Anal. Chem. 2000, 72, 3683-3688
Accelerated Articles
Accurate Mass Liquid Chromatography/Mass Spectrometry on Orthogonal Acceleration Time-of-Flight Mass Analyzers Using Switching between Separate Sample and Reference Sprays. 1. Proof of Concept Christine Eckers,*,† Jean-Claude Wolff,† Neville J. Haskins,† Ashley B. Sage,‡ Kevin Giles,‡ and Robert Bateman‡
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW, U.K., and Micromass UK Ltd., Floats Road, Wythenshawe, Manchester, M23 9LZ, U.K.
This paper describes the use of two separate electrosprays for introducing sample and reference for accurate mass liquid chromatography/mass spectrometry (LC/MS) on an orthogonal acceleration time-of-flight mass analyzer. This is carried out using an adaptation of the multiplexed electrospray ion source in which only two of the sprays are utilized. Results are shown for the positive ion detection of trace-level components in complex matrixes and good mass accuracies are obtained, even for very low level components. An example of accurate mass measurements obtained using negative ion LC/MS is also shown. To obtain additional structural information, an example of cone voltage fragmentation is included and shows that good mass accuracy can be obtained for both precursor and fragment ions.
The use of orthogonal acceleration time-of-flight (oa-TOF) mass analyzers for accurate mass measurement of small molecules is becoming a widespread technique, particularly for the characterization of trace level impurities.1 Generally the instrument is calibrated using a reference compound that contains known masses over the mass range at which a particular piece of analysis is to be carried out. Subsequent instrumental drift is corrected * Corresponding author: (fax) +44-(0)1279-627655; (e-mail)
[email protected]. † SmithKline Beecham Pharmaceuticals. ‡ Micromass UK Ltd. (1) Eckers, C.; Haskins, N.; Langridge, J. Rapid Commun. Mass Spectrom. 1997, 11, 1916-1922. 10.1021/ac000448i CCC: $19.00 Published on Web 07/12/2000
© 2000 American Chemical Society
for by the introduction of a single reference compound, which is used as a lock mass. The latter is introduced postcolumn when coupled to a liquid chromatography (LC) system. Ideally, a reference compound is chosen that gives a single peak (no cluster ions or in-source fragment ions) with a mass-to-charge ratio (m/z) higher than the expected sample ions. This removes the possibilities of mass interferences and provides a mass scale correction factor, which is most accurate for m/z values up to that of the lock mass used. When gradient LC is used as the separation technique, the changing eluent composition leads to enhancement or suppression of the ionization efficiency of the reference compound. This means that the signal from the reference compound may not be in the optimal intensity range for the best accurate mass measurements. Moreover, postcolumn addition can involve complex systems, involving “T”-piece and splitter arrangements. This can cause back-pressure problems and significant band broadening when used with narrow-bore LC systems. The presence of the reference signal within the eluent being introduced into the mass spectrometer also can have a deleterious effect on the appearance of the total ion current chromatogram (TIC) obtained for LC/MS and this may make it difficult to identify low-level components. In other circumstances, mass interferences may occur where the lock mass could be of the same nominal mass as an analyte, resulting in errors in mass measurement. In addition, when product ion MS/MS is carried out, the presence of the lock mass will have no benefit, since there will be no reference ions present in the MS/MS spectrum. Todate accurate mass MS/MS reported on ToF instruments work has used either a residual precursor ion of known mass or a Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3683
Table 1. Structures of Compounds Investigated
fragment ion of known mass as reference.1,2 This approach can work very well for samples for which some information is known but is of little use for total unknowns. Alternate methods for introduction of the reference material have therefore been sought. The use of separate sprayers for sample and reference has been suggested.3-6 These offer the advantage of nonmixing of the eluent and reference prior to the mass spectrometer, and therefore, there should be no effect on the chromatographic integrity. In addition, if the sample and reference are introduced into the mass spectrometer at separate (2) Hopfgartner, G.; Chernushevich, I. V.; Covey, T.; Plomley, J. B.; Bonner, R. J. Am. Soc. Mass Spectrom. 1999, 10, 1305-1314. (3) Eckers, C.; Wolff, J.-C.; Haskins, N.; Bateman, R.; Hoyes, J.; Preece, S. Presented at the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-18, 1999. (4) Dresch, T.; Keefe, T.; Park, M. Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-18, 1999; pp 1865-1866. (5) Andrien, B. A.; Whitehouse, C.; Sansone, M. A. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, May 31June 4, 1998; p 889. (6) Whitehouse, C.; Gulcicek, E.; Andrien, B.; Shen, S. Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-18, 1999; pp 454-455.
