Agilent Technologies Simultaneous Electrospray Atmospheric Pressure Chemical Ionization: The Science Behind the Agilent Multimode Ion Source service manual
Contents
1. 0 75 Time minute Select Plate View Threshold Peak 5 Target Mass 314 2 Expected Mass 315 2 MSD1 TIC MS File me 1043 3154 I SampleID 7 Target mass 754 Estimated Sample Purity 2 503 163 3585 M byMS I by M 6 4007 25 4 ies bern T A Purity E h l Average lan ay GS eS Dam GC GG aC i Gn Gt Oe at CC SG a Oa 100 150 200 250 3200 350 400 450 500 550 600 650 700 mz Sample Header info D SHPCHEM 1 DATASASMSO4A2 ASMSOT AEV 1571672004 3 48 PM Figure 13 High throughput analysis of progesterone 100 ng on column top trace positive mode TIC second from top trace 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 16 Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Sample Throughput 96 Well Plate E multimode source dedicated ESI and APCI sources with 1 8um column add overlapped injection minimize delay volume add alternating column regeneration 5 6 Time hours Table 1 Summary of responses for pharmaceutical compounds and intermediates analyzed using various ion sources left two columns analysis using a dedicated APCI source middle two columns using a dedicated ESI source right two columns2. Mobile phase Gradient Flow rate Column temperature Injection volume MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time filter SIM ion Fragmentor 30 x 2 1 mm ZORBAX SB C18 3 5 um Agilent part no 873700 902 25 75 water methanol with 5 mM ammonium formate None isocratic 0 4 mL min ESI and mixed mode 1 0 mL min APCI Room Tul Multimode positive APCl only ESI only or mixed mode 5 L min 20 psig APCI 60 psig ESI 40 psig mixed mode 350 C 250 C 3000 APCI 2000 V ESI and mixed mode 6 pA APCI 2 pA mixed mode 0 08 min ESI and mixed mode 0 04 min APCI On 609 3 m z 200 V While the instrument with a multimode source has excellent response for a single compound as shown 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 An example of this would happen if two compounds coeluted but each needed a significantly different vaporizer temperature for optimal response In this case one must select a compromise tempera ture for the vaporizer Note that this situation is no different than exists for a dedicated ion source With the greater number of compounds that may be ionized with a multimode source in mixed mode
3. 0 5 mL min at 45 02 min Time filter On 3 B 0 5 mL min at 50 min Scan 130 330 m z 3 B 0 3 mL min at 50 01 min Fragmentor 130 V positive and negative Stop run at 52 min Stepsize 0 1 m z Flow rate 0 3 mL min Column temperature 60 C Injection volume 5 uL Terbutylazine Secbumeton Siduron Simetryn Methiocarb Fluometuron Atrazine Propazine Chlorpropham ws E Diuron Ametryn Positive mixed mode TIC Carbaryl Siduron Barban i i primarily M H and M Na Atat P Mexacarbate Carbofuran opnam Prometryn Propoxur Linuron Diquat renun Prometon Terbutryn Paraquat Simazine Neburon Monuron Methomyl Oxamyl Siduron Neburon Negative mixed mode TIC primarily M H and M acetate 4 Nitrophenol Diuron responds best in APCI Fluometuron 2 4 D Linuron Dicamba Figure 16 Analysis of compounds of environmental interest 5 ng per component using the multimode source mixed mode with positive negative polarity switching A wide range of compound classes was represented bipyridyliums triazines phenylureas carbamates phenoxyacetic acids phenols Denotes impurity Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Conclusions The Agilent multimode source contains several novel design elements which are patented or in the process of being patented These elements and the
4. charg ing electrode instead of forward toward the mass spectrometer entrance In the thermal container space between the ESI and APCI zones the IR emitters convert the residual droplets to vapor while 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 of ESI and APCI ions is a function of the overall design of the source and the interaction of its unique parts If desired the multimode source also functions well in ESI only or APCI only modes To perform only ESI the APCI corona needle voltage is turned off while leaving the ESI charging electrode volt age on and only ESI ions are produced To per form only APCI the ESI charging electrode is turned off while leaving the APCI corona needle voltage on and only APCI ions are produced These voltages are under data system control and thus the instrument may be switched between ESI APCI and mixed mode operation manually with a click of the mouse automatically during an analy sis or automatically during a sequence Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Proof of concept ESI only APCl only and simulta neous mixed mode ESI and APCI The proof of concept for simultaneous ESI and APCI operation is shown in Figure 6 A mixture of bovine insulin and indole was in
