The standard deviation of the proportionality coefficient is 0.03 %. The % re1 std dev at the low signal levels increases as expected from small fluctuations in the circuit OAs. The ability of the dual channel system to average noise over a broad frequency spectrum is shown by the data in Table 111. When the frequency is varied from 100 Hz to 1 Megahertz for a 500 mV peak-to-peak sinewave, the average integrator output remains constant within 0.02%. However, at the low frequencies (100-200 Hz), the relative standard deviation increases from 0.02 to 0.2 %. This is expected because the integration period per pulse is only 8 milliseconds. Typical test
signals with superimposed sinewave noise are illustrated in Figure 8. The combination of the dual channel synchronous measurement system with the intermittent operation of hollow-cathode tubes provides A F sensitivities that are one to three orders of magnitude greater than by operation in the dc mode. RECEIVED for review June 7, 1972. Accepted July 31, 1972. Presented in part at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1972. Work supported in part by NSF Grant GP 18910.
Signal Enhancement in Real-Timefor High-ResolutionMass Spectra F. W. McLafferty, John A. Michnowicz, Rengachari Venkataraghavan, Peter Rogerson, and B. G. Giessner Department of Chemistry, Cornell University, Ithaca, N . Y . 14850 An on-line, real-time computerized method for effectively increasing the sensitivity, resolution, and mass measuring precision of a high-resolution mass spectrometer has been developed. This method for Signal Enhancement in Real Time (SERT) utilizes the relatively large vacant areas between peaks to rescan peaks in real-time under direct computer feedback control. The ensemble-averaged rescans have an increased signal/noise ratio when compared to the single scans and significantly increase the effective sensitivity, resolution, and mass measuring precision of the instrument without increasing the scanning time, in contrast to most methods for ensemble-averaging of spectral data.
THEDIRECT APPLICATION of computers to mass spectrometry has made possible many advances in the field of mass spectrometry. The power of the computer in acquiring, reducing, and displaying data has been well documented. Highresolution mass spectrometry especially has benefitted by the tremendous speed and efficiency of the computer in collecting data, computing exact masses, and relating these masses t o elemental compositions (1-6). The computer, however, has not, up to this time, been used in improving the rather inefficient process of magnetically scanning a high-resolution mass spectrum. When compared to photoplate detection which is a time-integrated process, magnetically scanned mass spectrometric data have a low information content per unit time (2). These methods have approximately comparable sensitivities only when a high-gain electron multiplier is used with the latter. At a resolution of 10,000, a m/e 100 peak in a magnetically scanned spectrum will be only 0.01 amu wide, and therefore a spectrum will exhibit peaks in less than 1 of (1) “Biochemical Applications of Mass Spectrometry,” G. Waller, Ed., John Wiley & Sons, New York, N.Y., 1972. (2) John Roboz, “Introduction to Mass Spectrometry, Instrumentation and Techniques,” Interscience Publishers, New York, N.Y., 1968, pp 361-4. (3) J. S. Halliday, in “Advances in Mass Spectrometry,” E. Kendrick, Ed., Vol. 4, Institute of Petroleum, London, England, 1967, p 239. (4) F. W. McLafferty, R. Venkataraghavan, J. E. Coutant, and B. G. Giessner, ANAL.CHEM., 43, 967 (1971). ( 5 ) R. J. Klimowski, R . Venkataraghavan, F. W. McLafferty, and E. B. Delany, Org. Moss Sprctrom., 4, 17 (1970). (6) D. H. Smith, R. W. Olsen, F. C. Walls, and A. L. Burlingame, ANAL.CHEM.,43, 1796 (1971). 2282
Figure 1. Effect of ESA potential on magnetic field required to focus the m/e 80 peak. The insert illustrates the incremental shifts in ESA potential which make possible the rescanning of the peak at a higher magnetic field the mass axis (3). The other 99% of the time is used waiting for a peak to appear on the detector. At a resolution of 100,000, 99.9% of the mass axis is “vacant” time. In a preliminary communication, it was suggested that these vacant regions between peaks can be used to collect useful data ( 4 ) . By placing the mass spectrometer under computer control and rescanning each peak repeatedly after it originally passes the detector, the time between peaks can be used to increase the information content of a spectrum. We report here how this method of signal enhancement in real-time (SERT) can effectively increase the sensitivity, resolution, and mass measuring precision of the mass spectrometer. A multiscan averaging technique has been used to improve mass measuring accuracy ( 5 , 6 ) , and other spectrometric techniques use similar methods of multiple scanning (7), but all require that the time for data (7) M. Silverstein and G. Bassler, “Spectrometric Identification of Organic Compounds,” J. Wiley & Sons, New York, N.Y., 1968, p 114.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1 9 7 2
