Frequency-Modulated Continuous Flow Analysis Electrospray

Subscriber access provided by MT ROYAL COLLEGE

Letter

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing Robert T. Filla, Adrian M Schrell, John B. Coulton, James L Edwards, and Michael G. Roper Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04669 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Letter to be submitted to Analytical Chemistry

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing

Robert T. Filla‖, Adrian M. Schrell†, John B. Coulton‖, James L. Edwards*‖‡ and Michael G. Roper*†‡

†Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL 32306

ǁDepartment of Chemistry and Biochemistry, Saint Louis University, 3501 Laclede Ave, Saint Louis, MO 63102 ‡ Authors contributed equally

*Address Correspondence to: Dr. James L. Edwards Department of Chemistry and Biochemistry Saint Louis University 3501 Laclede Ave Monsanto Hall St Louis, MO 63102 Ph 314-977-3624 E-mail: [email protected]

Dr. Michael G. Roper Department of Chemistry and Biochemistry Florida State University 95 Chieftain Way Dittmer Building Tallahassee, FL 32306 Ph 850-644-1846 E-mail: [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flowrate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration, encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described which have used modulation techniques to increase the signal-to-noise of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Due to the combination of the samples, there was little effect of salt on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed.


Page 2 of 16

Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION As sample complexity grows, added requirements are needed to maintain accuracy and precision of the final result. This often includes obtaining multiple calibration curves throughout the day as well as multiple control runs. One means to increase throughput and efficiency is to analyze multiple samples at the same time, so called multiplexing. Multiplexing is defined here as the analysis of multiple samples simultaneously. One technique particularly suited for analyzing multiple analytes in a single run is mass spectrometry (MS). One of the most common methods for multiplexing samples for analysis by MS is through isotope labeling or tagging of one, or multiple, samples.1 Stable isotope labeling by amino acids in cell culture2, SILAC, isotope coded affinity tags3, ICAT, and isobaric tags, such as isobaric tags for relative and absolute quantification4 (iTRAQ), and tandem mass tags5 (TMT) have all been used to analyze multiple analytes from multiple samples. Tagging schemes typically target primary amines on the peptides of interest, although different chemistries targeting alternate functional groups are available. iTRAQ and TMT kits offer multiplexing and relative quantitation of up to 8 and 10 different samples, respectively. However, drawbacks of these methods include the cost, labeling efficiency, isotope impurity, and ratio compression.1 The goal of this study was to develop a multiplexed MS method without the need for tagging the various samples. To achieve this goal, we examined methods to perform multiplexed sample analysis in optical spectroscopy where this type of analysis is common. “Colorblind” optical methods were of particular interest since they utilize a single detector to examine multiple analytes (analogous to a single mass spectrometer analyzing multiple samples). Frequency modulation is one colorblind method that has been applied to measure multiple samples in atomic absorption analysis6, DNA



sequencing7,

infrared-mediated

quantitative

polymerase

Page 4 of 16

chain

reaction8,

and

fluorescence anisotropy9. The common theme in these reports is the encoding of analyte signals at specific frequencies. Detection of the total signal results in a convoluted spectrum in the time domain, but spectral analysis reveals the individual contributions of each sample at their specific frequency. Importantly, the peak intensities in the frequency domain are related to the concentration of the individual samples. In this letter, we describe the analogous use of frequency modulation to multiplex mass spectrometric analysis. This newly-described method is not to be confused with earlier reports that have described modulated sample introduction techniques to analyze a single sample multiple times.10-18 A main goal in these methods is to increase S/N of a single sample, whereas in this new method the main goal is to analyze multiple samples simultaneously. As a proof-of-concept, two samples were multiplexed and 9 analytes common to both samples were quantified. We also demonstrate that the ability to quantitate is relatively independent of the salt concentration in either sample because they are mixed together and analyzed simultaneously.


Page 5 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION Proof of concept work was undertaken to determine if the method of frequency modulation could be applied to multiplex mass spectrometry analysis. In this method (Figure 1), the flow rates of different samples are sinusoidally modulated at nonharmonic frequencies of each other, but the total flow rate to the MS is held constant by inclusion of a make-up flow of buffer. After mixing, concentration pulses of each sample are produced and encoded at the same frequency. Extraction of each RIC yields timedomain data traces that are then decoded by a fast Fourier transform. The frequency domain trace indicates the sample from which the peak belongs (by the frequency at which it occurs) and its relative concentration (by the peak height). Therefore, numerous samples can be analyzed simultaneously, without tagging, by pulsing each at a unique frequency.

