heat inan uncontrolled fashion, and promote the thermaldegradation of these compounds under ESI condi-tions. Shown in Figure 11 is the analysis of thechemotherapy agent, taxol, a compound thatdegrades if exposed to too high a temperature.Using a vaporizer temperature of 150 C, taxolhad essentially no response in APCI mode (top). It was analyzed successfully by positive ESI-onlymode or positive mixed mode (middle and bot-tom). Only minor peaks appeared in the massspectra other than the protonated molecular ion,attesting to the lack of degradation products. Thegentleness of the heating by the IR emitters isattributable to the vapor temperature sensor andfeedback control which enable accurate, rapid andprecise adjustment of the vapor temperature atthe setpoint. Another example of the analysis of athermally labile compound was presented previ-ously in Figure 6, the spectrum of bovine insulin.

Higher HPLC flow rates: IR emitters and tempera-ture feedback

Typical analytical HPLC flow rates are in therealm of 0.250.4 mL/min using 2.1 mm columns.However, high throughput analysis is generallyperformed on 4.6 mm ID columns, injecting sev-eral hundred samples per day into an LC/MS, withcolumn flow rates between 12 mL/min. A dedi-cated ESI source normally has poor sensitivity atthese higher flow rates and is operated with apostcolumn split, diverting the majority of the

split flow to waste. This works but is inconvenientand needs additional parts in the system. Further-more, operation in APCI mode becomes moreattractive at higher flow rates as the analyte sig-nal generally increases at higher flows.

As mentioned previously, the IR emitters emitenough power to vaporize HPLC effluents at up to 2 mL/min flow rate. The data in Figure 9 wasobtained at 1 mL/min flow rate and illustratesthat the feedback control is rapid enough to tracka step change in temperature or mobile phasecomposition, coming to a steady state in a matterof 2030 seconds. Data shown in Figures 12 and13 were obtained at even higher flow rates, 1.5 mL/min.



5 m (Agilent part no. 860975-906)Mobile phase: A = water with 0.05% formic acid

B = acetonitrileGradient: 10% B at 0 min

95% B at 7 minStop run at 8 min

Flow rate: 0.4 mL/minColumn temperature: 30 CInjection volume: 1 L

MS ConditionsSource: Multimode, APCI-only, ESI-only

or mixed modeDrying gas flow: 5 L/minNebulizer: 20 psig APCI, 60 psig ESI, 40 psig mixed-mode Drying gas temp: 200 CVaporizer temp: 150 CCapillary voltage: 2000 VCorona current: 6 A APCI, 2 A mixed-modePeakwidth: 0.1 minTime filter: OnScan: 100 1000 m/zFragmentor: 150 VStepsize: 0.1 m/z

13


m/z200 400 600 800

854.2

854.2

876.2

854.2

876.1

min1 2 3 4 5 6 7

APCIMaximum: 1606

0

60000

Mixed ModeMaximum: 57783

0

60000

ESIMaximum: 57308

m/z200 400 600 800

m/z200 400 600 800

min1 2 3 4 5 6 7

min1 2 3 4 5 6 7

0

60000

0

60000

0

60000

0

60000

Figure 11. Extracted ion chromatograms of m/z 854.2 and spectra for the thermally labile compound taxol (1ng/L) using the multi-mode source. (top) APCI-only mode, (middle) ESI-only mode, (bottom) mixed mode operation. Vaporizer setpoint was 150 C. Theresponse in ESI-only and mixed mode was quite similar, and the spectra show few mass peaks at m/z values less than the [M+H]+

ion, signifying that there was little thermal degradation of taxol under these conditions.

14


Higher HPLC flow rates: Mixed mode operationyields better data for fast-eluting chromatographicpeaks

It is possible to analyze samples either by mixedmode ESI and APCI or by alternating between thetwo ionization techniques on a scan-to-scan basis.When acquiring data, the instrument uses eitherone signal for mixed mode or two signals for alter-nating operation. There is a difference in the dataquality between the two operational modes whichbecomes apparent when analyzing the narrowerchromatographic peaks obtained during fast chro-matography (Figure 12). Two compounds, taxoland progesterone, were analyzed using isocraticconditions in positive mixed mode (one signal, top trace) and in alternating ESI and APCI modes(two signals, the ESI signal is the middle trace).All other parameters: mass range, a/d samplingrate, etc. were held constant. (The signal from theUV detector is shown in the bottom trace for com-parison.) The effect on the data is obvious, asabout two times more scans across the peak wereobtained when using mixed mode than when alter-nating modes. The reduction in chromatographicpeak fidelity was caused by the fact that theinstrument was monitoring two signals instead of one. Looking at it another way, in alternatingmode operation, the instrument was not acquiringESI data half of the time and was not acquiringAPCI data during the other half of the time. But inmixed mode operation, the instrument was acquir-ing ESI data and APCI data all of the time. Note,the time it takes to switch between ESI and APCIis only a minor contributor to the differencesobserved. If the switching time were instanta-neous, the instrument would still acquire twice asmany scans across the peak in mixed mode opera-tion. Therefore, mixed mode is in general thepreferred mode of operation.

