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).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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' 61
LIDOCAINE
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Figure 1. Mass fragmentogram of a plasma extract from patient No. 1 containing 0.58 pg/ml MEGX and 4.2 pg/ml lidocaine. The intensities of ions with m / e 58, 86, and 120 were monitored with recorder attenuations of 1, 10, and 0.1 volts, respectively. The time scale is aligned with the pen recording m / e 86, the other pens being offset
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cal fluids (2, 3). But previously, the precision with which the ratios of ion intensities of standard compounds could be determined has not been used to confirm compound identification. This more rigorous application of the technique of mass fragmentography is illustrated in the present work in which plasma concentrations were measured of lidocaine and of monoethylglycinexylidide (MEGX), a desethylated metabolite of lidocaine that has been suspected of causing some of the toxicity associated with lidocaine therapy (4, 5 ) . This example also demonstrates the great versatility of the quadrupole mass spectrometer with respect to the range of mass of the fragment ions that can be selected for analysis by mass fragmentography . EXPERIMENTAL
Reference Compounds. Reference lidocaine hydrochloride monohydrate (mp 78 "C) and MEGX (mp 51 "C)were provided by Rune Sandberg (Astra Lakemedel AB, Sodertalje, Sweden). The trimecaine (mp 45-47 "C)was supplied by Melvin B. Meyer (Astra Pharmaceutical Products, Inc., Worcester, Mass.) and made up in benzene as a n internal standard solution with a concentration of 0.8 pgjml. Gas Chromatography-Mass Spectrometry. A Finnigan Model 3000 quadrupole gas chromatograph-mass spectrometer was used to obtain conventional mass spectra from approximately 0.2-mg samples of the reference compounds. The gas chromatograph was interfaced to the mass spectrometer with a glass jet separator. The chromatograph was fitted with a 2.0 m x 2 mm (i.d.) glass coil packed with 3 6 : l SE-30:OV-17 coated on an SO/lOO mesh Chromosorb
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(2) 0. Borgi, L. Palm&, A. Linnarsson, and B. Holmstedt, Aiiul. Left.,4,837 (1971). (3) , , L. Bertilsson. A. J. Atkinson. Jr., J. R. Althaus, A. Harfast, J.-E. Lindgren, and B. Holmstedt. ANAL. CHEM.,44, 1434 (1972). (4) A. H . Beckett, R. N. Boyes, and P. J. Appleton, J . Plrarm. Pliurmucol., 18,76S (1966). (5) R. N. Boyes. D. B. Scott, P. J. Jebson, M. J. Godman, and D. G . Julian, Clifi.Pliarmrrcol. Tlwr., 12, 105 (1971). 2288
Figure 2. Standard curves for the quantitation of ( A ) MEGX and ( B ) lidocaine in plasma, with trimecaine used as the internal standard solid support (Chemical Research Services, Addison, 111.). The flow of helium carrier gas was 20 mlimin. The temperature of the injection port was 250 "C,the column 210 "C, and the transfer line 180 "C. Mass spectra were taken at a n ionizing energy of 70 eV. For mass fragmentography this equipment was operated under the same conditions. The intensity of the selected fragment ions was monitored using the Finnigan Model 240-01 Automatic Peak Selector and a four-pen recorder (Model KA-40, Rikadenki Kogyo Co., Ltd., Tokyo, Japan). The peak selector was modified to include bucking control and 0.16 to 16.0 Hz filtering for the output of each channel. In the present work the ion intensities at mje 58, 86, and 120 were monitored on separate channels of the Automatic Peak Selector. In some instances, two channels were used to monitor m/e 120 to provide sufficient dynamic range when MEGX concentrations were low compared to lidocaine and trimecaine. A mass marker was used to center each channel on its assigned ion peak with a window width of less than 0.1 amu. The channel dwell time was 25 milliseconds. Procedure. Heparinized blood was obtained from patients whose lidocaine therapy, after several hours of constant intravenous infusion, was being monitored by plasma levels, determined by a modification of previous gas chromatographic methods (6, 7). The plasma was separated by centrifugation at 800 x g for 15 minutes and stored frozen until analyzed. To a 15-ml glass-stoppered, conical centrifuge tube were added 1 ml plasma, 5 ml internal standard solution in benzene, and 0.2 ml 5N NaOH. After mixing, the tube was centrifuged for 5 minutes at 800 X g. The benzene phase was transferred to a 5-ml pear-shaped flask and evaporated (6) G. Svinhufvud, B. Ortengren, and S.-E. Jacobson, Scnrid. J . Clin. Lab. I/?cesf..17, I62 (1965). (7) J. B. Keenaghan, Auestlwsiology, 29! 110 (1968).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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Table I. Determination of MEGX and Lidocaine in Plasma Samples Lidocaine MEGX mje 120 m/e 120mje 58 mie 86 Plasma sample pLg/ml pg/ml 0.021 + 9%" 0.3-5.0 0,5-10.0 Standard solutions 0.038 =t7 Z a 0.58 4.2 0.022 0.042 Patient No. 1 0.022 Patient No. 2 0.83 3.8 0.041 0.022 0.35 2.4 0.037 Patient No. 3 0.31 1.9 0.021 Patient No. 4 0,038 0.52 6.5 0.021 0.037 Patient No. 5 5.2 0.021 0.034 0.60 Patient No. 6 4.4 0.024 0,040 2.6 Patient No. 7 Std dev of ratios obtained on six separate analyses.
