and an emission wavelength of 450 nm are used to measure the fluorescence of the methadone fluorophore. Within a range of 80 to 120x of an anticipated concentration between 1 and 10 pg/ml of methadone, a nearly linear relationship exists between log fluorescence and log concentration at the analytical wavelengths of the fluorophore. A standard curve should be plotted for each series of determinations. Maximum fluorescence intensity was experimentally determined to result from using 0.1 ml of a 0.20% solution of formaldehyde/sulfuric acid. Care should be taken during the addition of the reagent to the residue to deliver the same amount of reagent to each successive sample. Precision was determined by comparing 18 determinations of a 5pg/ml standard methadone solution. Six determinations were made each day for 3 days. The relative standard deviation was 2.2 Chloroform solutions of morphine, codein:, heroin, cocaine, quinine, amphetamine, and meperidine were prepared at a concentration of 5 pg/ml. A 1.0-ml portion of each solution was treated according to the procedure and compared with an equal concentration of methadone in chloroform. No measurable fluorescence could be detected at the analytical excitation and emission wavelengths of the methadone fluorophore for morphine, codeine, heroin, or cocaine. Meperidine, quinine, and amphetamine fluoresce at these wavelengths, interfering with the determination of methadone. In addition, morphine, codeine, heroin, and cocaine when added to a solution of methadone at equal concentrations did not produce any quenching when carried through the procedure. Thus, methadone may be determined in the presence of an equal concentration of morphine, codeine, heroin, or cocaine without prior separation.
x.
Y
W A V E L E N G T H (n rn)
Figure 1. Excitation ( A ) and emission ( B ) spectra of the methadone fluorophore (uncorrected) perature. The fluorescence spectra possesses a single broad emission maximum at 450 nm and three excitation maxima: 410 nm, 384 nm, and 270 nm, respectively. The uncorrected spectra are shown in Figure 1. At methadone concentrations less than 5 pg/ml, sulfuric acid interferes with fluorescence at excitation wavelengths of 410 nm or 384 nm, but there is no interference at an excitation wavelength of 270 nm. Therefore, in a determination, an excitation wavelength of 270 nm
RECEIVED for review February 19, 1971. Accepted March 30, 1971.
CORRESPONDENCE
I
Increased Sensitivity in High-Resolution Mass Spectrometry Using an On-Line Computer SIR: Measurement of a high-resolution mass spectrum by magnetic scanning is a relatively inefficient process; a peak is in focus at the collector only a very small proportion of the time ( I ) . With a resolving power of 20,000, for which a peak at mass 200 is only 0.01 mass unit wide, a typical spectrum will exhibit peaks in less than 1 % of the mass axis. The sensitivity is directly dependent on the scan rate; for the detection of a particular peak, a statistically significant number of ions must fall on the detector. With direct digital data acquisition techniques (2-4), the exact mass ~
(1) J. H. Beynon. “Mass Spectrometry and its Applications to Organic Chemistry,” Elsevier Publishing Company, Amsterdam, 1960. (2) C. Merritt, Jr., P. Issenberg, M. L. Bazinet, B. N. Green, T. 0. Merron, and J. G. Murray, ANAL.CHEM.,37, 1037 (1965). (3) W. J. McMurray, B. N. Greene, and S. R. Lipsky, ibid., 38, 1194 (1966). (4) For a recent review, see R. Venkataraghavan, R . J. Klimowski, and F. W. McLafferty, Accounts Chem. Res., 3, 158 (1970).
