Ion Specific Detection of Internal Standards Labelled with Stable Isotopes Thomas E. Gaffney,l Carl-Gustaf Hammar,2 Bo Holmstedt, and Robert E. McMahon3 Department of Toxicology, Swedish Medical Research Council, Karolinska Institutet, 104 01 Stockholm 60, Sweden, and The Lilly Reseach Laboratories, Indianapolis, Ind.
The purpose of an internal standard in quantitative analytical techniques is to permit the analyst to correct for losses of the compound under study. This requires chemical similarity between the internal standard and the compound i n question. When conventional gas chromatographic detector systems are used, there must a t the same time be sufficient dissimilarity so that the peaks from the compound and the standard can be resolved. However, i n the study described in which the mass spectrometer is used as the detector for a gas chromatographic effluent, there is no need for any chemical differences, and mass differences alone are sufficient to discriminate between the compound being analyzed and the standard. This allows the use of the ideal internal standard, namely the actual compound under study modified to contain an increased mass by the introduction of stable isotopes. Examples of the use of 15N and/or deuterium labelled compounds as internal standards are given. THETECHNIQUE in which a mass spectrometer is used as an ion-specific detector for a gas chromatographic effluent has been used for the qualitative identification of drugs and drug
Present addresses, Division of Clinical Pharmacology, Departments of Pharmacology and Internal Medicine, University of Cincinnati, College of Medicine, Cincinnati, Ohio. * Present address, Research Department of the KABI Group, S-104 25 Stockholm 30, Sweden. Present address, The Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Ind. 46206.
metabolites in biological fluids (I). The sensitivity, gram, and ion-specificity of this technique and the need for sensitive, specific, and quantitative methods for studies in the biology and medical sciences have encouraged us to consider innovations which may help this technique serve in a quantitative as well as qualitative manner. Since internal standards provide the greatest accuracy in quantitative gas chromatography, we have tested the use of isotopically labelled internal standards (stable isotopes). Our reasoning was that the best internal standard might be the actual molecule under study modified to contain an increased mass. For example, the substitution of 15N for 14Nand/or deuterium for hydrogen in a molecule should not change its chemical characteristics but the increase in mass would allow the molecule to be easily distinguished from its nonisotopic counterpart. We have studied two possible internal standards for nortriptyline (NT), Le. deuterated nortriptyline (M 2 AMU) and deuterated and 16N labelled (M 3 AMU) nortriptyline. An increase of two AMU enabled us to avoid interference from the natural 13C present in the unlabelled NT. For comparison with isotopically labelled internal standards, we have used what has to date been our best unlabelled internal standard for NT, namely IBD 78.
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(1) C.-G. Hammar, B. Holmstedt, and R. Ryhage, Anal. Biochem., 25, 532 (1968).
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Figure 1. Mass spectra of nortriptyline-trifluoroacetylated(NT-TFA) deuterated, W-labelled NT-TFA and IBD-18-TFA ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971
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Figure 2. Recording of the molecular ions of nortriptyline- trifluoroacetylated (NT-TFA), M 359, doubly-deuterated NT-TFA, M 361, and deuterated and '5Nlabelled NT-TFA, M 362 Two-hundred nanograms of each compound were injected at zero time
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EXPERIMENTAL
Materials and Methods. The study was performed using the basic technique originally described by Hammar, Holmstedt, and Ryhage (1). The actual recordings were made, however, using the recently developed Multiple Ion Detector Accessory (MID) described by Hammar and Hessling (2). (2) C.-G.Hammar and R. Hessling, ANAL. CHEM.,43, 298
(1971).