v Teletype
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Reference Voltage to ESA Stepping Device
collection be increased in proportion t o the signal enhancement achieved. Three methods were considered for rescanning a peak once it has passed the detector in a magnetically scanned, doublefocusing, high-resolution mass spectrometer. A small opposing magnetic field may be introduced into the system, but the large time constants needed t o change magnetic fields makes this method impractical. A change in either the ion accelerating (IA) or electrostatic analyzer (ESA) voltage can also be used to rescan a peak. We initially investigated the ESA potential change method because of the smaller voltages present in the ESA circuit and also our laboratory had previous experience in computer control of the ESA potential, developed for defocused metastable analysis (8). The finite width of the @-slitbetween the ESA and magnetic sectors allows the ESA potential to be varied over a small range (720 f 4.4 V at maximum slit width in the Hitachi
RMH-2) without defocusing the main ion beam ( 9 ) . Thus as the magnet is continuously scanning, the ESA potential can be varied to keep a peak on the detector. The possible combinations of ESA and magnetic field values produce the “ion ridge” illustrated in Figure 1; as the magnetic field is increasing, a peak a t mje 80 will be in focus a t a n ESA potential range of 720.0 to 724.4 volts, making it possible to rescan the peak many times by incrementing the ESA voltage. At mass 80, the ion ridge extends for a half mass unit, and at mass 600, 3.3 mass units. There are major disadvantages to this method; maximum instrument resolution cannot be achieved with the &slit a t its maximum setting, the resolution changes across the ESA potential range, and only a limited degree of offset is possible. In a similar fashion, the same mass can be brought back into focus a t a higher magnetic field by increasing the IA relative to the ESA potential. The IA potential can be changed over a
(8) J. E. Coutant and F. W. McLafferty, Iut. J . Muss Spectrori?. Zorr Phys., 8, 323 (1972).
(9) John Roboz, “Introduction to Mass Spectrometry,” Interscience
Publishers, New York, N . Y . , 1968, p 61.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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Figure 5. Rescanned mje 77 peaks; abscissa is time (mass), ordinate is ion current. Bottom: first five and last five rescans. Top: ensemble-average of 45 rescans
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much wider range of values (at least *IO%), permitting a longer mass range to be rescanned and making dynamic peak matching possible. Also the P-slit can be closed to a setting which allows optimum instrumental operation despite the changing IA values. Since the IA/ESA stepping method appears t o be preferable, we have recently developed the circuitry to step the IA/ESA potential. EXPERIMENTAL
Instrumentation. A block diagram of the instrumentation is shown in Figure 2. The mass spectrometer is a Hitachi RMH-2 modified to allow computer control of the ESA or IA potential. It operated at a 9.2-kV accelerating potential and a scan speed of 60 seconds/decade. In the ESA stepping experiments the /3-slit was opened to its maximum, 3.7 mm, and the resolution was approximately 2284
10,000. For the IA/ESA stepping the P-slit was closed to approximately 0.90 mm and adjustment of other slits gave a resolution of 20,000. ESA Modifications. The existing ESA power supply was replaced by two battery stacks, one for each ESA plate. The low potential side of each stack is driven by a n operational amplifier as shown in Figure 3. The voltage supplied by a digital-to-analog converter (D/A) is applied t o the bottom of both battery stacks. An amplifier is used as a line driver to enhance the low output current of the D/A. The potential of the ESA plates can be changed in approximately 1 psec; a slight modulation extends the effective response time to S psec. IA Modifications. Stepping of the 9.2-kV accelerating voltage is achieved by adding the incremented D/'A voltage to the bottom of the RMH-2 Hitachi power supply. The normal ground of the power supply is disconnected, thus allowing the 10-kV supply to be incremented in voltage steps supplied by the D,'A. Grounding of the power supply is then made through the load resistor network of the stepping circuit, as seen in Figure 4. When used in the IA,%SA stepping mode, the voltage increments required for the ESA plates are obtained from the same circuit through a resistor network which