Anal. Chem. 1982, 54, 345-347
CONCLUSION While the power of liquid chromatography primarily lies in the ability to vary the mobile phase for optimizing selectivity, selection of the proper mobile phase requires an understanding of the behavior patterns of that particular column packing. Data obtained in this study illustrate the vastly differing chromatographic properties of commercially available reversed-phase packings. The factor compounds the difficulty in predicting and identifying the optimum module phase for a particular separation. The data presented here, while empirical, should provide a starting point for the better under-
345
standing of the interactive properties of commercially available LC columns. Continuing efforts in this regard are being carried out in our laboratory.
LITERATURE CITED (1) Horvath, C.; Melander, W. J. J . Chromafogr. Scl. 1977. 15, 393. (2) Melander, W.; Campbell, D. E.; Horvath, C. J . Chromafogr. 1978, 158, 215. (3) Scott, R. P. W.; Slmpson, C. F. J . Chromafogr. 1980, 797, 11. (4) Colln, H.; Gulochon, G. J . Chromafogr. 1978, 158, 183. (5) Colin, H.; Gulochon, G. J . Chromafogr. 1977, 141, 289.
RECENED for review July 6,1981. Accepted October 26,1981.
Interference in a Liquid Chromatographic Assay from Impurities in the Derivatization Reagent Robert W. Stotz” and Dennis H. Hasslng Control Analyiical Research and Development, The Upjohn Company, Kalamazoo, Mlchlgan 4900 1
The use of derivatization in high-performance liquid chromatographic analyses to improve chromatographic properties and to increase sensitivity to detection has become more prevalent in recent years (I, 2). For most published chromatographic procedures utilizing derivatization, the derivatization reagent is obtained from a commercial supplier and used without further purification. However, as this paper describes, the presence of low level impurities in the derivatization reagent, some of which are not removed by the more common methods of purification, can easily lead to some erroneous conclusions regarding the concentrations of impurities in the material being analyzed. Our laboratories currently employ several assay procedures for prostaglandins that involve esterification of the carboxylic acid moiety with a-bromo-2‘-acetonaphthone (a-BAN or 2naphthacyl bromide) prior to quantitation by HPLC. In general, the derivatization procedure is similar to the microscale derivatization method reported by Morozowich and Douglas (3) for the formation of p-nitrophenacyl esters of prostaglandins, viz., the Nfl-diisopropylethylamine assisted reaction of the derivatization reagent with the prostaglandin in an acetonitrile solution. During the development of a normal phase HPLC assay for dinoprostone (an E-type prostaglandin) that also involved quantitation of impurities, it was discovered that most lots of a-BAN purified by the generally recommended recrystallization procedure (carbon tetrachloride as solvent with carbon treatment) still contained significant levels of impurities. One of these impurities closely mimics a-BAN and forms a derivative of dinoprostone that elutes just prior to the a-BAN analogue. The peak arising from this impurity was initially thought to result from an impurity present in the dinoprostone because it was in a region of the chromatogram where peaks attributed to isomers of this prostaglandin appear. Although the interference problem described in this report was encountered in the development of only one specific assay, the problem would be expected to manifest itself in any HPLC assay that utilized impure a-BAN as a derivatization reagent.
EXPERIMENTAL SECTION Equipment and Apparatus. High-performance liquid chromatography data were obtained by using an Altex Model llOA HPLC pump with a Valco N-60 injector, two Varian Micro Pak, 5-p silica gel columns in series, and a Waters Model 440 dual0003-2700/82/0354-0345$0 1.25/0
channel UV detector operating at 254 nm interfaced to a Sargent-Welch DSRG-2dual pen recorder. Mass spectra were obtained via direct insertion probe into a VG Model 7070 mass spectrometer. Procedure. Separation of a-bromo-2’-acetonaphthone (aBAN) and its impurities was achieved by using a butyl chloride:hexane (7030) mobile phase and a flow rate of 0.6 mL/min. Samples of a-BAN were dissolved (100 pg/mL) in mobile phase and approximately 10 pL of the solution was injected into the HPLC system. Chromatographyof dinoprostone was accomplished by using a methylene ch1oride:acetonitrile:glacial acetic acid:water (900100205.5) mobile phase and a flow rate of 0.4 mL/min. Samples of the prostaglandin were derivatized by combining approximately 1mg of material with 0.5 mL of a freshly prepared acetonitrile solution containing 3 mg of a-BAN and 1.5 pL of N,N-diisopropylethylamine. The derivatization solution was allowed to stand at ambient temperatures, protected from light, for 2 h before evaporating to dryness with a stream of nitrogen and adding 10 mL of mobile phase. About 10 pL of this solution was injected into the HPLC system.