5. filter SIM ion Fragmentor Agilent Technologies 30 x 2 1 mm ZORBAX SB C18 3 5 um Agilent part no 873700 902 25 75 water methanol with 5 mM ammonium formate None isocratic 1 0 mL min Room 1 uL 5 injections at each vaporizer temperature Multimode APCl only 5 L min 20 psig 350 C 125 250 C in 25 C steps 3000 V 6 pA 0 1 min On 609 3 m z 200 V Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Agilent Technologies 3500 4 7 75 25 MeOH water 5 mM ammonium formate 7 mL min 3000 4 2500 4 2000 1500 1000 7 500 7 LI T T T T T T T 10 20 30 Vaporizer Relative Rel std dev temperature peak area 4 replicates 250 C 16 8 2 0 225 C 15 1 1 7 200 C 12 7 2 1 175 C 8 3 2 9 150 C 3 7 1 5 125 C 1 0 13 4 111 17 T 40 50 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 of the IR emitters to a 25 C step change in the setpoint Good sensitivity in ESI APCI and mixed mode The multimode source can achieve detection limits in 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 left side of t
6. the peak were obtained when using mixed mode than when alter nating modes The reduction in chromatographic peak fidelity was caused by the fact that the instrument was monitoring two signals instead of one Looking at it another way in alternating mode operation the instrument was not acquiring ESI data half of the time and was not acquiring APCI data during the other half of the time But in mixed 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 APCI is only a minor contributor to the differences observed If the switching time were instanta neous the instrument would still acquire twice as many scans across the peak in mixed mode opera tion Therefore mixed mode is in general the preferred mode of operation LC Conditions Column Mobile phase Gradient Flow rate Column temperature Injection volume Diode array detector MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time filter Scan Fragmentor Stepsize Agilent Technologies 15mm x 4 6 mm ZORBAX RRHT SB C18 1 8 um Agilent part no 821975 902 A water with 0 2 acetic acid B acetonitrile with 0 2 acetic acid None isocratic 45 A 55 B 1 5 mL min 40 C 1 uL Signal 240 10 nm reference 340 20 nm Multimode positive mixed mode or alternating ESI and APCI
7. vapor temperature at the setpoint Another example of the analysis of a thermally 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 the realm of 0 25 0 4 mL min using 2 1 mm columns However high throughput analysis is generally performed on 4 6 mm ID columns injecting sev eral hundred samples per day into an LC MS with column flow rates between 1 2 mL min A dedi cated ESI source normally has poor sensitivity at these higher flow rates and is operated with a postcolumn split diverting the majority of the LC Conditions Column Mobile phase Gradient Flow rate Column temperature Injection volume MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time filter Scan Fragmentor Stepsize Agilent Technologies 50 x 2 1 mm ZORBAX SB C8 5 um Agilent part no 860975 906 A water with 0 05 formic acid B acetonitrile 10 B at 0 min 95 B at 7 min Stop run at 8 min 0 4 mL min 30 C Tul Multimode APClI only ESl only or mixed mode 5 L min 20 psig APCI 60 psig ESI 40 psig mixed mode 200 C 150 C 2000 V 6 pA APCI 2 pA mixed mode 0 1 min On 100 1000 m z 150 V 0 1 m z split flow to waste This works but is inconvenient and needs additional part
8. 0 V effluent was converted essentially completely to the vapor state and the APCI response increased approximately five fold 1600000 1200000 800000 Vaporizer 250 C 500 400000 0 T T T T T T T T T T T T T T T T T T T T T T T T T T rs ee ee 2 4 6 8 10 12 14 min 1600000 1200000 Vaporizer 115 C 180 800000 400000 0 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T i 2 4 6 8 10 12 14 min 1600000 1200000 H o 0 sani Vaporizer 60 C 100 400000 0 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 2 4 6 8 10 12 14 min Figure 8 Improvement in diphenhydramine response with increasing IR emitter vaporizer temperature Response increased by 5X over the temperature range of the experiment Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source A more dramatic illustration of this effect is shown in Figure 9 The compound here is reser pine also a compound that responds well by both ESI and APCI and is generally used to test the sensitivity of LC MS systems Reserpine has a lower vapor pressure than diphenhydramine when compared at the same temperature so greater vaporizer temperatures are needed to convert the analyte into the gas phase The experiment con sisted of performing five consecutive injections of reserpine 10 pg each increasing the vaporizer temperature by 25 C p
9. 624 0 1000 4 MM APCI Indole cet Camere Ne e Ao Ne e AN A N T 0 1000 2000 m z Max 77149 MM Mixed 118 1 1912 1 Insulin 7 2867 8 T T T T 2000 m z L E of Figure 6 Proof of concept of simultaneous mixed mode ESI and APCI using a mixture of indole 50 pmol pL and bovine insulin 9 pmol pL top Source in ESl only mode exhibiting a response from insulin with a weak response from indole middle Source in APCI only mode with only indole showing a response bottom Source in mixed mode with both indole and insulin showing strong responses Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Another example of this performance is shown in Figure 7 This was a mixture of four compounds each of which responded most strongly in a single one of four possible modes negative ESI negative APCI positive ESI or positive APCI By setting the ionization parameters to one of these possibil ities e g positive ESI mode only the response of crystal violet was seen However injection of the same mixture using mixed mode operation and positive negative polarity switching allowed detec tion of all four compounds using a single injection and analysis A broad range of response like this is a welcome feature for analyses such as com pound screening and high throughput analysis in early drug disc
