ANALYTICAL CHEMISTRY, VOL. 51, NO. 2 , FEBRUARY 1979
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Gas Chro mat ographic- Mass Spect ro met r ic Determination of Etorphine with Stable Isotope Labeled Internal Standard Satya P. Jindal,
Theresa Lutz, and Per Vestergaard
Rockland Research Institute, Orangeburg, New York, 10962
A quantitative gas chromatographic-mass spectrometric assay was developed for the determination of etorphine in urine. Etorphine, on treatment with N,O-bis(trimethylsily1)trifluoroacetamide forms t w o etorphine trimethylsilyl derivatives-etorphine-TMS and etorphine(TMS),. Experimenial conditions were developed for selective and quantitative conversion of etorphine to etorphine-TMS. The assay utilizes selected ion focusing to monitor in a GLC effluent the molecular ion generated by electron impact ionization ( E I ) of etorphine-TMS. Etorphine-d, was synthesized and used as an internal standard. The assay can measure 2 ng/mL of the drug with about 5 % precision. The methodology is used for the urinary assay of etorphine of a rabbit given a single subcutaneous dose ( 1 pg/kg) of the drug.
Etorphine, a 6,14-endoetheno-tetrahydrothebaine derivative is a n analgesic of unprecedented high potency (1-3). When given subcutaneously, etorphine is 1000 t o 80 000 times as potent as morphine (4) and is characterized by its rapid onset and short duration of action. Its ability t o cause catatonia at very low dose levels has resulted in its use for t h e immobilization of game animals ( 5 ) . T h e fact t h a t it works sublingually and has abuse potential ( 6 ) raises the possibility that it may become a future threat in t h e drug abuse field. T h e low dose levels a t which etorphine is used (1 pglkg) demands a sensitive measurement technique for its determinations in biological fluids. Etorphine could not be detected in urine with commonly available methodology including gas-liquid chromatography after the administration of highly euphoronigenic doses in man ( 7 ) . Recently, in a preliminary communication (8) we reported a mass spectrometric assay of etorphine in urine using commercially available tritiated etorphine as an internal standard. T h e internal standard was a complex mixture of nontritiated, monotritiated, and ditritiated etorphine; consequently, t h e assay precision, particularly at low levels of etorphine, was unsatisfactory. This paper reports a mass fragmentographic assay of etorphine in urine using stable isotope-labeled etorphine as a n internal standard. Selective ion monitoring, t h e technique built on combined gas chromatography-mass spectrometry (9. 10) was used t o develop a very sensitive and specific assay of etorphine in urine utilizing site specific deuterium-labeled etorphine as a n internal standard. T h e methodology was used t o study t h e urinary excretion of free etorphine in rabbits after a single subcutaneous dose.
EXPERIMENTAL Materials. Analytical grade etorphine, N-desmethyl etorphine (Reckitt and Colman Pharmaceutical Division, Hull, U.K.); methyl iodide-d,, isotopic purity 99%, (International Chemical and Nuclear Corp., Irvine, Calif.); N,O-bis(dimethylsi1yl)acetamide (BDSA), Applied Science Laboratories, State College, Pa.); N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA, Pierce Chemical Co., Rockford, Ill.) were used without further purification. All solvents used were of ACS analytical grade (Pfaltz and Bauer, Flushing, N.Y.), silanized tubes with screw caps lined 0003-2700/79/0351-0269$01.00/0
with Teflon (Kimble, Owens, Chicago. Ill.) were used for extraction. Urine samples were processed as soon as obtained. Synthesis of Etorphine-d, (N-Methyl-d,). Etorphine-d, was synthesized by treatment of N-desmethyl etorphine with methyl iodide-d, using an established procedure for alkylation of secondary amines (11). The labeled compound gave satisfactory mass spectral (electron impact ionization, EI) characteristics. A selected ion detection analysis of etorphine-d, showed the presence of an ion equivalent to 98.7% f 0.15% ( n = 6 ) etorphine-d3and an ion equivalent to 1.17~f 0.15% ( n = 6) etorphine. Animals. All animals used in this work were male, New Zealand white rabbits. Extraction of Etorphine from Urine. T o 1 mL of urine was added an appropriate amount of etorphine-d, (typically 28 ng/mL) as an internal standard. The urine was adjusted to p H 9 with 1 N ",OH and extracted twice with 6 mL protions of N-butylchloride. The organic fractions were combined, 1 mL 0.1 N HC1 was added to it, and the solution