Putting it all together: applications examples

High throughput analysisHigh throughput analysis benefits from all thefeatures of the multimode source: good ESI sensi-tivity, good APCI sensitivity, ability to handlehigher HPLC flow rates, better chromatographicperformance with mixed mode operation andhigher HPLC flow rates, and better productivity in mixed mode operation. Faster injection-to-injection cycle time is obtained by combiningthese features with optimized HPLC techniques:short columns with 1.8 micron particle size,higher HPLC flow rates, minimized delay volume,overlapped injections, and alternating columnregeneration.[6] A test set composed of pharma-ceuticals and their intermediates was analyzed,


LC ConditionsColumn: 15mm x 4.6 mm ZORBAX RRHT SB-C18,

1.8 m (Agilent part no. 821975-902)Mobile phase: A = water with 0.2% acetic acid

B = acetonitrile with 0.2% acetic acidGradient: None (isocratic), 45% A, 55% BFlow rate: 1.5 mL/minColumn temperature: 40 CInjection volume: 1 LDiode-array detector: Signal 240, 10 nm; reference 340. 20 nm

MS ConditionsSource: Multimode, positive mixed mode

or alternating ESI and APCIDrying gas flow: 7 L/minNebulizer: 60 psigDrying gas temp: 200 CVaporizer temp: 175 CCapillary voltage: 1500 VCorona current: 2 APeakwidth: 0.04 minTime filter: OffScan: 200900 m/zFragmentor: 120 VStepsize: 0.1 m/z

15

and one result from this set is shown in Figure 13.This represents a single injection of 100 ng proges-terone analyzed in positive/negative mode andmixed mode operation. The top chromatogram isthe positive mode TIC, second chromatogram isthe negative mode TIC, the third chromatogram isthe target mass trace, and the fourth chromato-gram is the UV chromatogram. The spectrum atthe apex of the target mass chromatogram peak isshown at the bottom. A gradient of 15100%methanol in 0.75 minutes was used, thus reducing

the injection-to-injection cycle time to about 1.75 minutes. As Figure 14 shows, an entire 96-wellplate can be analyzed in under three hoursby using such a system. Of the compounds in thetest set (Table 1), 89% were detected using adedicated APCI source, and a different 89% weredetected using a dedicated ESI source. However,all test compounds were detected using the multi-mode source in positive/negative mode, attestingto the greater utility of the multimode source.


0

10

20

30

40

0.2

min0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0

100000

200000

300000

400000

500000

0.020 min

0.034 min

mAU

0

100000

200000

300000

400000

4 scans

12 scans10 scans across the peak

5 scans

Simultaneous mixed mode ESI+APCI, one signal

ESI/APCI switching using two signals

DAD

TaxolProgesterone

min0.3 0.4 0.5 0.6 0.7 0.8 0.9

min0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Figure 12. Simultaneous (mixed mode) operation vs. alternating mode in the analysis of taxol and progesterone. Using one signalfor mixed mode operation (top) resulted in better chromatographic peak fidelity than using two signals for alternating mode opera-tion (middle, only the ESI signal shown). The DAD signal is shown (bottom) for comparison. Even if the switching time were instan-taneous, mixed mode operation would yield twice the number of scans across the peak because only one signal is being monitored,not two.

16


LC ConditionsColumns: Two alternating 15 x 4.6 mm ZORBAX RRHT

SB-C18, 1.8 m (Agilent part no. 821975-902)Binary pump mobile A = water with 0.2% acetic acidphase: B = methanol with 0.2% acetic acidGradient: 15% B at 0 min

100% B at 0.75 min100% B at 1.00 min15% B at 1.01 minStop run at 1.50 min

Binary pump flow rate: 1.5 mL/minColumn temperature: 40 CIsocratic regeneration pump mobile phase: 85:15 water:methanol with 0.2% acetic acidIsocratic pump flow rate: 1.5 mL/minInjection volume: 1 L, overlapped injection and minimized

delay volumeDiode-array detector: Signal 250 nm, 10 nm; reference off


Figure 13. High throughput analysis of progesterone (100 ng on column). (top trace) positive mode TIC, (second from toptrace) negative mode TIC, (third from top trace) positive mode target ion m/z 315.2, (fourth from top trace) DAD signal,(bottom) spectrum from peak apex of target mass trace.