10. 7 L min 60 psig 200 C 175 C 1500 V 2 pA 0 04 min Off 200 900 m z 120 V 0 1 m z Putting it all together applications examples High throughput analysis High throughput analysis benefits from all the features of the multimode source good ESI sensi tivity good APCI sensitivity ability to handle higher HPLC flow rates better chromatographic performance with mixed mode operation and higher HPLC flow rates and better productivity in mixed mode operation Faster injection to injection cycle time is obtained by combining these features with optimized HPLC techniques short columns with 1 8 micron particle size higher HPLC flow rates minimized delay volume overlapped injections and alternating column regeneration A test set composed of pharma ceuticals and their intermediates was analyzed Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Agilent Technologies 500000 4 400000 300000 l 200000 7 100000 Simultaneous mixed mode ESI APCI one signal 10 scans across the peak 12 scans 400000 ESI APCI switching using two signals 300000 7 4 scans 5 scans 200000 100000 4 0 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 min mAU 40 DAD a 0 020 min Progesterone Figure 12 Simultaneous mixed mode operation vs alternating mode in the analysis of taxol and progesterone Usi
11. R emitters and vapor temperature sensor with respect to other parts of the source Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Counterelectrode pin ae ad a a 7 L N 7 N l M i C p 1 l I Corona needle N N 7 SN 7 s a X Figure 5 A close up diagram of the APCI zone of the multi mode source producing ions A separator in the middle of the source screens most of the ESI ions from reaction with the corona discharge present in the APCI zone The ESI ion stream on one side of the sepa rator and the APCI ion stream from the other side combine 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 the design of the multimode source solves the prob lems of maintaining separate but connected ESI and APCI regions in a simple yet elegant fashion The liquid from a single nebulizer is first charged and converted to an aerosol in the presence of an electrical field the conditions for optimal ESI ion formation Then the residual droplets and ESI ions are pushed past the reversing electrode into the heated region on their way to the APCI zone Agilent Technologies The reversing electrode is the electrical boundary between the ESI and APCI zones and without it ions would tend to migrate back toward the
12. Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Technical Overview Steven M Fischer and Patrick D Perkins Agilent Technologies Introduction The invention and commercialization of electro spray ionization ESI was revolutionary when applied to the field of organic analytical chem istry For the first time chemically and thermally fragile molecules proteins drugs environmental compounds to name a few classes could easily and reliably be analyzed in high sensitivity by LC MS techniques ESI is used today in thousands of laboratories around the world for routine high sensitivity analysis of these and other analytes As a testimony to the utility of this technique its inventor Professor John Fenn was one of the recipients of the Nobel Prize in chemistry in 2002 As remarkable as the technique of ESI is it does not ionize all organic compounds of interest that may be eluted from an HPLC It has been estimated that about 80 of all analytes respond by ESI leaving a significant fraction as undetectable This limitation was noticed early in the development of ESI and other ionization techniques were devel oped almost simultaneously to fill this need The technique of atmospheric pressure chemical ionization APCD and the related atmospheric pressure photoionization APPI are the best known alternatives Further study of ESI and APCI pointe
13. d up funda mental differences between them The spray conditions and voltage conditions for optimal ESI operation are very different than for optimal APCI operation In optimized ESI ion formation occurs in the following sequence 1 A surface charge is induced on the emerging liquid by imposition of an external electrical field 2 The charged liquid is converted to a spray of droplets 3 The droplets shrink due to solvent evapora tion which increases the surface charge density 4 Droplets undergo fission as a consequence of the high surface field strength 5 When a droplet is small enough ions are ejected from the droplet due to the high field strength at the surface of the droplet Agilent Technologies Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Under optimal APCI conditions ion formation occurs 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 molecules pass through a corona discharge 4 The corona discharge ionizes the solvent mole cules creating solvent ions 5 The solvent ions transfer charge to the analyte molecules creating analyte ions From the above lists it should be apparent that the spray formation and timing of ion generation are different in ESI and APCI As a r