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times, then it will be possible to acquire the sample information excluding any effect from the reference. The reference, however, needs to be sampled sufficiently closely in time to the sample to be able to obtain exact mass measurements. This could be accomplished in a number of ways using a dual-spray system. One option would be to switch the electrospray voltage on and off to the separate sprays. However, often a residual ion current is observed in electrospray for a few seconds after switching off the current, presumably due to ions already generated within the flow, and thus would be impractical for on-line LC/MS. An alternate would be to physically prevent the sample and reference sprays being presented to the MS at the same time. This could be accomplished simply by switching the flow on and off; however, this is not practical for LC/MS. A method of diverting either the sample or reference flow from the MS would solve the problem, and in this paper we describe an adaptation of the use of the multiplexed electrospray source (MUX)7,8 coupled to the Z-Spray interface of an LCT orthogonal time-of-flight mass spectrometer (Micromass Ltd., Wythenshawe, Manchester, U.K.). This system was devised to allow four or eight LC streams to be analyzed in parallel using a single mass spectrometer with negligible interchannel crosstalk. However, in this work, we used only two inlet channels along with modified stepper motor control to allow either sample or reference to be introduced into the ion source of the mass spectrometer. This system would have the same benefits for both MS and MS/MS since separate sprays are used for sample and reference. Examples of results obtained with this system will be shown, including the accurate mass measurements obtained for the identification of trace impurities and the sensitivity of the measurements. The use of in-source cone voltage fragmentation will also show an indication of the applicability of this technique for MS/MS. Also shown are some preliminary results using negative ion LC/MS. EXPERIMENTAL SECTION Reagents, Calibrants, and Compounds Investigated. All reagents and calibrants were obtained from Sigma-Aldrich (SigmaAldrich Co. Ltd., Poole, Dorset, U.K.), unless otherwise stated. Solvents were from BDH (Poole, Dorset, U.K.). The compounds investigated are listed in Table 1. Mass Spectrometry. Experiments were carried out using a Micromass LCT orthogonal acceleration time-of-flight mass spectrometer (Micromass UK Ltd.), which was equipped with an eightsprayer MUX.7 The MUX source was operated in either positive or negative ion electrospray mode, depending on the samples under investigation, with only two of the eight sprayers being used to carry out the experiments. For all experiments, the first sprayer was used to introduce the reference compound for lock mass correction and subsequent exact mass measurement, while the second sprayer was used to admit the LC eluent. Each of the sprayers within the MUX source is indexed using an optical position sensor and selected using a programmable stepper motor, which is controlled through the MassLynx data system. The (7) De Biasi, V.; Haskins, N.; Organ, A.; Bateman, R.; Giles, K.; Jarvis, S. Rapid Commun. Mass Spectrom. 1999, 13, 1165-1168.
Figure 1. Total ion current chromatogram (TIC) and mass chromatograms for SKF-92452 (m/z 269), SKF-82964 (m/z 315), SKF105779 (m/z 329), and SKF-88964 (m/z 423) in cimetidine drug substance.
