Agilent Technologies Advantages of Orthogonal Nebulizer Orientation in Electrospray for Quadrupole Ion Trap Mass Spectrometers service manual (1)
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1. 5988 1949EN RE Agilent Technologies2. the course of this experiment the system exhibited a constant response preserving the precision and accuracy necessary for quantitation of the analyte Hydrolysis Sample Internal Standard Area 1 2 965 x 10 70 2 984 x 10 150 2 932 x 108 300 2 935 x 108 500 2 926 x 10 800 2 901 x 10 Average internal standard area 2 992 x 10 Standard deviation n 800 2 6 400000 Area 2 969 x 108 w Q Q oO 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 Area 2 960 x 108 Run1 Run 200 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 Area 2 869 x 108 N ee co 10 11 Time minutes Run 750 12 Figure 5 Selected ion chromatograms for the internal standard demonstrate virtually no performance dropoff even after 750 injections Advantages of Orthogonal Nebulizer Orientation in Electrospray Agilent Technologies for Quadrupole and lon Trap Mass Spectrometers Orthogonal and nonorthogonal charged particles Figure 6 shows the comparison nebulization in an ion trap of A the full scan ion trap analysis off axis spray of a plasma sample B the MS MS ion trap analysis off axis spray of a spiked plasma sample 30 ng ml C the MS MS ion trap analysis on axis spray of a spiked solvent standard 30 ng ml The conditions chosen to analyze the dihydroxy vitamin D3 in plasma represent high throughput retenti
3. 32 829 837 3 Doroshenko V M Cotter R J J Mass Spec trom 1997 32 602 615 4 Voyksner R D Lee H Anal Chem 1999 71 Orthogonal e On xis 1441 1447 Relative Intensiy Author Number of Sample Analysis Dr Robert D Voyksner Figure 8 Comparison of ruggedness of on axis and off axis LCMS Limited nebulization P O Box 13348 Durham NC 27709 Conclusion Nondesolvated charged residues created during the electrospray process negatively affect the performance of mass spectrometers Compared to on axis nebulization off axis nebulization reduces the number of nondesolvated residues transported into the mass analyzer and reduces the contamina tion of the sampling orifice and mass analyzer Thus sensitivity precision accuracy and ruggedness are improved The performance of the system remains nearly constant as required for quantitative analyses www agilent com chem Copyright 2001 Agilent Technologies 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 All rights reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Printed in the U S A March 15 2001
4. al nebulization reduces the negative effects of matrix contami nation of the sampling orifice and ion optics compared to conventional on axis nebulization Orthogonal nebulization also reduces the total number of charged particles that enter the sampling orifice Figure 2 This is particularly important in maintaining ion trap precision dynamic range and sensitivity Data from two different experiments are presented here The first experiment is a long term stability study involving decomposition of a potassium salt It demonstrates the stability of a single quadrupole mass spectrometer using orthoganol nebulizer orientation The second experiment involves the detection of dihydroxyvitamin D3 in plasma It compares the results of on axis and off axis orthoganol nebulization in an ion trap mass spectrometer and contrasts those results with results from the same analysis performed using a triple quadrupole mass spectrometer Agilent Technologies 25 20 10 7 Mt 7 Ain in in S l l l A E aeae tech beste ae Pte tt 20 40 60 80 100 140 a from Nebulize degrees Current pA a Nz nebulization gas 70 psi 3 KV r 25cm _ 0 2 mL min H20 X Fd Figure 2 Spray currents measured at atmospheric pressure at various nebulizer tip angles Experimental In the first experiment the kinetics of decomposition of a potassium salt m z 915 were studied through a series of hydrolysis e
5. f axis and on axis nebulization configurations with an ion trap mass analyzer from Finnigan MAT Figure 4 shows the schematic of this HPLC ion trap system For comparative purposes identical dihydroxyvitamin D3 plasma extracts were analyzed under the same LC conditions using an Agilent 1100 Series HPLC system and a Micromass Quattro II triple quadrupole mass spectrometer HPLC inlet a Capillary Octopole Nebulizer con OS Fragm TIX B gt zone CID Lenses 6 x 10 4 i s Quadrupole Figure 3 Schematic of the LC MSD system with electrospray ion source eS Figure 4 Schematic of the ion trap LC MS system Nebulizer a Octopole Capillary vy eo Lenses yar lon S HPLC inlet Advantages of Orthogonal Nebulizer Orientation in Electrospray for Quadrupole and lon Trap Mass Spectrometers Results and Discussion Orthogonal nebulization spray using the single quadrupole LC MSD The results from the analysis of 800 hydrolysis samples run over 15 days shows the ruggedness of the orthogonal spray nebulization configuration The average standard deviation Table 1 was found to be only 2 6 This is significantly better than the Agilent Technologies Table 1 Ruggedness of the LC MSD using orthogonal nebulization 5 8 that is typical for mass spectrometric detection Figure 5 shows the potassium salt chromatograms from samples 1 200 and 750 Over