14. em comprised of a binary pump autosampler thermostatted column com partment and an Agilent 1100 Series LC MSD SL G1956D quadrupole mass spectrometer The LC MSD was equipped with a multimode source dedicated ESI source or dedicated APCI source as needed The sources for the LC MSD are all inter changeable The multimode source is currently supported on G1946 LC MSD instruments models B C and D and on all G1956 LC MSD instru ments models A and B However some of these models need both hardware and software upgrades to be able to control the multimode source see your 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 appropriate section Results and Discussion The Agilent multimode source is a compact ion source that fits on the left side of the LC MSD quadrupole mass spectrometer Some distinguish ing features of the source are highlighted Figure 1 Concepts behind simultaneous mixed mode ESI and APCI operation A functional view of this source is given in Fig ure 2 The top part of the source is the ESI zone where the conditions exist for optimal electro spray ion formation The bottom part of the source contains the APCI zone where optimal conditions exist for this ionization process Some of the functional elements of this source are also shown and are discussed in detail be
15. erforming another five injections and so forth up to the temperature limit Then the vaporizer was allowed to cool to its starting temperature while injections of reser pine were continued The flow rate and mobile phase during this experiment was 1 0 mL min of 75 25 methanol water with 5 mM ammonium for mate typical for APCI which performs better at higher flow rates The signal response for reserpine increased almost 17 times as the vaporizer temperature was increased Furthermore the response of the emit ters was quite rapid The command to raise the temperature occurred simultaneously with an injection In the 20 seconds that elapsed while the analyte traversed the tubing and column from the ALS to the ion source the gas temperature essen tially reached and stabilized at the new value 25 C higher The source also cooled reasonably rapidly dropping from 250 C to 125 C in about three minutes A final point of this figure is that the increased signal also resulted in better repro ducibility of the measured response Data obtained at the lowest vaporizer temperature 125 C had a higher RSD value of 13 four replicates com pared to the data taken at higher temperatures LC Conditions Column Mobile phase Gradient Flow rate Column temperature Injection volume MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time
16. esult of these differences all MS manufacturers have developed dedicated optimized ion sources for each tech nique Users who want to analyze a sample using both techniques generally must run the analysis using one source first and if there is no response run the sample on the instrument using the other source This significantly decreases the number of samples per day that may be analyzed on a given instrument In response to this diminished throughput caused by the fundamental differences between ESI and APCI novel ion sources have been developed to combine the two ionization techniques into one source l45 The differences in performance of these combination also known as dual mode or multimode sources may be traced to how they address the incompatibilities between ESI and APCI Do they perform the ionization in the same region of space or do they separate the ionization regions and attempt to optimize the performance of each And if the ionization regions are sepa rate how is the sample delivered to the two regions and how is the separation maintained This document describes the features of the Agilent multimode ionization source and how it addresses these incompatibilities permitting high flow high sensitivity ESI and APCI to be per formed independently or simultaneously on a single injection of a sample Agilent Technologies Experimental All experiments were performed using an Agilent 1100 Series LC syst
17. fused into the multimode source mounted on the LC MSD quadrupole system Bovine insulin like all pro teins possessing multiple basic sites yields a char acteristic multiple ion spectrum in positive ESI mode it has no response in APCI mode Indole being a small molecule with a weakly basic site protonates poorly under ESI conditions but responds well in positive APCI mode When a mix ture of the two was analyzed using the multimode source bovine insulin had a strong response in ESI only mode whereas indole had a very weak response The opposite was true in APCI only mode with indole having a strong response Both compounds responded well when the simultane ous ionization mode was enabled This and other tests indicate that the multimode source produces ESI and APCI spectra similar to those of the dedi cated sources and that the two ionization modes may be switched off on or combined for simulta neous ionization LC Conditions Column None infusion 0 1 acetic acid Flow rate 0 1 mL min MS Conditions Source Multimode positive ESI only APCl only or mixed mode Drying gas flow 5 L min Nebulizer 40 psig Drying gas temp 350 C Vaporizer temp 200 C Capillary voltage 2500 V Corona current 2 pA Peakwidth 0 2 min Time filter On Scan 100 3000 m z Fragmentor 150 V Stepsize 0 1 m z Agilent Technologies Max 103701 1912 0 a Insulin 7 MM ESI m z 2000 118 1 Max 90