RESULTS AND DISCUSSION The initial chromatograms obtained in the dinoprostone assay development work contained two minor component peaks that appeared just in front of the peak arising from the prostaglandin. Both peaks were resolved from the major component peak but were incompletely resolved from each other. On the basis of comparisons of relative retention times vs. an authentic sample, the peak nearest the dinoprostone peak was determined to result from one of its common isomers. The other minor peak that represented a concentration of about 1% could not be assigned to any one of the known isomers or degradation products of the prostaglandin. Its original assignment as being the result of a previously unresolved impurity of dinoprostone became suspect as the development work progressed. While attempting to further refine the chromatography, it was observed that for a given lot of prostaglandin, the relative intensity of the “impurity” peak was dependent upon the source and/or history of the a-BAN utilized for the derivatization. For example, when dinoprostone was derivatized with an “as received” sample of CY-BANobtained from The Aldrich Chemical Co., Inc., the intensity of the “impurity” peak corresponded to a concentration of about 1.2%. Recrystallization of this material and subsequent derivatization of the 0 1982 American Chemical Society
348
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
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Flgure 1. Chromatogramsof as-received a-brom*2'-acetonaphthone from (a) Aldrich Chemical Co., Inc. (A) impurky I, (B) impurity 11, (C) a-bromo-2'-acetonaphthone, and (b) Pfaltz & Bauer, Inc. (A) impurity 111, (8)Impurity I, (C) impurity 11, (D) a-bromo-2'-acetonaphthone.
same lot of prostaglandin yielded a chromatogram where the concentration of the "impurity" was approximately 0.9%. The most marked effect was noted when a sample of a-BAN obtained from another source (Pfaltz & Bauer, Inc.) was recrystallized and subsequently utilized in the derivatization. In this case the "impurity" peak was almost negligible. Several samples of a-BAN with different histories were eventually chromatographed by the HPLC assay procedure outlined in the Experimental Section of this paper. A chromatogram (Figure la) of as-received a-BAN from Aldrich Chemical Co. indicates the presence of two major impurities. (A chromatogram of as-received material purchased from a third source, Columbia Chemical Co., was essentially identical.) Assuming equal response for the two impurities vs. the major component, impurity I (peak A) and impurity I1 (peak B) correspond to concentrations of approximately 0.6% and 0.5%, respectively. Recrystallization of this material reduces the concentration of impurity I to about 0.2%) but does not
significantly alter the concentration of impurity I1 (0.4% vs. 0.5%). A chromatogram (Figure lb) of as-received a-BAN from Pfaltz & Bauer shows the presence of one significant impurity (impurity 111) and two others that can be considered negligible. The two negligible peaks, peaks B and C of Figure lb, correspond to impurities I and 11, respectively, as seen previously in the sample obtained from Aldrich. Impurity I11 could not be identified; however, one recrystallization with carbon treatment of Pfaltz & Bauer's a-BAN essentially eliminates this one significant impurity. Impurities I and I1 were isolated by a semipreparative HPLC procedure and used to derivatize samples of dinoprostone. Subsequent chromatography demonstrated that impurity I1 (peak B in Figure la) yields the extraneous peak in chramatograms of dinoprostone derivatized with Aldrich's a-BAN. Impurity I gave a chromatogram containing several peaks. The major peak had a relative retention time equal to that of the usual a-BAN derivative of the prostaglandin. Mass spectra were obtained on samples of pure a-BAN as well as on impurities I and 11. The spectra of pure a-BAN (Figure 2a) exhibits molecular ions of nearly equal intensity at 248 and 250 amu, and major fragment ions at 155 and 127 amu. The fragment ions correspond to losses of CHzBr and COCH2Br,respectively. The appearance of a molecular ion pair having approximately equal intensities and separated by two mass units is characteristic of a molecule containing one bromine atom. This is a result of the natural abundances of the two major bromine isotopes (79 and 81, a 2 mass unit difference, and 50.5% and 49.5% abundances, respectively). The mass spectrum of impurity I (Figure 2b) shows three molecular ions at 326,328, and 330 m u with an intensity ratio of about 1:21and fragment ions at 248/250,155, and 127 amu. The appearance of the three molecular ions is characteristic of a molecule containing two bromine atoms, and the molecular weights of these ions correspond to the addition of a second bromine atom to an a-BAN molecule. The fragment ions are the same as those obtained from pure a-BAN and probably correspond to losses of Br (with replacement by H), CHBr2,and COCHBg On the basis of the mass spectral data, impurity I was tentatively identified as a,a'-dibrom0-2'acetonaphthone. One literature source (5) cited this compound as being a probable impurity formed in the preparation of