uses a 500-ohm potentiometer for fine adjustment of the IA/ESA ratio. The IA can presently be stepped a maximum of 100 V, although a 1000-V stepping circuit is being planned for our future system. Rise time of the IA step varies from a minimum of 5 psec for steps of less than 5 V to a maximum of 13 psec for a 100-V step. Additional Modifications. The existing electron multiplier and signal amplifier were replaced with a Bendix Spiraltron Model 4219-X in-line electron multiplier whose output is connected to a Keithley 421 current amplifier. Hardware. The mass spectrometer was coupled to a Digital Equipment Corporation PDP-8 computer with 4K of core, two 32K random access disk units, a high speed paper tape punch and reader, and a Shepard 880 high speed line printer. The output of the Keithley amplifier is connected to the computer via a 12-bit unsigned analog-to-digital converter (A/D) with a sample-and-hold amplifier, a 24-bit binary counter driven by a 20-kHz crystal oscillator, a 24-bit buffer register, and logic for automatic sequencing of all interface functions; operation of this system for normal high-resolution data collection has been described previously ( 5 ) . Output from the computer to the stepping circuits is made through a 12-bit D/A developed for automated metastable ion analysis (8). All data were collected at 10 kHz, which with a 60 sec/decade scan speed gave approximately 20 data points per peak a t 10,000 resolution. Software and System Operation. The system operation and programming techniques for SERT will be demonstrated by outlining the operations required t o rescan selected peaks in a spectrum by incrementing the ESA voltage. A scheme for applying SERT to a n entire spectrum will then be discussed. At a resolution of 10,000, a peak at nile 80 will have a width of 8 millimass units (mmu) and contain approximately 25 data points at a 10-kHz digitization rate. (Thus if a valley definition of resolution is used. the peak width will be 25 data points at 5 of its height.) With these conditions, the computer calculates that a shift of 0.129 V in the ESA potential will cause a displacement in the mass scale equivalent to one peak width, permitting the m / e 80 ion to be rescanned. With the total ESA potential range of 4.4 V available in the RMH-2, 4.4 V 10.129 V or 34 rescans of the m / e 80 ion are possible. A graphical representation of these rescans is shown in Figure 1. At a resolution of 50:000, the peak width is reduced to 0.0016 amu and 170 rescans could be obtained. However, the peak would contain only four data points at a 10-KHz digitization rate. If 20 data points are desired for adequate identification of the peak profile ( 3 ) : a SO-kHz digitization rate would be required. By stepping the IA/
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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Figure 6. Left: CsH$J.+ peak. Right: 5 rescans of base line and (Top) ensemble-average of 40 baseline rescans
Figure 7. Left: C&N peak. Right: 5 rescans of 13CC5H6.+ion and (Top) ensemble-average of 40 rescans +
ESA voltage, the same m/e 80 peak a t a 10,000 resolution can be rescanned by changing the IA 1.25 volts. The present IA voltage range of 100 V permits 80 rescans to be performed. The limited 4K of core in our PDP-8 necessitates that the program be divided into two phases. The first phase calculates the necessary IAiESA or ESA voltage increments, collects data, and, when time becomes available, transfers it to the disk. Phase two retrieves the data from the disk, sums all the individual rescans of an ion ridge, and outputs the composite profile and centroid time. RESULTS AND DISCUSSION
Alignment and Signal Enhancement. The exact mass range covered in each rescan must be invariant for proper averaging of the scans; for this the computer must calculate the exact offset potential required to rescan a peak. Figure 5 shows rescanned peaks containing 15 data points (approximately 5 mmu) from the m/e 77 ion of benzene produced by stepping the ESA potential during magnetic scanning. The first five and the last five of 45 rescans show similar peak shapes, indicating that alignment has been achieved. The high frequency response of the amplifier produces peaks which are not electrically smoothed, but the random noise produced in the signal is almost completely eliminated in the composite of the 45 rescans, resulting in a smooth, Gaussianshaped peak profile. Sensitivity. By ensemble-averaging the data of multiple rescans of a particular portion of a spectrum and thereby increasing the signal/noise ratio, it should be possible t o improve the sensitivity of the mass spectrometer and detect peaks previously lost in base-line noise. To test this concept, mixtures with varying concentrations of benzene and pyridine were made and the doublet a t m/e 79 (m/Am = 9,745) was rescanned.