18. g 350 C 200 C 1500 V positive and negative 4 pA positive and negative 0 12 min On 165 600 m z 130 V positive and negative 0 1 m z Positive mixed mode BPC primarily M H and M Na Corticosterone Hydrocortisone Prednisone Triamcinolone 2 isomers 11 c Hydroxyprogesterone Testosterone Negative mixed mode BPC primarily M H and M acetate Progesterone Chenodeoxycholic acid 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 Simultaneous Electrospray and Atmospheric Pressure Chemical Agilent Technologies lonization The Science Behind the Agilent Multimode lon Source LC Conditions MS Conditions Sample 1 ng per component dissolved in Source Multimode mixed mode with positive negative 80 20 water methanol with 1 acetic acid switching Column 150 x 2 1 mm ZORBAX Eclipse XDB C18 Drying gas flow 6 L min 3 5 um Agilent part no 930990 902 Nebulizer 40 psig Mobile phase A water with 1 mM ammonium acetate Drying gas temp 300 C B methanol with 1 mM ammonium acetate Vaporizer temp 150 C Gradient 3 B 0 3 mL min at 0 min Capillary voltage 1000 V positive and negative 90 B 0 3 mL min at 45 min Corona current 2 pA positive and negative 3 B 0 3 mL min at 45 01 min Peakwidth 0 3 min 3 B
19. he figure the measured sensitivity was 36 in positive APCI mode top 32 in positive ESI mode 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 a dedicated mode Furthermore these sensitivity values were similar to a dedicated ESI or dedi cated APCI ion source on the same instrument data not shown current sensitivity specifications for this instrument are 20 1 signal noise with either a dedicated ESI or dedicated APCI source There is typically no compromise in sensitivity for the analysis of a single compound analyzed under the same conditions Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Agilent Technologies MM APCI 1 pg Reserpine S N 36 MM ESI 1 pg Reserpine S N 32 MM Mixed 1 pg Reserpine S N 36 1600 eetet 1200 5 16 18 18000 14000 10000 5 6000 20 22 24 26 16 18 oe oe 3000 T 16 18 20 22 24 26 20 22 24 26 Figure 10 Typical sensitivity data for the multimode source in positive polarity operation repetitive 1 pg reserpine injections top APCl only mode middle ESI only mode bottom mixed mode operation LC Conditions Column
20. he multimode source whereas 12 14 L min is usual for a standard ESI source The power to the IR emitters is controlled by feedback from a vapor temperature sensor located in the exit gas flow stream of the multimode source As the flow and or mobile phase composition changes during Agilent Technologies an LC MS analysis or during restoration of the initial gradient conditions the emitter power changes to maintain the same vapor temperature the vaporizer temperature as set in the soft ware This feedback control maintains ESI and APCI at the conditions for optimal performance and minimizes decomposition of thermally sensi tive analytes that may be present APCI zone with IR emitters corona needle and APCI counter electrode The droplets continue down into the thermal con tainer that surrounds the APCI zone of the source Here radiation from the IR emitters completely converts the droplets to solvent and analyte vapor to create the conditions for optimal APCI A corona discharge is struck between the APCI needle and a counter electrode Figure 5 This discharge ion izes the solvent molecules which subsequently transfer their charges to the analyte molecules HPLC on P Nebulizer Charging electrode Reversing electrode Thermal container Corona needle Separator IR emitters Vapor temperature sensor Figure 4 Diagram of the multimode source showing the rela tionship of the I
21. inclair I Southern L J Combined electrospray ionization atmospheric pressure chemical ionization source for use in high throughput LC MS applications Anal Chem 2003 75 973 977 6 Huber U High throughput HPLC Alternating column regeneration with the Agilent 1100 Series valve solutions Agilent Technologies application note 2002 publication number 5988 783 1EN 7 Higashi T Shimada K Derivatization of neu tral steroids to enhance their detection charac teristics in liquid chromatography mass spec trometry Anal Bioanal Chem 2004 378 875 882 8 Thurman E M Ferrer I Barcelo D Choosing between Atmospheric Pressure Chemical Ion ization and Electrospray Ionization Interfaces for the HPLC MS Analysis of Pesticides Anal Chem 2001 73 5441 5449 9 US patent 6 646 257 others pending Authors Steven M Fischer is a senior research chemist and Patrick D Perkins is a senior applications chemist at Agilent Technologies Inc in Santa Clara California U S A www agilent com chem Agilent Technologies Inc 2005 Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Printed in the U S A April 21 2005 5989 2935EN j Agilent Technologies