to dryness at 25 mm Hg in a 25 "C water bath. After dissolving the residue in 50 ~1 of benzene, a 2-pl aliquot was analyzed by mass fragmentography to give a record such as that shown in Figure 1. Standard plasma samples containing known concentrations of lidocaine and MEGX were also analyzed by this procedure. These results were used to prepare standard curves relating the concentrations of these standards to the ratio of the heights of mje 86 for lidocaine and mJe 58 for MEGX to the mje 86 trimecaine peak (Figure 2 ) . These standard curves were used t o determine lidocaine and MEGX concentrations from similar ratios calculated from the analysis of plasma samples from patients. Confirmation of the identity of the measured compounds was provided by checking the ratio of rnje 120 to m!e 58 for MEGX, mje 120 to nile 86 for lidocaine, and mja 120 to mje 86 for trimecaine against the ratios found with reference compounds. The standard curves also were used to determine the efficiency of lidocaine and MEGX extraction. Plasma solutions of these compounds were extracted with 0.2 ml 5 N NaOH and 5 ml benzene without internal standard. After mixing and centrifugation, the benzene phase was discarded, 5 ml of internal standard solution added to the aqueous phase, and the analytical procedure carried out to determine the amount of lidocaine and MEGX remaining after the initial benzene extraction. In this way it was determined that the extraction efficiency was greater than 90 for both lidocaine and MEGX.
60.
MEGX
Trimecaine rnje 120 rnje 86 0.012 i 7 x 0 0.012 0.012 0.011 0.011 0.011 0.011 0.011
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RESULTS AND DISCUSSION The present work was begun by obtaining the conventional spectra of lidocaine, MEGX and trimecaine shown in Figure 3. The base peak for each compound arises from cleavage of the carbon-carbon bond /Ito the substituted amine nitrogen atom. This is characteristic of aliphatic amines (8), and is expected since the free electron pair of the nitrogen atom stabilizes a positive charge o n the adjacent carbon atom (9). Mass fragmentography of these compounds is made difficult by the fact that the intensity of other fragment ions is small relative t o the base peak. However, a fragment ion a t mje 120 is present for each of these compounds and was chosen for monitoring. For lidocaine and MEGX, this fragment results from cleavage of the amide bond. But for trimecaine, the analogous fragment has an mie of 134, and the origin of the nz,'e 120 ion is unclear. The channels of the Automatic Peak Selector were used to record the ion current a t the m/e corresponding to the base ~__._______
(8) R. S. Gohlke and F. W. McLafferty, ANAL.CHEX, 34, 1281 ( I 962). (9) K . Biemann. "Mass Spectrometry: Organic Chemical Applications," McGraw-Hill Book Co., New York, N . Y . . 1962, p 87.
m/e Figure 3. Mass spectra of reference trimecaine, lidocaine, and monoethylglpcinexylidide (MEGX) peak of each compound that was analyzed, mje 58 for MEGX and m/e 86 for lidocaine and trimecaine, and the mjc 120 ion present in these compounds. The ratio of the intensity of the mje 120 to the base peak signal from each compound was used to check that another plasma constituent with the same retention time was not contributing to the recorded intensity of either ion. This was done after calculating the precision with which this ratio could be determined using standard plasma solutions of each compound in the concentration range that was to be analyzed (Table I). In this way, identification of the compounds measured in the patient specimens was established in accordance with predetermined statistical criteria. Although previous applications of mass fragmentography have not used the full rigor of this approach, this
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
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should be an essential prerequisite to exclude co-chromatography before attempting quantitative analysis of compounds in biological materials that have as many varied constituents as blood plasma. If such co-chromatography were found, it would be necessary to monitor alternate fragment ions or change the chromatographic column. The intensity of the base peak signals from each compound was used for quantitation of MEGX and lidocaine by the internal standard method. The precision of this analytical technique was estimated by measuring lidocaine and MEGX concentrations in standard plasma solutions and was found to average 3.1 % for lidocaine in the range of 0.5-10 pg/ml (20400 ng injected o n column), and 7.4% for MEGX in the range of 0.3-5 kg/m1(12-200 ng injected on column). The feasibility of using quadrupole mass fragmentography for the quantitative analysis of pesticides has been demonstrated recently, but biological samples were not used (10). We have shown that drugs and drug metabolites can be measured in blood plasma by this technique. Indeed, for this application the quadrupole mass spectrometer has an important advantage over currently available magnetic instruments in that the range of fragment ions that can be selected for monitoring is less restricted. This results from the fact that the intensity of ions whose m/e is not contiguous often must be recorded at intervals of several milliseconds. In