MS ConditionsSource: Multimode, mixed mode with positive/

negative switchingDrying gas flow: 5 L/minNebulizer: 40 psigDrying gas temperature: 350 CVaporizer temperature: 200 CCapillary voltage: 1500 V (positive and negative)Corona current: 4 A (positive and negative)Peakwidth: 0.07 minTime filter: OffFast scan mode: EnabledScan: 1001150 m/zFragmentor: 120 V (positive and negative)Stepsize: 0.3 m/z

17


Compound Positive Negative Positive Negative Positive NegativeAcetazolamide D D DButyl 4-aminobenzoate D D D Cortisone D D D D DGemfibrozil D D DHexahydro-...-dione D D D Hydroflumethiazide D D DIndole D D Iodipamide D D DLabetalol D D D D D DLidocaine D D D Morin D D D D D DPaclitaxel D D Phenylbutazone D D D D D DProcainamide D D D Progesterone D D D Sulfamethoxazole D D D D D DTolazamide D D D D D DUracil D D

Detected by polarity 67% 56% 67% 50% 78% 61%

APCI Source ESI Source Multimode Source

Detected by source 89% 89% 100%D = compound detected

Sample Throughput, 96-Well Plate

0 1 2 3 4 5 6 7 8 9 10 11 12

add alternating columnregeneration

add overlapped injection,minimize delay volume

with 1.8m column

Time (hours)

multimode source dedicated ESI and APCI sources

Figure 14. Improvement in sam-ple throughput by combininganalysis techniques and hard-ware. An entire 96-well plate canbe analyzed in under three hoursby combining all improvements.

Table 1. Summary of responses for pharmaceutical compounds and intermediates analyzedusing various ion sources: (left two columns) analysis using a dedicated APCI source, (middletwo columns) using a dedicated ESI source, (right two columns) using a multimode source inmixed mode. The multimode source detected all compounds in the test suite, whereas the ded-icated sources did not.

*Hexahydro-dione = Hexahydro-2,6-

bis(2,2,6,6-tetramethyl-4-piperidinyl)-

1H,4H,5H,8H-2,3a,4a,6,7a,8a-hexaaza-

cyclopenta[def]fluorene-4,8-dione

18



3.5 m (Agilent part no. 873700-902)Mobile phase: A = water with 0.2% acetic acid

B = methanol with 0.2% acetic acidGradient: 10% B at 0 min

10% B at 0.5 min100% B at 9 minStop run at 12 min


MS ConditionsSource: Positive/negative switching multimode,

mixed modeDrying gas flow: 5 L/minNebulizer: 40 psigDrying gas temp: 350 CVaporizer temp: 200 CCapillary voltage: 1500 V (positive and negative)Corona current: 4 A (positive and negative)Peakwidth: 0.12 minTime filter: OnScan: 165600 m/zFragmentor: 130 V (positive and negative)Stepsize: 0.1 m/z


Steroids analysisSteroids are known to be difficult to analyze;some respond well in ESI, and others respondbetter in APCI mode. Investigators sometimesderivatize them in an effort to improve theirdetection limits.[7] Several steroids with differentfunctional groups (acid, ketone, fluoride, hydrox-yl) were analyzed successfully, showing a strongresponse under positive/negative mixed modeconditions (Figure 15).

Environmental compound screeningFigure 16 shows the analysis of several classes of compounds of interest in environmental com-pound screening.[8] Representative pesticides,herbicides and insecticides (bipyridyliums, tri-azines, phenylureas, carbamates, phenoxyaceticacids, phenols) were analyzed at the 5 ng level inpositive/negative mixed mode. All compoundsgave a response that was visible in the TIC. Thisis especially noteworthy as the broad range ofcompound types, chromatography, and coelutiondictate that the pH and vaporization conditionswere not optimal for every analyte in the sample.

min5 6 7 8 9

Chenodeoxycholic acid

Corticosterone

11--Hydroxyprogesterone

Hydrocortisone

Prednisone ProgesteroneTestosterone

Triamcinolone(2 isomers)

Positive mixed mode BPC,primarily [M+H]+ and [M+Na]+

Negative mixed mode BPC,primarily [MH] and [M+acetate]

min5 6 7 8 9

Figure 15. Analysis of underivatized steroids (100 ng per component) using the multimode source, mixed mode with positive/nega-tive polarity switching. A wide range of functional groups was represented.