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The focusing of individual channels of single ions was achieved by first focusing with the manual magnetic field control on a reference ion introduced via the direct inlet probe. The remaining ions were then brought into focus on the other channels by the addition or subtraction of accelerating voltages while the magnetic field was held constant. The accuracy of focusing was confirmed during the elution of the compounds from the column. An all glass chromatographic column was used containing 0.5% XE 60 and 0.25 % DC LSX on silanized Chromosorb G ; the internal diameter was 1.5 mm and the length about 1.2 meters. The flash heater and column were held at 220 and 190 "C, respectively. Helium flow was about 20 ml/minute. Conditions included an ion source temperature of 310 "C and a separator temperature of 250 "C. All recordings were made at an ionization energy of 20 electron volts. Chemicals Used. NT was obtained as the hydrochloride salt from the Lilly Company, U S A . Deuterium and 15N labelled NT were synthesized and provided as the hydrochloride salts by Dr. Fred Marshall and Dr. Bill Lacefiled of the Lilly Research Laboratories, Indianapolis, Ind. The side-chains were labeled as -CHCH2CD2NHCH3,(the M 2 compound) and =CHCH2CH2lSNHCH3(the M 3 compound). IBD 78, whose formula is shown in Figure 1, was provided by Sandoz Laboratories in Switzerland. Derivatives were prepared with trifluoroacetic anhydride as the reagent and dimethylformamide as the catalyst. RESULTS AND DISCUSSION
Mass spectra confirmed the chemical identity, Figure 1 , of each compound studied. The simultaneous but easily resolved elution of an equimolar mixture of the nortriptylines,
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Figure 3. Recordings of M 359, M 362, and IBD-lbTFA, M 365 Two-hundred nanograms of specified compounds were injected at zero time. All three compounds were injected in 3 A and 3 E , but M 359, NT-TFA, was omitted from the injection shown in 3 C. The capacity of individual channel ampliation systems to normalize peak heights is shown by a comparison of 3 A and 3 B . The specificity of the technique is emphasized by comparison of 3 A with 3 C 308
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Figure 4. Base peak fragmentograms of 100 picograms (A) and 50 picograms ( B ) of NT-TFA, M-232,deuterated NT-TFA, M 234, and IBD 18,M 238 Minimal disorption produced by the injection of solvent is shown in panel C. Notice the reduced signals from IBD 18-TFAcompared to that produced by the same concentrationsof the nortriptyline, M 232 and M 234
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Figure 5. Mass fragmentograms of trifluoroacetylated extracts of human plasma without (A) and with (B) NT, M 359, and its deuterated counterpart M 361. Injection is marked as zero time ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971
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i.e. M, M+2 and M+3, is shown in Figures 2 and 3. The elution characteristics of the three molecular ions emphasize the chemical similarity between NT and its labelled counterparts, either one of which could function as an internal standard for NT. The impressive resolving power of the technique as compared to the more commonly used chromatographic detection systems is also evident. The chromatographic characteristics of IBD 78 as compared to NT and M+3 is also shown in Figure 3. IBD 78 has different chromatographic characteristics. Performance of Internal Standards Near the Limits of Sensitivity. It is apparent that the use of the base peak offers the highest sensitivity. For example, the elution of 100 and even 50 picograms of NT-TFA is easily distinguished as the base peak from the same concentrations of its isotopically labelled counterpart, Figure 4. Even at this concentration, the chromatographic characteristics of NT-TFA and M+2 are indistinguishable. In contrast, 100 and 50 picograms of IBD 78-TFA produce much lower signals than the same concentrations of NT-TFA or M f 2 . This difference between IBD 78-TFA and the nortriptylines suggest that more IBD 78-TFA is lost by column absorption. Such a difference in column absorption between a compound and the substance serving as its internal standard is only one example of how the measured recovery of most internal standards probably does not reflect the recovery of the compound under study. In the example shown in Figure 4, the differential loss of IBD 78-TFA over NT-TFA would result in an overestimation of the concentration of NT that is present.