stepper motor controls the position of a sampling aperture in the electrospray source, which is used to admit ions from one spray at a time to the sampling cone of the LCT. To acquire data from the reference spray and eluent spray sequentially, the stepper motor was “flipped” between the two sprayers, with the electrospray ion current sampled from each channel being acquired into its own discrete data file. Acquisition times per spray were set to 0.5 s, with the interspray switch time being set to 0.05 s. This produced a data point for each spray every 0.55 s. For the sets of samples investigated, the instrument was operated in either positive or negative ion electrospray, with capillary voltages of 3.2 and 3.0 kV being used, respectively. A desolvation temperature of 320 °C and a source temperature of 110 °C were used for all experiments. The nitrogen desolvation and nebulizer gas flow rates were set to 1100 and 300 L/h, respectively. Prior to performing all experiments, the instrument was calibrated in both ionization modes over a 100-800 Da massrange, using a 20 ng/µL solution of poly(DL-alanine) dissolved in methanol. This solution produces singly charged reference ions in both ion modes and is ideal as a calibrant for low molecular weight analysis. A separate calibration line was used for each ion mode, and the 100-800 Da mass range was subsequently used to acquire all experimental data. Chromatography. LC was performed using a Waters 2690 solvent delivery system (Waters Corp., Milford, MA). For all experiments, prior to entering the MUX source, the LC eluent was split down to ∼100 µL/min via a zero dead volume T-piece (Valco Instruments Co. Inc., Houston, TX). Chromatographic Conditions for the Analysis of a Cimetidine Sample and the Measurement of SKF-88964 in SKF-92058. The HPLC column was a Waters NovaPak C18 (3.9 × 150 mm). The flow rate was 1 mL/min, and reequilibration time was 10 min. (8) Bateman, R.; Jarvis, S.; Giles K.; Organ, A.; de Biasi, V.; Haskins, N. Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-18, 1999; pp 1865-1866
The gradients used for the two measurements were slightly different. For the measurement of SKF-88964 in SKF-92058, the run was started at 100% 0.05 M ammonium acetate in deionized water (HiPerSolv for HPLC, BDH). The acetonitrile content was increased linearly from 0% over 25 min to 30% and then over 8 min to 60%. For the analysis of cimetidine, the gradient was as follows: the run started at 100% 0.05 M ammonium acetate, was held for 3 min, and then the acetonitrile content was programmed linearly from 0 to 60% over 30 min. Chromatographic Conditions for a Sample Consisting of SB243213, SKF-105409, and Cimetidine. The HPLC column was a Waters Symmetry C18 (3.9 × 150 mm). The flow rate was 1 mL/ min, and reequilibration time was 5 min. The run was started at 70% 0.05 M ammonium acetate in deionized water. The acetonitrile content was increased linearly from 30% over 10 min to 70% and then held for 5 min at 70%. Chromatographic Conditions for the Separation of Dibenzoyl Tartaric Acid and Di-p-toluyl Tartaric Acid in a Main Matrix Component. The separation was carried out isocratically using a Waters Spherisorb NH2 (100 × 2.1 mm) column. The eluent composition was 50 mM ammonium acetate in acetonitrile/water (70:30 volume fraction), the flow rate 0.2 mL/min, and the column temperature 40 °C. Lock Mass Reference Compound Introduction. The reference solution(s) used for lock mass correction was introduced into sprayer 1 of the MUX source using a Harvard 22 syringe pump (Harvard Apparatus Inc., South Natick, MA) and silica transfer line. For all experiments, 0.1 ng/µL solutions of reference compound in acetonitrile/water (50:50 volume fraction) were infused. Depending on the reference compound used, the infusion flow rate (typically between 5 and 10 µL/min) was adjusted in order to obtain an ion current between 400 and 500 counts per second. For the positive ion electrospray experiments, the reference compounds used for lock mass correction were leucine enkephalin ([M + H]+ ) 556.2771 Da) and terfenadine ([M + H]+ ) 472.3215 Da). For negative ion experiments, a small peptide, Val-Tyr-Val ([M - H]- ) 378.2029 Da), was used. No interchannel crosstalk was observed between samples and reference ions in any of the analyses carried out in this work. Procedure Used for “Mass Measuring” Sample Components. All data were acquired in continuum mode, and hence before any mass measurement, the reference and sample mass spectra were centered using the top 80% of the peak area. Then, the mass of the reference (e.g., m/z 472.2679 for the protonated molecule of terfenadine in Figure 4) in the centroid spectrum was compared to its theoretical exact mass (e.g., m/z 472.3215 for protonated terfenadine). A factor (1.000 113 49), being the ratio between the two masses, was calculated. This factor was then applied to the mass of the sample in the centroid spectrum using the “Modify Calibration” feature of MassLynx. This gives the “accurate” mass which is within a few millidaltons (or ppm) of the exact mass. RESULTS AND DISCUSSION Accurate Mass Measurement of Trace-Level Impurities in Cimetidine Drug Substance. Figure 1 shows the TIC obtained for a sample of cimetidine drug substance. The major peak observed at ∼10 min is due to cimetidine itself. Close observation of the baseline shows that a number of small Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
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Figure 2. (a) Raw data (noncentered and noncorrected) mass spectrum for peak B (consisting of SKF-82964, SKF-105779, and SKF-88964) and (b) raw data (noncentered) mass spectrum for reference compound leucine enkephalin.