magnetic instruments, hysteresis of the magnetic field is too great to permit rapid enough alternation in the strength of this field for repetitive scanning. Therefore, mass fragmentography has been accomplished by changing the accelerating voltage in order to switch focus rapidly between the desired ions. Published reports indicate that the range of mass selection by this means is limited (11, 12). Thus with an LKB 9000 mass spectrometer the intensity of ions could only be recorded within a range of 20% below the m/e of the ion with highest mje by adding up to 700 volts to the basic accelerating voltage of 3.5 kV (11). More recently, the m/e range that can be monitored by accelerating voltage alternation has been extended to more than 30%, but a special ultrastable high voltage power supply with separate lens controls for each mass was required to prevent degradation of ion optics (12). Although further refinements may make it possible to monitor ions over an even wider range without degradation of ion optics, this capability has yet to be demonstrated. On the other hand, mass selection of ions formed in a quadrupole mass spectrometer is accomplished by a hyperbolic electrical field produced by direct current and radio frequency voltages applied to four cylindrical rods, or poles. Since it is possible to switch the amplitude of the rod voltages rapidly enough to record the intensities of only the few ions selected for analysis, any ions within the mass range of the quadrupole mass spectrometer can be selected for mass fragmentography . This flexibility was essential for the present work in which the intensities of the m/e 58, 86, and 120 ions were recorded. This constitutes a range of 52 % from the nz/e 120 ion. The lidocaine concentrations shown in Table I are representative of those found in patients being treated for cardiac (10) E. J. Ronelli, ANAL.CHEM., 44,603 (1972). (11) C.-G. Hammar and R. Hessling, ibid.,43, 298 (1971). (12) P. D. Klein, J. R. Haumann, and W. J. Eisler, ibid., 44, 490 (1 972).
2290
arrhythmias with this drug. Plasma concentrations of MEGX have not been measured in patients previously. Plasma MEGX levels were determined in the present work because this metabolite may contribute to the toxic side-effects observed in some patients treated with lidocaine ( 4 , 5). Like lidocaine, MEGX has local anesthetic activity (13), and preliminary studies in this laboratory indicate that in rats it also acts on the central nervous system to produce symptoms . similar to those seen in patients with lidocaine toxicity. In patients one through four, the plasma concentrations of lidocaine fell within the accepted therapeutic range of 1.2 to 6.0 pg/ml ( I d ) , and no drug toxicity was suspected on clinical grounds. MEGX concentrations averaged one-sixth of the measured concentrations of lidocaine in these patients. Patients five and six were agitated and confused at the time their blood was obtained for analysis, and central nervous system toxicity due to lidocaine was suspected. Plasma concentrations of lidocaine were higher in these patients than in the first four patients and were thought to account for the altered central nervous system function, as has been observed with plasma levels of 5.29 + 0.55 pg/ml or more (15). The concentration of MEGX in the plasma of these patients averaged only one-tenth the concentration of lidocaine. A third pattern of altered lidocaine metabolism was found in patient seven. He was also confused and had visual hallucinations at the time his blood was sampled for analysis. The concentration of lidocaine in his plasma of 4.4 pg/ml was relatively low, but there was a marked increase in the concentration of MEGX, which at 2.6 pg/ml was greater than half the lidocaine level. Thus it is probable that MEGX contributed to the central nervous system toxicity that was observed in this patient. More generally, this example suggests that analytical methods intended to help guide the therapy of patients should be designed to include measurement of active metabolites as well as the parent drug. ACKNOWLEDGMENT
We thank Bo Holmstedt, Department of Toxicology of the Karolinska Institutet, for instruction in the principles and application of the technique of mass fragmentography, and William Fies of the Finnigan Corporation for help in modifying the Automatic Peak Selector that was used. Arne Astrom, Department of Physiology I of the Karolinska Institutet, arranged for the reference compounds to be provided for this work. RECEIVED for review May 15, 1972. Accepted August 24, 1972. This project was supported by Fellowship GM-52, 667-01 from the National Institute of General Medical Sciences, National Institutes of Health and by a Pharmaceutical Manufacturers Association Foundation Faculty Development Award in Clinical Pharmacology. (13) N. Lofgren, “Studies on Local Anesthetics: Xylocaine a New Synthetic Drug,’’ Ivar Haggstroms Boktryckeri, A. B., Stockholm. 1948, p 48. (14) D. C. Herrison, R. E. Stenson, and R. T. Constantino in: “Symposium on Cardiac Arrhythmias,” E. Sandpe, E. FlenstedJensen, and K. H . Olesen, Ed., A. B. Astra, Sodertalje, Sweden, 1970, p 427. (15) F. F. Foldes. R. Molloy, P. G. McNall. and L. R. Koukal, J . Ai7wr. Med. Ass., 172, 1493 (1960).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