22. it is more likely that one will have to use a compromise setting for a parameter howev er the multimode source is designed to reduce the impact of those compromises Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Analysis of thermally labile compounds IR emitters and temperature feedback ESI is renowned for its ability to ionize thermally labile substances such as proteins and drugs which in part gives rise to its great utility As stated previously the multimode source uses IR emitters to aid in drying the droplets during the ESI process A natural concern is that the emitters may introduce enough heat or introduce heat in an uncontrolled fashion and promote the thermal degradation of these compounds under ESI condi tions Shown in Figure 11 is the analysis of the chemotherapy agent taxol a compound that degrades if exposed to too high a temperature Using a vaporizer temperature of 150 C taxol had essentially no response in APCI mode top It was analyzed successfully by positive ESI only mode or positive mixed mode middle and bot tom Only minor peaks appeared in the mass spectra other than the protonated molecular ion attesting to the lack of degradation products The gentleness of the heating by the IR emitters is attributable to the vapor temperature sensor and feedback control which enable accurate rapid and precise adjustment of the
23. ity 50 Instrument HITHRUPUT 0405126 DataFile DAHPCHEMINDATASASMS04A21ASM50103 D 3 040512H Method File MMM 0405121 Target Mass 2 040512 aj Operator pdp Date 51312004 Time 1 24PM ChemStation Location 040512 Time Area Target Mass Peak Purity Area Start Time End Time Signal amp 040512M DADI 250 MSD1TIC MSD2TIC MSD1 315 DAD1250 MSD1 TIC 040512N 2 059 0405120 3 A 0405134 a Fa 0405138 ASMSO4AI ASMSO4A2 469 10 61 6 45 100 00 6 0 93 565 2 466 0 54E7 0 33E7 314 2 a a EE 037 0 60 075 060 075 MSD2 TIC MS F 2 TK w ih MSD2 TIC MS F ASMSO1 ASMSO4A4 MSD1 TIC MS File MSD2 TIC MS Filef MSD1 315 EIC 314 9 315 9 DAD1 A Sig 250 10 Ref off Overlay AIl Stack ASMSO445 ASMSO4A6 ASMSO447 ASMS0448 MSD1 TIC MS File MSD2 TIC MS File DAD1 A Sig 250 10 Refroff ASMSO4AS ASMS04B0 ASMSO4B1 ASMS04B3 ASMSO4B7 ASMS04B8 ASMSO4BS ASMSOACO ASMSOACI ASMSPNSC 0 MMENVOT 1 0 16 43 77 2 0 65E6 314 2 a o S065 8 jiitiitiiiit Hrrrhrrt a o 3S MSD1 315 EIC 314 9 315 9 5 0 91 56 23 1 2 34E6 314 2 9 4 8 1 85E6 314 2 1 0 16 16 18 Pure 0 64E2 314 2
24. low Simultaneous Electrospray and Atmospheric Pressure Chemical Agilent Technologies lonization The Science Behind the Agilent Multimode lon Source Nebulizer APCI counterelectrode IR emitters APCI corona needle Sensor for vapor temperature Field shaping electrodes Figure 1 left The Agilent multimode ion source mounted on the MSD quadrupole mass spectrometer right Source opened to show a few of the inner parts ESI zone with nebulizer charging electrode HPLC Nebulizer inlet ea and reversing electrode This source uses the same patented orthogonal ESI geometry with a grounded nebulizer as is found on Agilent s dedicated ESI source This design has proven to be extremely robust direct peta ing the vast majority of solvent and spent droplets to waste while selectively diverting the ions to the Reversing capillary entrance of the mass spectrometer The electrode grounded nebulizer permits the mass spectrome ESI Zone APCI Zone Thermal ter to be interfaced directly to other devices e g container capillary electrophoresis instruments without APCI fear of electrical interactions or special isolation counterelectrode precautions The elements in the nebulizer are carefully optimized to provide the proper droplet size and size distribution for maximum produc Corona Drying tion of ions The charging electrode in the ESI zone Figure 3 induces the surface charge on the liquid to start the io
25. method of their use allow the multimode source to perform ESI APCI or simultaneous ESI and APCI at higher HPLC flow rates with compa rable sensitivity to dedicated ion sources Switch ing between modes is as simple as clicking the mouse or may be programmed automatically Mixed mode operation permits higher quality data to be obtained on narrow chromatographic peaks such as those found in high throughput analysis Nitrogen gas consumption is reduced significantly using this source rather than a dedicated ESI source The ultimate benefits of the source are increased sample throughput better scheduling of samples better use of instrument time lower operating costs and less time spent reanalyzing samples References 1 Cody R B Electrospray Ionization Mass Spec trometry History Theory and Instrumenta tion In Practical Spectroscopy Series Volume 32 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 32 677 688 3 Niessen W M A Tinke A P Liquid chromatog raphy mass spectrometry general principles and instrumentation J Chrom A 1995 703 37 57 Agilent Technologies 4 Siegel M M Tabei K Lambert F Candela L Zoltan B J Am Soc Mass Spectrom 1998 9 1196 1203 5 Gallagher R T Balogh M P Davey P Jack son M R S