Figure 1. Experimental set up for FM-CFA-ESI-MS. Two samples are placed in separate syringe pumps and the flow rates are varied at unique frequencies (VS1(t) and VS2(t), respectively). These solutions meet at a cross with flow from a third syringe pump that has a time-dependent velocity used to ensure the total flow rate is kept constant at 40 µL/min. The top and bottom insets show the time-dependent flow rates from the two pumps, while the middle inset shows the timedependent concentration profile that is produced. The combined solution is then subjected to electrospray ionization and the mass spectrometer used to monitor particular m/z. Individual selected ion currents (SIC) are subjected to a fast Fourier transform (FFT) which produces two peaks in the frequency domain. The magnitude of the peaks in the frequency domain is indicative of the analyte concentration and the frequency of each peak encodes the sample identification.

To demonstrate this method (experimental details given in the Supporting Information), caffeine was used as a model analyte with one syringe set to a period of 97



s (0.01 Hz) and at a concentration of 100 µM while the second syringe was set to a period of 43 s (0.023 Hz) and varying concentrations from 50-300 µM. Data were collected for 30 min. Figure 2 shows the calibration curve obtained by plotting the ratio of peak intensities in the frequency domain against the ratio of caffeine concentrations in each syringe. The data show good linearity with a slope ~1 and a correlation coefficient > 0.99 indicating that this system is capable of quantitating co-infused analytes at different concentrations. The linear dynamic range saturated above a concentration ratio of ~3, which was due to the difficulty in measuring disparate peak heights. We expect that further optimization of the method and analysis technique will allow a larger linear dynamic range to be employed.

Figure 2. Calibration curve of caffeine using FM-CFA-ESI-MS. Syringe one held 100 µM caffeine while syringe 2 contained variable caffeine concentrations from 50 to 300 µM. The experimentally measured ratios of the peak heights in the frequency domain are shown on the y-axis while the expected concentration ratio is shown on the x-axis. The insets show representative traces from the frequency domain at various concentration ratios.

Examination of the frequency domain data indicated low frequency noise, which was to be expected. Other frequencies, such as from the oscillatory movement of the syringe pump, were not observed. All periods were chosen to be prime numbers to ensure frequency harmonics would not complicate the data analysis. The peak at lower frequency had a larger peak width in the frequency domain due to the reduced number of cycles analyzed relative to the higher frequency sample.


Page 6 of 16

Page 7 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Critical to success of these experiments was the proper alignment of the capillaries at the mixing tee. When tubing was not flush and properly seated in the tee, one syringe would pull fluid from the second syringe. This attenuated the signals and diminished the quality of the calibration curve. To diagnose this effect, theophylline was added to the sample in syringe 2, and after each analysis, the RIC of theophylline was analyzed. For properly aligned connections, theophylline signal was only present at the frequency of syringe 2 and absent at the frequency of syringe 1. Data which contained theophylline signal at both syringe frequencies were discarded and connections realigned. Effect of period length and acquisition time To gain a better understanding of the method, the effects of several experimental variables were examined. Equal concentrations of caffeine were placed in both syringes and syringe 1 and 2 were oscillated with a 227 s and 157 s period, respectively. By increasing the acquisition time of the experiment from 25 to 50 min, a decrease in the frequency-domain peak widths occurred (Figure S-1A-D). While this effect was expected due to increased sampling of the concentration pulses, no noticeable changes in the ratio of peak heights were observed (Figure S-1E). Another experimental variable tested was the effect of the syringe oscillation periods on the measured peak height ratios in the frequency domain. Caffeine was placed at a 1:1 concentration ratio in both syringe 1 and 2. Syringe 1 was oscillated with a period of 97 s and the period of syringe 2 was varied between 7 and 61 s. Higher periods in syringe 2 trended to a ratio of 1 in the frequency domain, while lower periods showed a marked decrease in this ratio (Figure S-2). These data suggest that the higher frequency concentration pulses were attenuated, potentially due to limitations with the syringe pumps or with broadening of these pulses in the fluid path as they were delivered to the mass spectrometer. The attenuation of high frequency pulses has been