19

LC ConditionsSample: 1 ng per component, dissolved in

80:20 water:methanol with 1% acetic acidColumn: 150 x 2.1 mm ZORBAX Eclipse XDB-C18,

3.5 m (Agilent part no. 930990-902)Mobile phase: A = water with 1 mM ammonium acetate

B = methanol with 1 mM ammonium acetateGradient: 3% B, 0.3 mL/min at 0 min

90% B, 0.3 mL/min at 45 min3% B, 0.3 mL/min at 45.01 min3% B, 0.5 mL/min at 45.02 min3% B, 0.5 mL/min at 50 min3% B, 0.3 mL/min at 50.01 minStop run at 52 min



Positive mixed mode TIC,primarily [M+H]+ and [M+Na]+

min5 10 15 20 25 30 35

DiquatParaquat

(responds best in APCI)

OxamylMethomyl

Fenuron

Monuron

*

Simazine

Propoxur

AtratonPropham

Fluometuron

Carbaryl

AminocarbCarbofuran

TerbutylazineSiduron

Methiocarb PropazineAmetrynSiduron

Secbumeton

LinuronPrometon

SimetrynAtrazine

Diuron

Prometryn

TerbutrynNeburon

ChlorprophamBarban

Mexacarbate

Negative mixed mode TIC,primarily [MH] and [M+acetate]

4-Nitrophenol

Dicamba2,4-D

Monuron2,4,5-T

** * **

Fluometuron

Diuron

Linuron

NeburonSiduron

min5 10 15 20 25 30 35

MS ConditionsSource: Multimode, mixed mode with positive/negative

switchingDrying gas flow: 6 L/minNebulizer: 40 psigDrying gas temp: 300 CVaporizer temp: 150 CCapillary voltage: 1000 V (positive and negative)Corona current: 2 A (positive and negative)Peakwidth: 0.3 minTime filter: OnScan: 130330 m/zFragmentor: 130 V (positive and negative)Stepsize: 0.1 m/z

Figure 16. Analysis of compounds of environmental interest (5 ng per component) using the multimode source, mixed mode withpositive/negative polarity switching. A wide range of compound classes was represented (bipyridyliums, triazines, phenylureas,carbamates, phenoxyacetic acids, phenols). *Denotes impurity

Conclusions

The Agilent multimode source contains severalnovel design elements which are patented or inthe process of being patented.[9] These elementsand the method of their use allow the multimodesource to perform ESI, APCI or simultaneous ESIand APCI at higher HPLC flow rates with compa-rable sensitivity to dedicated ion sources. Switch-ing between modes is as simple as clicking themouse or may be programmed automatically.Mixed mode operation permits higher quality datato be obtained on narrow chromatographic peaks,such as those found in high throughput analysis.Nitrogen gas consumption is reduced significantlyusing this source rather than a dedicated ESIsource. The ultimate benefits of the source areincreased sample throughput, better scheduling of samples, better use of instrument time, loweroperating costs, and less time spent reanalyzingsamples.

References

1. Cody, R.B. Electrospray Ionization Mass Spec-trometry: History, Theory, and Instrumenta-tion. In Practical Spectroscopy Series, Volume32, Applied Electrospray Mass Spectrometry,Pramanik, B.N.; Ganguly, A.K.; Gross, M.L.,Eds.; Marcel Dekker, Inc.: New York, NY, 2002;chapter 1.

2. Gaskell, S.J. Electrospray: Principles and Prac-tice. J. Mass Spectrom. 1997, 32, 677688.

3. Niessen, W.M.A.; Tinke, A.P. Liquid chromatog-raphy-mass spectrometry general principlesand instrumentation. J. Chrom. A 1995, 703,3757.

4. Siegel, M.M.; Tabei, K; Lambert, F.; Candela, L.;Zoltan, B. J. Am. Soc. Mass Spectrom. 1998, 9,11961203.

5. Gallagher, R.T.; Balogh, M.P.; Davey, P.; Jack-son, M.R.; Sinclair, I.; Southern, L.J. Combinedelectrospray ionization-atmospheric pressurechemical ionization source for use in high-throughput LC-MS applications. Anal. Chem.2003, 75, 973977.

6. Huber, U. High throughput HPLCAlternatingcolumn regeneration with the Agilent 1100Series valve solutions. Agilent Technologiesapplication note 2002, publication number5988-7831EN.

7. Higashi, T.; Shimada, K. Derivatization of neu-tral steroids to enhance their detection charac-teristics in liquid chromatographymass spec-trometry. Anal. Bioanal. Chem. 2004, 378,875882.

8. Thurman, E.M.; Ferrer, I.; Barcelo, D. Choosingbetween Atmospheric Pressure Chemical Ion-ization and Electrospray Ionization Interfacesfor the HPLC/MS Analysis of Pesticides. Anal.Chem. 2001, 73, 54415449.

9. US patent 6,646,257; others pending.

Authors

Steven M. Fischer is a senior research chemistand Patrick D. Perkins is a senior applicationschemist at Agilent Technologies, Inc. in SantaClara, California U.S.A.

www.agilent.com/chem

Agilent Technologies, Inc. 2005

Information, descriptions and specifications in this publication aresubject to change without notice. Agilent Technologies shall not be liablefor errors contained herein or for incidental or consequential damages inconnection with the furnishing, performance or use of this material.

Printed in the U.S.A. April 21, 20055989-2935EN


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