Drugs and Internal Standards in Biological Materials. Biological samples present problems in that they must be shown to be free of the substance or in this case, the mass which is chosen as the “marker” for the internal standard, Figure 5 shows a study of plasma before and after the addition of NT and a possible internal standard-i.e., M f 3 . The plasma is first shown to be free of NT and M+3, and other substances with comparable retention times containing the same masses. Figure 5 , A and B demonstrate the elution and resolution of the two substances after their addition to the plasma. Notice that even though the background recorded from plasma (Figure 5 ) is greater than is seen with the injection of methanolic solutions of reference material (Figures 2-4), the quality of the resolution of the two nortriptylines is adequate. Notice that had IBD 78-TFA been used as the internal standard for this plasma sample, the broad peaks present in the plasma extract, which represent the same masses as are found in IBD 78-TFA, could have interfered with the resolution of IBD-78-TFA. This example of the potential interference by non-drug substances in biological samples emphasize the usefulness of internal standards which have a retention time identical to the substance under study. RECEIVED for review June 8, 1970. Accepted December 1, 1970. This research was supported by the Clinical Pharmacology Fund of Cincinnati, Ohio, NIH-USPHS Grants HE 07392, GM13 978, NIH3RoI MH, 12007-0381, and by a grant from the Wallenberg Foundation and Riksbankens Jubileumsfoud, Stockholm, Sweden, Swedish Medical Research Council NO,B70-40X-2629-01-02.
Multivariable Analysis of Quantitative X-Ray Emission Data The System Zirconium Oxide-Aluminum Oxide-Silicon Oxide-Calcium Oxide-Cerium Oxide Donald A. Stephenson Research and Deuelopment Laboratories, Corning Glass Works, Corning, N . Y. 14830 Guidelines for the correct use of statistical methods for quantitative X-ray emission analysis of multicomponent systems are set forth, and a general review and discussion of the technique is presented. The regression model first proposed by Alley and Myers is the only model, among many, that seems both physically and statistically adequate for general application, and its use is recommended before more elaborate models are tried. A new procedure for the selection of synthetic reference standards covering any region of interest in any n-component system in a homogeneous fashion is described and applied to the standardization of part of the system ZrOz (35-85 wt %)-AlpOa (5-55 w t %)-Sios (1-10 wt %)-CaO (5-15 w t %)-CeOz (5-15 W! %). The residual error in the prediction of the composition of the synthetic standards is around 1 to 2% relative, which compares favorably with other methods of analysis.
THE MATHEMATICAL INTERPRETATION of quantitative X-ray emission data has steadily gained popularity during the last decade. The spate of activity in this area is due largely to the widespread availability of large-scale computing facilities 310
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necessary to perform the complex calculations associated with various techniques for treating the data. Numerous authors have proposed equations that relate the concentration of a particular component in an unknown sample to the observed intensity of characteristic X-rays of that and/or other components through coefficients obtained from known standards by either regression analysis-Lucas-Tooth and Price ( I ) , Sugimoto (2,3), Lamborn and Sorenson (49, Lucas-Tooth and Pyne (5), Alley and Myers (6, 7), Miller and Galletta (8), (1) H. J. Lucas-Tooth and B. J. Price, Metallurgiu, 64, 149(1961). (2) M. Sugimoto, Bunseki-Kuguku, 11, 1168 (1962). (3) h i d . , 12, 475 (1963). (4) R . E. Lamborn and F. J. Sorenson, Adoan. X-Ray h a / . , 6,422 (1963). (5) H. J. Lucas-Tooth and C. Pyne, ibid., 7,523 (1964). (6) B. J. Alley and R . H. Myers, ANAL.CHEM., 37, 1685 (1965). (7) B. J. Alley and R. H. Myers, Norelco Rep., XV, 87 (1968). (8) L. D. Miller and F. A. Galletta, “Application of Computer
Techniques in the X-Ray Fluorescence Analysis of Iron Ore Sinters,” Denver Conference on the Applications of X-Ray Analysis, Denver, Colo., 1967.