Figure 3. Mass chromatogram for SKF-88964 (a)sthe signal-tonoise ratio being ∼6:1 (see magnified noise in chromatogram)sand total ion current chromatogram (b). Table 2. Accurate Mass Measurement Results for the Impurities Eluting in Peaks A (Retention Time 8.1 min) and B (Retention Time 13.3 min) from Figure 1 impurity and mol formula for[M + H]+ SKF-92452 C10H17N6OS SKF-82964 C10H19N8S2 SKF-105779 C11H21N8S2 SKF-88964 C16H27N10S2
retention time, min
theor mass for [M + H]+
measd mass, corr
deviation, mDa (ppm)
8.11
269.1186
269.1185
0.1 (0.4)
13.36
315.1174
315.1166
0.8 (2.5)
13.32
329.1331
329.1335
0.4 (1.2)
13.34
423.1862
423.1859
0.3 (0.7)
peaks can be observed, and Figure 1 also shows mass chromatograms for the protonated molecule for four minor impurities, i.e., SKF-92452, SKF-82964, SKF-105779, and SKF-88964. SKF-92452 (peak A) elutes just prior to cimetidine at ∼8 min, and the three others (labeled as peak B) almost coelute just after the main peak. Figure 2a shows the noncorrected mass spectrum obtained for mixed peak B, while Figure 2b shows the noncorrected mass spectrum for the reference, leucine enkephalin, as an example. The leucine enkephalin reference data were used to correct the masses for the impurities under peaks A and B as described in the Experimental Section. The data are shown in Table 2. All the measured masses are within 1 mDa or 3 ppm of the theoretical 3686 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
Figure 4. Centered and corrected mass spectrum of SKF-88964 (a) and centered reference spectrum of terfenadine (b).
values. There are other background ions observed in both spectra, for example the ion at m/z 505. Sensitivity of the System and Accuracy of Mass Measurements at Limit of Detection Levels of Trace Compounds. Figure 3 shows the mass chromatogram for SKF-88964, which was spiked at a level of 10 ppm mass fraction (relative to the matrixcompound) into SKF-92058. This demonstrates that despite switching between reference and sample sprayer, good sensitivity is achieved. The sensitivity was similar to a previous analysis carried out on the same sample using a quadrupole orthogonal acceleration time-of-flight instrument (Q-ToF).9 It shows that even at the edge of the detection limit, i.e., having as few as 21 counts and a signal-to-noise ratio of only 6:1 (Figure 3), mass measurements with good accuracy can be performed. For the present example, terfenadine was used as reference and the deviation from the exact mass of the [M + H]+ ion for SKF-88964 was found to be 3.6 mDa (8.5 ppm) (refer to Figure 4). The accuracy of the mass measurement is comparable with other results shown in this work. Use of the Dual-Sprayer MUX System for Providing a Reference for Accurate Mass Measurement of Fragment Ions. As this investigative work was carried out on a singleanalyzer ToF system, in-source fragmentation was used to
Figure 5. Centered and corrected mass spectrum of SB-243213 using a cone voltage of 80 V.