The averaged base-line noise that could be expected was first determined using a sample of pure pyridine. Figure 6 shows the normal C5H3N.+ion and an 8.8 mmu, portion of base line where the l:C12C5HG-ion of benzene would appear if it were present. Time requirements allow approximately 40 rescans, 5 of which are illustrated along with the ensemble average of the 40 rescans. A numerical analysis indicates a 3.5-fold increase in the S/N ratio of the composite. The theoretical increase should be proportional to the square root of the number of rescans, or 6.3. The occurrence of 60 Hz noise, clearly present in the base-line computer printouts, is most likely responsible for the discrepancy. Figure 7 shows 5 of the 40 base-line rescans of a pyridine : benzene mixture which should produce a 125/1 peak-height ratio. Although some rescans show apparent peaks, these can be misleading; however, from the composite of the 40 scans, there is n o doubt as to the existence and location of the second component. The observed mass difference between the two peaks is 8.7 mmu; theoretical separation is 8.3 mmu. Experimental peak height ratio is lower than theoretical, but well within our experimental limits. A lO00/l peak-height ratio was found to be the lower limit of detectability under these conditions using 40 rescans. Resolution. Figure 8 shows five ESA rescans cf the *3CCaH7+:CiHs+doublet ( m / h = 20,595) scanned at a resolution of approximately 5,000. The poor resolution and noise present in the signal obviously make recognition of the doublet and subsequent attempts to deconvolute (IO,1 I ) any single scan Impossible. However, the addition of 27 of these ( I O ) R . Venkataraghavan, F. W. McLafferty, and J. W. Amy, ANAL.CHEM., 39, 178 (1967). (11) D. D. Tunnicliff and P. A . Wadsworth, ibid., 40, 1826 (1968).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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rescans produces a composite (Figure 8) which clearly indicates the presence of a doublet. This composite profile was deconvoluted (IO) to the doublet shown in Figure 9. The experimental separation measured between the deconvoluted peaks is 4.56 mmu; the theoretical value is 4.47 mmu. Similar results were obtained from rescans obtained a t 7,000 and 10,000 resolution. Mass Measuring Precision, The increase in mass measuring precision utilizing a composite peak was investigated by rescanning the m / e 77, 78, and 79 ions of benzene. Centroid times were then computed for the composites of the rescanned 77, 78, and 79 peaks and also for a randomly-picked single scan from each of the rescanned peaks. A linear relationship between the centroid times and exact masses of the 77 and 78 ions was then used to calculate the mass of the 79 ion. Separate experiments in both the ESA and IA modes were performed 10 times with the minimum number of rescans for a peak being approximately 50. The results are shown in Table I. The theoretical decrease in the deviation should be or 7.1. Our results are high by a factor of 2.5. Again it is believed that the discrepancy is due to a periodic noise signal, which unlike random noise will not be averaged in the composite. This hypothesis is supported by the fact that when 80 of the rescans in the ESA experiments are omitted, the deviation increases to only k0.00027 amu. It should be possible to effect further improvements in mass accuracy by dynamic peak matching. Thus if the unknown peak and a known reference peak are rescanned alternatively by proper offsetting of the IA potential, periodic fluctuations which would have occurred during the time necessary to scan
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between them magnetically will be reduced by averaging. Preliminary experiments support the feasibility of this concept. Proposed System. The results obtained o n narrow regions of a spectrum indicate that SERT can be applied t o scan a complete high resolution spectrum. Experience with the present hardware indicates that the software can be written to scan a mass range of 60-600 in 60 sec utilizing a small computer system like the PDP-8 with 8K of memory, a bulk storage device like a 32-K disk, and a n appropriate mass spectrometer/computer interface. In our proposed general system, a low-resolution, high sensitivity scan is first taken from which the computer finds the time a t which each peak appears in the spectrum and locates broad peaks which will require a higher number of data points per scan. The computer checks to see if any adjacent peaks in the spectrum are separated by a greater mass ratio than can be accommodated by the IA stepping range; if they are not, the full 60-sec scan time can be utilized in rescanning. The number of rescans possible per peak is then calculated from the time available for rescanning, taking into account the number of peaks and the time required to manipulate the data between peaks. The high-resolution SERT mass spectrum is then run. Before each peak is scanned, the computer calculates the IA offset required by dividing the total IA range by the number of rescans. Each peak scan is directly ensemble-averaged by setting up buffer tables in the computer memory. If a peak is 15 data points wide, 15 core locations are set up, and the corresponding points from each rescan are added using multiple precision arithmetic to the previous totals to prepare an
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
averaged peak envelope. The centroid and area of the envelope will be computed and stored if the peak is a singlet, or the entire profile will be saved for later deconvolution if the peak is an unresolved multiplet. Since the time required to move information from core to a bulk storage device like a disk is relatively high (-40 msec), all of the data will be held in core until the spectral scan is complete; for a normal spectrum, a core of 8K should be sufficient to accomplish this. It also appears possible to utilize SERT under more demanding conditions, such as a scan speed of 6 sec/decade at 10,000 resolution or 60 sec/decade at 100,000 resolution. Such a system has been designed based on a computer with a 400-nsec memory cycle time (DEC PDP 11/45) and an interface with a high speed A/D or a pulse counter and digital logic for various control operations such as thresholding, multiple sampling of the A/D, and automatic updating of the D/A. Data transfers between the interface and the computer will be accomplished through high speed channels without any program supervision. It may also be possible to obtain the necessary peak information without the preliminary low-resolution scan. If only a limited combination of elements is possible, substantial regions of the spectrum (especially at lower masses) cannot contain peaks and can thus be used for rescanning. Further, it may be feasible to predict in real time the elemental compositions possible at higher masses from those found to be present at lower masses. Special problems, such as the sequencing of peptide mixtures (12), required the detection and exact mass (12) F. W. McLafferty, R. Venkataraghavan, and P. Irving, Biodiem. Biop/iys. Res. Commun., 39, 274 (1970).