26. mical Agilent Technologies lonization The Science Behind the Agilent Multimode lon Source Infrared emitters provide conditions for optimal APCI LC Conditions Column 30 x 2 1 mm ZORBAX SB C18 APCI requires different spray and voltage condi 3 5 pm Agilent part no 873700 902 tions than ESI for maximum production of ions It Mobile phase 50 50 water acetonitrile Gradient None isocratic has been known for many years that the best APCI f i Flow rate 0 4 mL min performance is obtained when both the solvent Column temperature Room and the analyte are in the vapor state All instru Injection volume Tub ment manufacturers including Agilent have addi tional heating on their dedicated APCI sources or MS Conditions ae S Multimode itive APCl onl APCI probes to achieve these conditions The sea Na a a ae j Drying gas flow 5 L min infrared emitters of the multimode source accom Nebulizer 20 psig plish the same feat completely vaporizing the Drying gas temp 350 C HPLC effluent and any analytes it contains to Vaporizer temp 60 115 250 C produce this vapor Figure 8 shows replicate Capillary voltage 2500 V _ Corona current 6 pA injections of diphenhydramine a compound that Peakwidth 0 05 min responds well by ESI and APCI at an HPLC flow Time filter On rate of 0 4 mL min As the vaporizer temperature SIM ions 167 1 256 2 m z was increased from 60 C to 250 C the HPLC Fragmentor 12
27. n formation and ESI ions are ae generated here The nebulizing gas flow pushes the droplets and ESI ions past the reversing elec IR emitters trode towards the APCI zone below Figure 2 An overview diagram of the multimode source Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Nebulizer Charging electrode Reversing electrode Figure 3 A close up diagram of the ESI zone of the multimode source Infrared emitters A major innovation in this ion source is a set of infrared IR emitters Figure 4 to aid in drying the aerosol These emitters give off light in a broadband emission whose maximum is at 3 um the absorption wavelength of the hydrogen bonded hydroxyl group of water About 40 of the emit ter s power occurs in this absorption band and the emitter s maximum power output is 270 W enough to completely and instantly vaporize 2 mL min of 100 water and maintain the vapor temperature at 250 C For production of ESI ions the emitters act in a similar role as heated drying gas 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 a dedicated ESI source using only heated drying gas Typical nitrogen consumption is 5 7 L min using t
28. ng one signal for 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 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 and mixed mode operation The top chromatogram is the positive mode TIC second chromatogram is the negative mode TIC the third chromatogram is the target mass trace and the fourth chromato gram is the UV chromatogram The spectrum at the apex of the target mass chromatogram peak is shown at the bottom A gradient of 15 100 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 hours by using such a system Of the compounds in the test set Table 1 89 were detected using a dedicated APCI source and a different 89 were detected using a dedicated ESI source However all test compounds were detected using the multi mode source in positive negative mode attesting to the greater utility of the multimode source Simultaneous Electrospray and Atmospheric Press
29. overy s Hexanesulfonic acid ESI m z 165 0 we g OH Negative lon a Crystal Violet ESI m z 372 a Carbazole LC Conditions Column Mobile phase Gradient Flow rate Column temp Injection volume MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time filter SIM ions positive mode negative mode Fragmentor positive mode negative mode 9 Phenanthrol APCI m z 193 Agilent Technologies 30 x 2 1 mm ZORBAX SB C18 3 5 um Agilent part no 873700 902 35 65 water methanol with 0 2 acetic acid None isocratic 0 4 mL min Room Tul Multimode mixed mode with positive negative switching 5 L min 60 psig 350 C 200 C 1000 V positive and negative 2 pA 0 2 min On 372 2 m z crystal violet 168 1 m z carbazole 165 1 m z hexanesulfonic acid 193 1 m z 9 phenanthrol 160 V at 168 1 m z 180 V at 372 2 m z 140 V at 165 1 m z 160 V at 193 1 m z APCI m z 168 Figure 7 Proof of concept of mixed mode operation with positive negative polarity switching using the multimode source demo sample These four compounds each respond pri marily in only one ionization mode and only one polarity but min may be detected in a single analysis Simultaneous Electrospray and Atmospheric Pressure Che
30. p APCl only mode middle ESI only mode bottom mixed mode operation Vaporizer setpoint was 150 C The response 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 Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Higher HPLC flow rates Mixed mode operation yields better data for fast eluting chromatographic peaks It is possible to analyze samples either by mixed mode ESI and APCI or by alternating between the two ionization techniques on a scan to scan basis When acquiring data the instrument uses either one signal for mixed mode or two signals for alter nating operation There is a difference in the data quality between the two operational modes which becomes apparent when analyzing the narrower chromatographic peaks obtained during fast chro matography Figure 12 Two compounds taxol and progesterone were analyzed using isocratic conditions 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 sampling rate etc were held constant The signal from the UV detector is shown in the bottom trace for com parison The effect on the data is obvious as about two times more scans across