observed in other fluid systems and can be mitigated with appropriate choice of tubing lengths and flow rates. Multiple analytes To test the ability of the new FM-CFA-ESI-MS method to quantify multiple analytes, nine solutes (caffeine (Caff), arginine (Arg), lysine (Lys), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), valine (Val), aspartate (Asp), histidine (His)) were placed in syringe 1 and 2 (theophylline was also included in syringe 2). Figure S-3 gives the MRM transitions monitored for these various analytes. Three experiments were performed where the concentrations of the analytes at different ratios were tested. In experiment 1 (Figure 3), all analytes were held at a 1:1 concentration ratio in syringe 1:syringe 2. Experiment 2 kept 3 analytes (Phe, Tyr, and Caff) at a 1:1 concentration ratio in both syringes, while the concentration ratio was increased to 3:2 for another 3 analytes (Trp, Lys, Arg), and decreased to 2:3 for the remaining 3 analytes (Val, Asp, His). Experiment 3 was similar to experiment 2 but used a larger range of concentration ratios, 5:2 and 2:5, for the increased and decreased samples, respectively. Figure 3 summarizes the results obtained. The data are plotted by experiment, where Figure 3A shows the data from Experiment 1, Figure 3B from Experiment 2, and Figure 3C from Experiment 3. For all analytes tested, the peak ratios in the frequency domain followed the expected trend. These results give confidence that the FM-CFAESI-MS method can quantify multiple analytes from multiple samples.


Page 8 of 16

Page 9 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Quantitation of multiple analytes using FM-CFA-ESI-MS. The measured frequency domain peak ratios are plotted with the expected concentration ratio shown above the bars given as analyte concentration in syringe 1: analyte concentration in syringe 2. In syringe 1, the concentrations of the analytes were held constant at: Val, Asp, and His at 25 µM each, while Phe, Tyr, Caff, Trp, Lys, and Arg were 10 µM each. The black bars indicate the expected ratio for each subset of analytes in each experiment. A. Syringe 2 held the same concentrations as syringe 1 producing a 1:1 ratio for all analytes. B. A 2:3 ratio of Val, Asp, and His were produced by using 16.75 µM for these 3 analytes in syringe 2. A 1:1 ratio of Phe, Tyr, Caff were made by placing 10 µM each into syringe 2, and a 3:2 ratio was produced by using 16.75 µM of Trp, Lys, and Arg in syringe 2. C. A 2:5 ratio was generated by placing 10 µM Val, Asp, and His in syringe 2. A 1:1 ratio was made by using 10 µM Phe, Tyr, and Caff in syringe 2, while a 5:2 ratio was made by placing 25 µM Trp, Lys, and Arg in syringe 2. Theophylline was kept in syringe 2 to ensure flow pattern fidelity.

Effect of salt on ionization suppression To investigate the ability of this system to quantify under more difficult conditions, signal suppression from salting was tested. Three high proton affinity analytes, Lys, Arg,



and Caff were analyzed simultaneously by both flow injection analysis-ESI-MS and the new FM-CFA-ESI-MS method. Figure 4A shows direct injection of these three analytes at 25 µM concentration in 50:50 acetonitrile:water containing either 0, 20, or 40 mM ammonium formate as the salt. Lys showed the highest signal with no ammonium formate, but a ~85% decline in signal upon addition of salt. Arg showed a similar trend as Lys. Caff was the most resistant to salting effects by direct injection as there was no difference in signal intensity between 0 and 20 mM ammonium formate, but a ~35% decrease upon addition of 40 mM salt. The new FM-CFA-ESI-MS method was performed by placing 25 µM Lys, Arg, and Caff in 50:50 acetonitrile:water into both syringes, and holding syringe 1 at 0 mM ammonium formate while varying the concentration from 0 – 40 mM in syringe 2. When the signals from these analytes were examined, the ratios of Lys and Arg were equivalent across all salt concentrations. The Caff signal intensity trended downward slightly as salt concentrations increased. These data suggest that FM-CFA-ESI-MS is resistant to ion suppression from salt at the concentration ranges tested. This effect is attributed to analytes in both high and low salt syringes being exposed to the same environment during electrospray.