Table I. Precision of 10 Separate Mass Determinations from Single Scan us. Rescan Measurements Mode Single scan Rescan ESA 79.04323i O.OOO62 79.04402C O.OOO24 IA 79.04296i 0.00080 79.04308i 0.00030
determination of peaks at only a limited number of possible masses, so that a much larger number of rescans and a concomitant increase in sensitivity should be possible for each peak. CONCLUSION
The signal enhancement of high-resolution mass spectral data using SERT is unique in that it is performed on-line, in real time, and does not increase the time requirements for a spectrum. Previously mentioned methods for increasing S/N ratios (5-7) require many spectra to be obtained, an obvious disadvantage when dealing with the small sample sizes often required in mass spectral studies. This technique should be applicable to the recording of other spectra and chromatograms in which a substantial proportion of the base line does not contain any real data. RECEIVED for review June 6, 1972. Accepted July 27, 1972. Financial support for this work was provided by National Institutes of Health Grant G M 16609.
Simultaneous Measurement of Plasma Concentrations of Lidocaine and Its Desethylated Metabolite by Mass Fragmentography John M. Strong and Arthur J. Atkinson, Jr. Clinical Pharmacology Laboratory, Division of Medicine, Passavant Memorial Hospital, and the Departments of Medicine and Pharmacology, Northwestern Unioersit).Medical School, Chicago, Ill. Lidocaine and its pharmacologically active metabolite, monoethylglycinexylidide (MEGX), have been measured in samples of blood plasma by the technique of quadrupole mass fragmentography. The standard deviation of the method was 3.1% for lidocaine and 7.4% for MEGX over the range of concentrations usually encountered in clinical practice. The technique of mass fragmentography was extended to include rigorous criteria for compound identification based on statistical analysis of the ratio of two fragment ions present in each of these compounds and in the trimecaine added to the plasma samples as an internal standard. These ratios were reproducible with a standard deviation of less than 10%. The quadrupole mass spectrometer was found to be a suitable instrument for quantitative mass fragmentography, and offered an important advantage over presently available magnetic instruments with respect to the range of m / e of the fragment ions that could be recorded.
THETHERAPEUTIC AND TOXIC effects of many drugs are related to the concentration of these drugs in the plasma of the patients that are being treated. This has led to an increasing demand for rapid and specific analytical methods that are sensitive enough to measure plasma concentrations of these
drugs as an adjunct to patient therapy. Chromatographic identification is not entirely satisfactory because co-chromatography cannot be excluded with certainty. Use of a mass spectrometer as the detector for a gas chromatograph has the theoretical possibility of eliminating this uncertainty, but plasma samples often contain insufficient material to permit a complete mass spectrum to be recorded. However, if the signal from only a few ions is recorded, a partial mass spectrum can be obtained with a markedly enhanced sensitivity. This technique, basing compound identification on the relative intensity of selected mass spectral ions in combination with gas chromatographic retention time data is called mass fragmentography and was first used for qualitative analysis of chlorpromazine metabolites in human blood ( I ) . Recently, the internal standard method for quantitative analysis by gas chromatography has been applied to mass fragmentography, making possible measurement as well as identification of drugs and endogenous metabolites in biologi( I ) C.-G. Hammar. B. Holmstedt, and R. Ryhage, A m / . Biochrm., 25,532(1968).
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