6. o 2 o Advantages of Orthogonal Nebulizer E Pg v ee Orientation in Electrospray PN for Quadrupole and lon Trap o o Mass Spectrometers o e e e e e Technical Note Dr Robert D Voyksner LCMS Limited Introduction With the increasing popularity of atmospheric Rabat pi j ipari pressure ionization API mass spectrometric ini al Oo G lt 2 charged techniques electrospray ionization has become ere po a widespread tool for quantitative determinations te_0 7O xe Om OD When the sample matrix is complex electro spray ionization can result in the formation g of nondesolvated charged residues as well as Drying ia analyte ions see Figure 1 These nondesolvated D Gas i rm residues contaminate the ion optics reducing p amp ie signal and thus system performance They also ae ic Residue ES contribute to high background noise in mass Evaporation noise vacuum spectra again reducing system performance In addition desolvated residue ions reduce sensi tivity dynamic range and performance of ion trap Figure 1 APl electrospray mechanism mass spectrometers through space charging gt RE Agilent Technologies Advantages of Orthogonal Nebulizer Orientation in Electrospray for Quadrupole and lon Trap Mass Spectrometers Optimal performance of the mass spectrometric system is required when performing quantitative analyses with numerous samples in complex matrices Off axis orthogon
7. on time 1 9 minutes but poor separation of the analyte from interferences matrix ions and Figure 6 LC ion trap electro spray of dihydroxyvitamin D in plasma comparing off axis z d on axis nebulizati 4 and on axis nebulization J Full Scan MS 2 0 5 TIC Sis MS x 4 R T Vitamin D3 1 S J Z 10 7 05 4 00 i i l r 207 B lon trap MS MS Off axis nebulization 2 Spiked plasma sample 30 ng ml z J 2 107 D 0 0 7 T T T m AN ntl tA 300 4 c lon trap MS MS On axis nebulization 200 Spiked solvent standard 30 ng ml 100 a alana hl 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 Time minutes Advantages of Orthogonal Nebulizer Orientation in Electrospray for Quadrupole and lon Trap Mass Spectrometers The sensitivity using the off axis nebulization is also shown in Figure 7 which compares the chromatograms of a 3 ng ml sample of dihydroxy vitamin D3 in plasma using the A off axis ion trap CID m z 399 gt 381 with the B triple quadrupole system CID m z 399 gt 135 Off axis nebulization also offers improved precision compared to the on axis configuration see Table 2 and comparable precision to the triple quadrupole Agilent Technologies Ruggedness is shown in Figure 8 which compare signal response for a constant level of dihydroxy vitamin D3 between off axis and on axis nebulization for the analysis of 60 plasma extracts The off axis re
8. sults show less than 10 variance in measured peak area over the 60 samples The on axis results show a loss in signal by a factor of 3 4 This loss is due to the buildup of residues on the capillary orifice and end plate reducing sampling efficiency Figure 7 LC MS MS CID 8000 A lon trap MS MS m z 399 gt 381 0 sd 6000 Off axis nebulization 5 Dihydroxyvitamin D3 4000 in plasma 3 ng ml of dihydroxyvitamin D3 in plasma comparing ion trap off axis nebulization with a triple quadrupole Time minutes 100 B J Triple quadrupole MS MS m z 399 gt 135 Dihydroxyvitamin D3 in plasma 30 ng ml o 0 CRP ERER et pba er PE EEEE ATEN AECE ere EEN EET es tps ys 0 5 1 0 1 5 2 0 2 5 3 0 3 5 Table 2 Comparison of precision for the determination of dihydroxyvitamin D3 in plasma using off axis and on axis nebulization electrospray on an ion trap mass spectrometer Instrument Configuration Relative Standard Deviation 3 ng ml 30 ng ml MS MS lon trap on axis ND 14 1 MS MS lon trap off axis 7 1 5 6 ND not detected Advantages of Orthogonal Nebulizer Orientation in Electrospray Agilent Technologies for Quadrupole and lon Trap Mass Spectrometers References 100 1 March R E J Mass Spectrom 1997 32 351 369 2 Mathurin J C Gregoire S Brunot A Tablet J C March R E Catinella S 50 Traldi P J Mass Spectrom 1997
9. xperiments in which pH and temperature were varied A dilute solution of potas sium salt in buffer was incubated in a temperature controlled environment with constant agitation for selected periods of time A portion of the solution was then quenched with methanol The resulting mixture was separated using an Agilent 1100 Series HPLC with a Keystone Betasil C1 reversed phase HPLC column 100 mm x 2 0 mm 5 um particle size and an ammonium acetate methanol solvent gradient Advantages of Orthogonal Nebulizer Orientation in Electrospray for Quadrupole and lon Trap Mass Spectrometers The separated species were detected using an Agilent 1100 Series LC MSD single quadrupole mass spectrometer operated in selected ion monitoring SIM mode using orthogonal negative ion electro spray Figure 3 shows the orthogonal nebulization configuration of the LC MS system For the second experiment rat plasma was spiked with dihydroxyvitamin D3 and mixed with an equal volume of chlorobutane The dihydroxyvitamin D3 was partitioned into the chlorobutane phase which was removed and evaporated to dryness The extract was reconstituted with 70 acetonitrile in water Agilent Technologies Separation was performed using an Agilent 1090 HPLC with a Zorbax XDB Cy reversed phase HPLC column 50 mm x 2 1 mm under isocratic conditions of 70 acetonitrile with 1 acetic acid Mass detec tion was performed using an experimental ion trap that included both of
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