347
Anal. Chem. 1902, 5 4 , 347-349
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a-BAN from 2‘-acetonaphthone. Another source (6)reported that the dibromo species can be prepared from the same starting material (2’-acetonaphthone) by following essentially the same procedure as for the monobromo species (4) and simply adding a sufficient excess of brominating agent. The mass spectrum of impurity I1 (Figure 2 4 , like impurity I, exhibits three molecular ions at 326, 328, and 330 amu indicative of a molecule containing two bromine atoms (Le., addition of a second bromine atom to an a-BAN molecule); however, its fragmentation pattern is significantly different. An ion pair at 248/250 amu is common to all the spectra, but pairs at 2331235 and 2051207 amu as well as a major fragment peak at 126 amu are unique features of this compound. The ion pair at 2331235 amu could correspond to the loss of a CHzBr fragment if one assumes that the second bromine is attached to the naphthalene ring of an a-BAN molecule. Attachment of a bromine atom to the naphthalene ring is strongly supported by the 2051207 amu pair, which would correspond to the loss of a COCHzBr fragment, and by the fragment appearing at 126 amu. This fragment (CloHG+) would correspond to the losses of both a COCHzBr moiety
and a Br atom from the ring. Therefore, impurity I1 is probably a mono ring brominated derivative of a-BAN. This structure assignment is consistent with the observed reactivity of this compound with dinoprostone and the chromatographic properties of its ester. Proton and carbon-13 nuclear magnetic resonance spectra were also obtained on impurities I and 11. Although their spectra were consistent with their assigned structures, they did not prove these assignments. The minute amounts of impurities isolated, contamination of these samples with residual solvents, and the lack of availability of authentic reference materials and/or spectra precluded a more definitive structure assignment. The observations presented in this report emphasize the importance of verifying the purity of reagents used for derivatization especially when an analyte’s impurities are also to be quantitated. We were fortunate in being able to observe the peak arising from impurity I1 in our initial work because the particular set of conditions (conditions not suitable for a routine assay) selected for the chromatography gave a very long retention time for the major component and relatively wide separations between the impurity peaks. However, under the final set of conditions selected for the dinoprostone assay, the absorption arising from the ester of impurity I1 is no longer resolved from a peak attributed to one of the isomers of the prostaglandin. A typical chromatogram (Figure 3), obtained by the final version of the dinoprostone assay procedure outlined in the Experimental Section, exhibits a minor peak (peak F) attributed to this isomer just in front of the major component peak. By substitution of impure a-BAN containing impurity I1 in the derivatization step, the chromatogram obtained is almost identical. The only significant difference is an apparent increase in the relative intensity of this minor peak.
ACKNOWLEDGMENT D. H. Rineveld is acknowledged for his efforta in isolating the a-BAN impurities. W. K. Duholke is acknowledged for obtaining and interpreting the mass spectral data presented in this report. LITERATURE CITED Morozowlch, W.; Cho, M. J . Chrotnatogr. Sci. 1978, (Series 9), 209. Ross, M. S. F. J . Chromatogr. 1977, 741, 107. Morozowlch, W.; Douglas, S. L. Prostaglandins 1975, 10, 19. Immedlata, T.; Day, A. R. J . Urg. Chern. 1940, 5 , 512. (5) Tavlor. Sac B . 1987. 904. -,- P. J. - J- . Chern. - (6) Kravets, V. P.; Chervenyuk, G. I.; Gin& G. V. Zh. Org. Khim. 1988, 2 . 1244 Chern. Abstr. 1987, 66, 85231h. (1) (2) (3) (4)
.-,
___._
RECEIVED for review August 3, 1981. Accepted October 29, 1981.
Detection of 1-Piperidlnocyclohexanecarbonitrile in Illicit Phencyclidine Samples John K. Baker Depatiment of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University, Mississippi 38677
The majority of “street” phencyclidine is manufactured in clandestine laboratories using 1-piperidinocyclohexanecarbonitrile (PCC) as an intermediate which can only be removed from the final product with some difficulty (I). The toxicity of PCC has been shown to be about three times greater than phencyclidine and there is a lethal synergism of PCC and phencyclidine (2,3).Previous reports on the analysis of 0003-2700/82/0354-0347$0 1.25/0
illicit phencyclidine samples have indicated that 25-33 % of the preparations were contaminated with PCC and that PCC represented from 1 to 70% of the active ingredients (4,5). Previous studies have shown that PCC decomposes almost completely during gas chromatographic analysis and the decomposition will also occur in thin layer analysis unless the TLC plate is deactivated (6). More recent gas chromato0 1982 American Chemical Soclety
