The application of deuterated analogues of drugs as internal standards is a common practice for quantitation using gas chromatographic-mass spectrometric ...
Journal of Analytical Toxicologv Vol. 21, May/June 1997
I Letter to the Editor
LargeAmountsof DrugsMay ConsiderablyInfluence the PeakAreasof Their Coinjected Deuterated AnaloguesMeasuredwith APCI-LC-MS To the Editor: The application of deuterated analogues of drugs as internal standards is a common practice for quantitation using gas chromatographic-mass spectrometric (GC-MS) or liquid chromatographic-mass spectrometric (LC-MS) procedures. Several authors postulated the use of analogues with the highest possible extent of deuteration in order to prevent the crosscontribution of fragments common in drugs and standards (1,2). Another aspect of this problem, particularly relevant in chemical ionization mass spectrometry, is a contribution of stable isotopes to the molecular mass of a given substance. So-called "isotope peaks" or rn/z286 4:11 "satellite peaks", usually showing 5058244 morphine 500 ng M A abundances (A+I) to (A+3), are commonly present in mass / l O" chromatograms obtained by means of m/z287 4:10 chemical ionization in positive mode. This M+l i00 phenomenon may be of practical value in the everyday casework. When analyzing 0 ,m~ 288 biological extracts on morphine by means 4:11 M+2 131~oo 100 of atmospheric pressure chemical I:18 A { ionization (APCI)-LC-MS,we have 21927 /'--h . . . . observed that the peak areas of the m/z 289 4 :12 protonated molecular ion of morphine-d:~ 1:2o M+3 19o20 (m/z 289). which was used as an internal ioo standard (IS), were distinctly larger in o - r " " ~ ' F ~ ' ~ ' . ~ ~...^- /'~.?/-~,.--.-/',-*,,-~_~_,v,.. ,--~ ~ _^ _....-~^ samples containing high concentrations i00 200 300 400 500 of morphine. This prompted us to carry out some experimental runs to elucidate rn/z290 M 248~08 the extent and practical relevance of this 50 benzoylecgonine 500 ng phenomenon. o The following drugs and their 7-'0 m/z291 deuterated analogues were examined: M+l 41~4 morphine and morphine-d3; benzoylecgonine (BZE), BZE-d3, and 10050I m/z292 M+2 s~/~ BZE-ds; methylenedioxyethylamphetamine (MDE) and MDE-d7;and 0 9 " methylenedioxymethamphetamine l O 0 4 re~z293 7:/~D r (MDMA) and MDMA-d5.The drugs were supplied by HSPC (Inglewood, CA) and Sigma Chemical (St. Louis, MO). '00 t rn/z298 M+8 [ Nondeuterated drugs were injected into ' ~ 1 ~7 6 the APCI-LC-MS in the amount of 500 ng. 200 400 600 800 1000 Also, the amounts of 10 or 500 ng of each drug were injected together with 10 ng of Figure I. APCI-LC-MS mass chromatograms of morphine and benzoylecgonine (500 ng injected). its respective deuterated analogue. M: mass-to-charge ratio of protonated molecular ion, (M+I) to (M+3): isotope peaks. Note absenceof The drugs were separated on the any peak at m/z 298, which corresponds to BZE-ds. Superspher Select B column
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Journal of Analytical Toxicology, Vol. 21, May/June 1997
(]25 x 3 ram) using acetonitrile-ammonium formate (0.05 raM, pH 3.0) as a mobile phase. The acetonitrile content was 5% for morphine and 15% for other drugs; the flow rate was 0.6 mL/min. An SSQ 7000 APCI-LC-MS instrument (Finnigan MAT,San Jose, CA) was used in APCI mode (positive ionization). The scan ranges applied were as follows: for morphine/morphine-d3, m/z 285 > 290; for BZE-BZE-d3-BZE-ds,m/z 289 > 299; for MDE-MDE-dT,m/z 206 > 215; and MDMAand for MDMA-ds,m/z 193 > 200. The mass chromatograms of all nondeuterated drugs, which were injected in 500 ng amounts, showed fairly high areas of the "isotope peaks" (M§ to (M+3). The percent contributions of (M+3) peaks, which were measured as percent of peak areas of protonated molecular peaks, were as follows: morphine, 0.4%; BZE, 0.3%; MDE, 0.1%; and MDMA, 0.1%. As expected, no isotopic contribution was observed for the masses (M+5) and higher, which corresponded to the masses of deuterated analogues of BZE, MDMA,or MDE. Figure I shows the chromatograms of morphine and BZE (500 ng each). The peak-area ratios (standard-IS), calculated for morphine-morphine-d3 and for BZE-BZE-d3,were lower than expected for standards containing 500 ng of drug. Calibration graphs calculated for BZE-BZE-d3and for BZE-BZE-d8 showed different slopes. The observed phenomenon is very well-known. The isotopic contributions for the elements involved are as follows: 13C= 1.1%, 2H= 0.015%, 15N=0.37%, 170= 0.04%, 180= 0.2%. The isotopic contribution for carbon and hydrogen for C16-C17hydrocarbons (corresponding to morphine or BZE) was estimated at 0.1% for the abundance (A+3) (3). Such a value may be of practical importance. Assuming the apparent concentration ratio standard-IS is 100, the isotope peak may diminish the ratio to 90.9. In the case of a ratio of 200, the value would be reduced to 166.6. As a solution of this problem, the second assay with diluted aliquot may be required, as suggested by Liu et al.(2). An even better solution is to apply, whenever available, the analogues with the highest possible extent of deuteration as internal standards for APCI-LC-MS. In the case of morphine, however, only @analogues are currently commercially available. Maciej J. Bogusz Institute of Forensic Medicine Aachen University of Technology Klinikum 52057 Aachen Germany
References 1. S. Valtier and J.T. Cody. Evaluation of internal standards for the analysis of amphetamine and methamphetamine. J. Anal. Toxicol. 19" 375-80 (1995). 2. R.H. Liu, G. Foster, E.J.Cone, and S.D. Kurnar. Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse--pentobarbital example. J. Forensic ScL 40:983-89 (1995). 3. E.W. McLafferty and F. Turecek. Interpretation of Mass Spectra. University Science Books, Mill Valley, CA, 1993.
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