Page 10 of 16

Page 11 of 16

A 0 mM

20 mM

40 mM

Arginine

Caffeine

20 mM

40 mM

Arginine

Caffeine

4.0

6

Signal Intensity (x10 )

4.5

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

B 1.8 Measured Peak Height Ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lysine 0 mM

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Lysine

Figure 4. The effect of salt on quantitation. A. 25 µM samples of Lys, Arg, and Caff were directly injected into an ESI needle and analyzed by MRM with the ammonium formate concentration shown in the legend. B. The same analytes were placed in both syringes 1 and 2, while the salt concentration in syringe 2 was varied as shown in the legend and analyzed via FM-CFA-ESI-MS. The concentration ratio for each analyte was 1:1. The error bars indicate +/- 1 SD.

Future Considerations The method described describes a new path toward label-free relative quantitation in MS analysis. In FM-CFA-ESI-MS, the number of compounds in a given sample that can be analyzed is dependent on the resolution of the mass spectrometer. Since a triple quadrupole was used in this report, we limited the number of analytes to 9 MRMs as a proof of concept. As is the case with the majority of MS analyses, the use of higher resolution mass analyzers would enable analysis of a higher number of compounds in a given sample. Another feature that plays a role in the number of analytes that can be quantified is the acquisition frequency of the MS. High acquisition



frequencies would ensure sampling of all ions at a rate that enables complete sampling of the concentration pulses. As for the number of samples that may be employed, we demonstrated two as a proof of concept. However, additional samples can be easily increased by using more pumps. This would come at the cost of increased complexity in the plumbing of the tubing lines, but microfluidic systems may be a simple solution to this complexity. It is not difficult to imagine multiplexing a similar number of samples that can currently be interrogated in the commercial assays that utilize chemical tags. A limiting feature of the number of samples that can be analyzed simultaneously is the dynamic range of the mass spectrometer. The total number of ions must be within the linear range of the mass analyzer, otherwise the amplitude of the signal will not be proportional to the concentration of each individual sample. This can be remedied by sample dilution, and with improvements in the dynamic range of mass spectrometers. CONCLUSIONS FM-CFA-ESI-MS has been successfully implemented to multiplex two samples with up to 9 analytes. This system demonstrates resistance to diminished quantitation due to salting effects, likely due to mixing of the samples before ESI which results in similar salt concentrations for all samples under analysis. While this proof of concept is promising, future investigations will focus on optimizing the system with regards to both analyte and sample number.


Page 12 of 16

Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGEMENTS Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental methods, results of acquisition time on frequency domain peak widths and peak height ratios, results of syringe pump oscillation period on peak height ratios, MRM transitions monitored for 9-plex analysis (PDF) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. JLE and MGR are co-corresponding authors. Notes JLE and MGR have filed a provisional patent on this work.



REFERENCES 1. Rauniyar, N.; Yates III, J. R. J. Proteome. Res. 2014, 13, 5293-5309. 2. Hoedt, E.; Zhang, G.; Neubert, T.A. Adv. Exp. Med. Biol. 2014, 806, 93-106. 3. Booy, A. T.; Haddow, J. D.; Ohlund, L. B.; Hardie, D. B.; Olafson, R. W. J. Proteome Res. 2005, 4, 325-334. 4. Chen, Z.; Wang, Q.; Lin, L.; Tang, Q.; Edwards, J. L.; Li, S.; Liu, S. Anal. Chem. 2012, 84, 2908-2915. 5. Werner, T.; Becher, I.; Sweetman, G.; Doce, C.; Savitski, M. M.; Bantscheff, M. Anal. Chem. 2012, 84, 7188-7194. 6. Edel, H.; Quick, L.; Cammann, K. Fresenius J. Anal. Chem. 1995, 351, 479-483. 7. Dongre, C.; van Weerd, J.; Besselink, G. A. J.; Vazquez, R. M.; Osellame, R.; Cerullo, G.; van Weeghel, R.; van den Vlekkert, H. H.; Hoekstra, H. J. W. M.; Pollnau, M. Lab Chip 2011, 11, 679-683. 8. Schrell, A. M.; Roper, M. G. Analyst 2014, 139, 2695-2701. 9. Schrell, A. M.; Mukhitov, N.; Roper, M. G. Anal. Chem. 2016, 88, 7910-7915. 10. Allen, P. B.; Doepker, B. R.; Chiu, D. T. Anal. Chem. 2007, 79, 6807-6815. 11. Chen, Y. H.; Siems, W. F.; Hill, H. H. Anal. Chim. Acta 1996, 334, 75-84. 12. Fernandez, F. M.; Vadillo, J. M.; Kimmel, J. R.; Wetterhall, M.; Markides, K.; Rodriguez, N.; Zare, R. N. Anal. Chem. 2002, 74, 1611-1617. 13. Kaneta, T.; Yamaguchi, Y.; Imasaka, T. Anal. Chem. 1999, 71, 5444-5446. 14. Kwok, Y. C.; Manz, A. Electrophoresis 2001, 22, 222-229. 15. Shen, H. L.; Jia, X.; Meng, Q. Y.; Liu, W. J.; Hill, H. H. RSC Adv. 2017, 7, 78367842. 16. Trudgett, M. J.; Guiochon, G.; Shalliker, R. A. J. Chromatogr. A, 2011, 1218, 35453554. 17. Yoon, O. K.; Zuleta, I. A.; Kimmel, J. R.; Robbins, M. D.; Zare, R. N. J. Am. Soc. Mass Spectrom. 2005, 16, 1888-1901. 18. Zare, R. N.; Fernandez, F. M.; Kimmel, J. R. Angew. Chem. Int. Ed. Engl. 2003, 42, 30-35.