measurement of a particular peak requires a minimum of approximately ten ions. The accuracy of both mass measurement and relative abundance determination is increased by slowing the scan speed or by multiscan (ensemble) averaging (4-6). On-line data acquisition systems currently in use in high-resolution mass spectrometry require 40 to 60 seconds per decade scan time for reasonable sensitivity at a resolving power of 10,000 (4-6); at higher resolutions, longer scan times are necessary. This time requirement is a severe limitation in some applications, particularly for the direct scanning of gas chromatographic effluents; for this, photoplate recording of high-resolution data appears to be
(5) A. L. Burlingame in “Recent Developments in Mass Spectroscopy,” K. Ogata and T. Hayakawa, Ed., University Park Press, Baltimore, Md., 1970. ( 6 ) R. J. Klimowski, R . Venkataraghavan, F. W. McLafferty, and E. B. Delany, Org. Mass Spectrom.. 4, 17 (1970). ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
967
730.0
73 ESA Potential , v
Figure 1. Effect of ESA potential on magnetic field required to focus the m/e 160 peak. The inset illustrates the incremental shifts in ESA potential which make possible the rescanning of the peak at a higher magnetic field. The actual experimental data show a slight deviation from linearity the method of choice (7). We propose that the vacant regions between peaks can be utilized to make multiple rescans of each peak under on-line computer control; this will increase the statistical significance of the data from the peak in proportion to the number of rescans, making possible higher sensitivity, higher mass-measuring accuracy, and increased capability for the deconvolution of overlapping doublets. If the spectrum is recorded by continuously changing the magnetic field, rescanning a particular peak should be possible after it passes the collector slit by inducing a small opposing field with an auxiliary magnet or by making a small incremental change in the ion accelerating potential ( 1 ) ; these techniques are employed in “peak matching” for exact mass measurements. We find that this can also be accomplished by an incremental change in the potential on the electrostatic analyzer (ESA); this method has the advantages that such potentials are generally 500-750 V, in comparison to ion accelerating potentials of 8-10 kV, and that the ESA potential shifts can be precisely controlled with the computer interface system developed for defocused metastables (8). The ESA potential can be varied over a small range without defocusing the main ion beam because of the finite width of the P-slit between the electrostatic and magnetic analyzers (9). The P-slit of the Hitachi RMH-2 mass spectrometer can be opened to a width of 4 mm without greatly degrading the instrument’s resolving power; maximum adjustment of the ESA potential at this width will cause a peak to be displaced by an amount equivalent to one mass unit (amu) at mje 160. The possible combinations of ESA and mag-
(7) K. Biemann in “Topics in Organic Mass Spectrometry,” A. L. Burlingame, Ed., Wiley-Interscience Publishers, New York, N. Y., 1970. (8) J. E. Coutant and F. W. McLafferty, unpublished data, J. E.
Coutant, Ph.D. Thesis, Cornell University, Ithaca, N. Y., 1971. (9) H. A. Duckworth and S. N. Ghoshal in “Mass Spectrometry,” C. A. McDowell, Ed., McGraw-Hill, New York, N. Y., 1963. 968
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, J U N E 1971
netic field values produces the “ion-ridge” illustrated in Figure 1; thus at an ESA potential of 734.4 V, the m / e 160.0 peak will be in focus at the same magnetic field as would be required to focus an mje 161.0 peak at an ESA potential of 730.0 V. [Defocused metastable ions produce similar ion ridges at other ESA values; a technique has been developed for a stepwise scan in the ESA axis of these through a similar computer control of the ESA potential (@.I At a resolving power of 16,000, the width of this peak is 0.01 amu, SO that a shift of 0.044 V in ESA potential will cause a displacement in the mass scale equivalence to one peak width. We find, as predicted, that under these conditions such a m / e 160 peak can be scanned 100 times while the magnetic field is swept through this range. Just after the completion of each peak scan, the value of the ESA potential is increased by 0.044 V, refocusing the start of the peak. Ion focusing conditions can be found for which the peak shapes produced (including those of unresolved doublets) remain constant within experimental error across the 4.4-V range of ESA potential values; however, the maximum obtainable resolution is affected by the ratio of the ESA and ion accelerating potentials. At 40 secldecade the scan of one such peak requires approximately 10-3 sec (thus providing 20 data points by sampling at 20 KHz); computer-controlled resetting of the ESA potential to within 1% of a new value requires