Figure 6. Centered and corrected mass spectrum of SKF-105409 using a cone voltage of 80 V. Table 3. Accurate Mass Measurement for SKF-105409 (Refer as well to Figure 6) structure and molecular formula C12H23N8S2 for
[M+H]+
theor mass
measd mass
deviation, mDa and (ppm)
343.1487 245.0895
343.1501 245.0906
1.4 (4) 1.1 (4)
157.0548
157.0546
0.2 (1)
C9H17N4S2
C5H9N4S
investigate the use of the dual-sprayer MUX approach for obtaining accurate mass measurement on fragment ions. SB-243213, SKF105409, and cimetidine were used to illustrate this application. The mixture was first analyzed by LC/MS using a low cone voltage (20 V) to obtain molecular weight information. The analysis was then repeated using a high cone voltage (80 V), which was found to generate a number of fragment ions. These fragment (9) Wolff, J.-C.; Barr, L.; Moss, P. Rapid Commun. Mass Spectrom. 1999, 13, 2376-2381.
ions were consistent with those previously observed using MS/ MS techniques. The protonated molecule of leucine enkephalin was used as reference standard for both the low and high cone voltage experiments. Mass measurement of SB-243213 (429.1535 Da for the protonated molecule) at cone voltage 80 V (Figure 5) was within 0.3 mDa (corresponding to 0.7 ppm) of the theoretical mass for the protonated molecule, i.e., 429.1538 Da. The molecule gives mainly one fragment ion (measured mass 228.0753 Da) which could Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
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correspond to the two isobaric portions attached to the carbonyl entity, i.e., C12H10N3O2 (theoretical mass 228.0773 Da) (I) and
C11H9NOF3 (theoretical mass 228.0636) (II). The mass measurement of the fragment ion solves the ambiguity, structure I being within 2.0 mDa compared to structure II, which is within 11.7 mDa of the theoretical mass for the fragment. This precludes the isobaric species II, which results are in accordance with those previously reported for that ion.10 SKF-105409 gave essentially two fragment ions using the insource fragmentation at a cone voltage 80 V (Figure 6). Massmeasured protonated molecule and fragment ions all were within 1.5 mDa of the theoretical mass. Data are summarized in Table 3. Negative Ion LC/MS. All of the above data were obtained using positive ion LC/MS. Consequently, an investigation was also carried out to show the applicability of this technique for negative ion work. A mixture of di-p-toluyltartaric acid (DTTA) and dibenzoyltartaric acid (DBTA) was prepared at levels of ∼5 ppm mass fraction relative to the matrix, which does not respond in negative ion. The reference compound, Val-Tyr-Val, gave a large ion at m/z 378 in negative ion. For DTTA, the measured mass was 357.0590 Da, which is within 2.1 mDa (or 6 ppm) of the theoretical mass of the deprotonated molecule [M - H]-, i.e., 357.0611 Da. The result of the mass measurement of DBTA was (10) Wolff, J.-C.; Monte´, S.; Haskins, N.; Bell, D. Rapid Commun. Mass Spectrom. 1999, 13, 1797-1802.
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very similar, i.e., within 2.0 mDa (i.e., 5 ppm) (measured mass 385.0903 Da and theoretical mass for the deprotonated molecule 385.0923 Da). The results obtained in negative ion mode compare well with those in positive ion mode. CONCLUSION This work illustrates that the proposed system provides accurate mass measurements within a few millidaltons of the theoretical value for both protonated and deprotonated molecules and fragment ions. No interchannel crosstalk is observed in this work; this has the additional benefit that the total ion current chromatograms obtained are free from interference from the ions of the reference standard and no suppression of ionization of either sample or standard is observed. At present the work has been carried out using a MUX system, which was adapted to switch between only two of the eight sprays and clearly illustrates the proof of the concept. However, there are a number of refinements that could be made for routine use including simplification of the system to only two sprayers. Also, with this system there was no opportunity to select the frequency and length of sampling times for the reference spray, all of which need further investigation. In this work, we demonstrate that it is possible to get good mass measurement on fragment ions generated by cone voltage fragmentation. Since these spectra are similar to those observed by product ion MS/MS for the compounds analyzed, then it would seem likely that this method of mass measurement would also be applicable to MS/MS. However, the interface would have to be adapted for use on a quadrupole orthogonal acceleration timeof-flight mass spectrometer in order to demonstrate the utility of this approach for product ion MS/MS. Received for review April 18, 2000. Accepted June 6, 2000. AC000448I