31. s in the system Further more operation in APCI mode becomes more attractive at higher flow rates as the analyte sig nal generally increases at higher flows As mentioned previously the IR emitters emit enough power to vaporize HPLC effluents at up to 2 mL min flow rate The data in Figure 9 was obtained at 1 mL min flow rate and illustrates that the feedback control is rapid enough to track a step change in temperature or mobile phase composition coming to a steady state in a matter of 20 30 seconds Data shown in Figures 12 and 13 were obtained at even higher flow rates 1 5 mL min Simultaneous Electrospray and Atmospheric Pressure Chemical Agilent Technologies lonization The Science Behind the Agilent Multimode lon Source 60000 60000 1 APCI 1 Maximum 1606 854 2 04 04 a D o A aha TOI a E a E E E E E E E T E S a T S E E Y 1 2 3 4 5 6 7mh 200 400 600 800 m z 60000 60000 854 2 4 ESI 4 Maximum 57308 J i 876 2 0 CERET TET b d ra ey i Soa OG OS Ge riti as nan anne Sar Coan Ca Ga cas CT Caen me Ss Ga a ae Ca a 2 ee A So e Tin 200 400 600 800 m z 60000 600004 854 2 1 Mixed Mode i Maximum 57783 i 876 1 0 ET T T E E E S Pout Bhd wun tpt E e E e E e a r e 1 2 3 4 5 6 7min 200 400 600 800 m z Figure 11 Extracted ion chromatograms of m z 854 2 and spectra for the thermally labile compound taxol 1ng pL using the multi mode source to
32. ure Chemical lonization The Science Behind the Agilent Multimode lon Source LC Conditions Columns Two alternating 15 x 4 6 mm ZORBAX RRHT SB C18 1 8 um Agilent part no 821975 902 A water with 0 2 acetic acid B methanol with 0 2 acetic acid 15 B at 0 min 100 B at 0 75 min 100 B at 1 00 min 15 B at 1 01 min Stop run at 1 50 min 1 5 mL min 40 C Binary pump mobile phase Gradient Binary pump flow rate Column temperature lsocratic regeneration pump mobile phase 85 15 water methanol with 0 2 acetic acid lsocratic pump flow rate 1 5 mL min Injection volume 1 uL overlapped injection and minimized delay volume Signal 250 nm 10 nm reference off Diode array detector X ChemStation Data Browser File Edit view Tools Window Help Agilent Technologies MS Conditions Source Multimode mixed mode with positive negative switching Drying gas flow 5 L min Nebulizer 40 psig Drying gas temperature 350 C Vaporizer temperature 200 C Capillary voltage Corona current 1500 V positive and negative 4 pA positive and negative Peakwidth 0 07 min Time filter Off Fast scan mode Enabled Scan 100 1150 m z Fragmentor 120 V positive and negative Stepsize 0 3 m z el lt gt at rT mE ak e el 0405128 4 sampiew 3 0405120 P1 A 03 3 0405120 Sample ID Progesterone amp 040512E DAQTime min 152 Software 040512F Qualifier Pur
33. using a multimode source in mixed mode The multimode source detected all compounds in the test suite whereas the ded icated sources did not APCI Source ESI Source Multimode Source Compound Positive Negative Positive Negative Positive Negative Acetazolamide SS Butyl 4 aminobenzoate D Sear D Cortisone D D D Gemfibrozil EE EEE EE Hexahydro dione D E D Hydroflumethiazide M pD M D Indole es gt lodipamide es gt fC Labetalol D D D D D D Lidocaine i o Morin D D D D D D Paclitaxel E o E o Phenylbutazone D D D D D D Procainamide D mE D Fa D Ss Progesterone D a D Sea D Des Sulfamethoxazole D D D D D D Tolazamide D D D D D D Uracil E O E Detected by polarity 67 56 67 50 78 61 Detected by source 89 89 100 D compound detected Agilent Technologies Figure 14 Improvement in sam ple throughput by combining analysis techniques and hard ware An entire 96 well plate can be analyzed in under three hours by combining all improvements 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 Simultaneous Electrospray and Atmospheric Pressure Chemical lonization The Science Behind the Agilent Multimode lon Source Steroids analysis Steroids are known to be difficult to analyze some respond well in ESI and others respond better in APCI mode Investigators sometimes derivatize them in an effort to impro
34. ve their detection limits Several steroids with different functional groups acid ketone fluoride hydrox yl were analyzed successfully showing a strong response under positive negative mixed mode conditions Figure 15 Environmental compound screening Figure 16 shows the analysis of several classes of compounds of interest in environmental com pound screening Representative pesticides herbicides and insecticides bipyridyliums tri azines phenylureas carbamates phenoxyacetic acids phenols were analyzed at the 5 ng level in positive negative mixed mode All compounds gave a response that was visible in the TIC This is especially noteworthy as the broad range of compound types chromatography and coelution dictate that the pH and vaporization conditions were not optimal for every analyte in the sample LC Conditions Column Mobile phase Gradient Flow rate Column temperature Injection volume MS Conditions Source Drying gas flow Nebulizer Drying gas temp Vaporizer temp Capillary voltage Corona current Peakwidth Time filter Scan Fragmentor Stepsize Agilent Technologies 30 x 2 1 mm ZORBAX SB C18 3 5 um Agilent part no 873700 902 A water with 0 2 acetic acid B methanol with 0 2 acetic acid 10 B at 0 min 10 B at 0 5 min 100 B at 9 min Stop run at 12 min 0 4 mL min 40 C Tul Positive negative switching multimode mixed mode 5 L min 40 psi
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