Page 14 of 16

Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions Figure 1. Experimental set up for FM-CFA-ESI-MS. Two samples are placed in separate syringe pumps and the flow rates are varied at unique frequencies (VS1(t) and VS2(t), respectively). These solutions meet at a cross with flow from a third syringe pump that has a time-dependent velocity used to ensure the total flow rate is kept constant at 40 µL/min. The top and bottom insets show the time-dependent flow rate velocities from the two pumps, while the middle inset shows the time-dependent concentration profile that is produced. The combined solution is then subjected to electrospray ionization and the mass spectrometer used to monitor particular m/z. Individual selected ion currents (SIC) are subjected to a fast Fourier transform (FFT) which produces two peaks in the frequency domain. The magnitude of the peaks in the frequency domain is indicative of the analyte concentration and the frequency of each peak encodes the sample identification. Figure 2. Calibration curve of caffeine using FM-CFA-ESI-MS. Syringe one held 100 µM caffeine while syringe 2 contained variable caffeine concentrations from 50 to 300 µM. The experimentally measured ratios of the peak heights in the frequency domain are shown on the y-axis while the expected concentration ratio is shown on the x-axis. The insets show representative traces from the frequency domain at various concentration ratios. Figure 3. Quantitation of multiple analytes using FM-CFA-ESI-MS. The measured frequency domain peak ratios are plotted with the expected concentration ratio shown above the bars given as analyte concentration in syringe 1: analyte concentration in syringe 2. In syringe 1, the concentrations of the analytes were held constant at: Val, Asp, and His at 25 µM each, while Phe, Tyr, Caff, Trp, Lys, and Arg were 10 µM each. The black bars indicate the expected ratio for each subset of analytes in each experiment. A. Syringe 2 held the same concentrations as syringe 1 producing a 1:1 ratio for all analytes. B. A 2:3 ratio of Val, Asp, and His were produced by using 16.75 µM for these 3 analytes in syringe 2. A 1:1 ratio of Phe, Tyr, Caff were made by placing 10 µM each into syringe 2, and a 3:2 ratio was produced by using 16.75 µM of Trp, Lys, and Arg in syringe 2. C. A 2:5 ratio was generated by placing 10 µM Val, Asp, and His in syringe 2. A 1:1 ratio was made by using 10 µM Phe, Tyr, and Caff in syringe 2, while a 5:2 ratio was made by placing 25 µM Trp, Lys, and Arg in syringe 2. Theophylline was kept in syringe 2 to ensure flow pattern fidelity. Figure 4. The effect of salt on quantitation. A. 25 µM samples of Lys, Arg, and Caff were directly injected into an ESI needle and analyzed by MRM with the ammonium formate concentration shown in the legend. B. The same analytes were placed in both syringes 1 and 2, while the salt concentration in syringe 2 was varied as shown in the legend and analyzed via FM-CFA-ESI-MS. The concentration ratio for each analyte was 1:1. The error bars indicate +/- 1 SD.



For TOC only


Page 16 of 16

Frequency-Modulated Continuous Flow Analysis Electrospray

Recommend Documents