was shaken for 15 min and centrifuged. The organic layer was discarded, the aqueous phase was adjusted t o pH 9 with 1 N ",OH and extracted twice with 3 mL portions of N-butylchloride. The organic fractions were combined, dried with sodium sulfate, filtered, and the solvent was evaporated at 50 "Cunder a gentle stream of nitrogen. Recovery of etorphine, added to control urine, was studied at the 5 ng/mL level. Formation of Derivatives. Etorphine has two hydroxyl functions; either both of these or a t least one of these must be selectively and quantitatively derivatized for the molecule to have good GLC characteristics. Similar compounds (12).on treatment with BSTFA, are known to give a complex mixture of derivatives. Multiderivatization in the area of biopharmaceutical analyses is a serious problem, which could adversely affect the sensitivity and possibly specificity of the mass spectrometric assay. Consequently experimental conditions were worked out for the quantitative preparation of etorphine-TMS-1 and etorphine(TMS)2-2from etorphine. Etorphine-TMS-1. Etorphine (100 ng) was taken in a Reacti-Vial, to this was added 150 pL of BSTFA. The mixture was heated at 60 "C for 1 h. After this period, the solution was cooled to room temperature, excess reagent was removed a t 40 "C under a gentle stream of Ne, the residue was reconstituted in 10 pL of benzene; an aliquot of this solution was injected into the GLC-mass spectrometer. The total ion current trace showed the material to be homogenous on two different columns (1.5% OV-1 and 1% OV-17) and its mass spectrum (Figure 1) is in agreement with the structure. Etorphine-(TMS),-2. Etorphine (100 ng) was taken in a Reacti-Vial, to this was added 50 pL of pyridine and 150 FL of BSTFA. The material was heated a t 120 "C for 2 h. After this period, the reagents were removed at 40 "C under a gentle stream of N2; the residue was reconstituted in 10 pL of benzene. An aliquot of this solution was injected into the GLC-mass spectrometer. Again the material was found to be homogenous on two different columns and its mass spectrum (Figure 2) confirms the structure. Instrumentation. Mass spectrometry was done using an LKB 9000 GC-MS system (9. 10) equipped with the multiple ion detectorlpeak matcher accessory (MID/PM). Gas chromatography was performed on a 1.8-m glass column (2-mm i.d.) packed with 1.57~OV-1 on gas chrom Q 100-200 mesh. The column temperature was maintained a t 225 "C, flash heater a t 235 O C , separator at 240 "C and the ion source at 250 "C. The accelerating voltage was 3.;i kV in the scan mode and 3.0 kV in the MID mode, the ionization potential was 70 e\'. and the trap current was set 'C 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
Scheme I. Proposed Fragmentation Mechanism for Electron Impact Ionization of Etorphine-TMS 396
8 354
'O01
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Figure 1. Mass spectrum of etorphine-TMS
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Figure 2. Mass spectrum of etorphine-(TMS),
at 60 PA. The magnetic field was kept constant by focusing the background ion (column bleed) at m / e 429 and the additional voltages were 108 V and 89 V for measuring the ion intensities at m l e 483 and 486, respectively. R E S U L T S AND DISCUSSION T h e mass spectrum of etorphine-TMS-1 (Figure 1) shows a molecular ion a t mle 483, a base peak a t mle 272 and other major peaks a t mle 354 and 396, respectively. A priori, one would expect some localization of charge on nitrogen atom in t h e molecular ion, to be followed by cleavage a t the three carbon-carbon bonds @ to t h e nitrogen atom (13,140 leading to radical ions a, b, and c shown in Scheme I. The ions b and c with radical sites a t bridge head and at primary C-13, respectively, are rather unstable; consequently they are expected to be of minor importance. T h e ion a, resulting from the cleavage of the C9-Clo bond, is a benzylic radical and must be of major importance in the electron impact fragmentation of t h e molecule. T h e ion a, probably, is in equilibrium with ion d, a n isomeric benzylic radical ion, arising by H-transfer from C-16 t o C-10 followed by C13-CI5 bond cleavage. This kind of H-atom migration is stereochemically feasible and has been well documented in electron impact ionization of morphine a n d similar compounds (14-16). T h e intense molecular ion peak a t m / e 483 must be attributed to the great stability of these benzylic radical ions. Reasonable mechanism for the fragmentation pattern (16, 17) is proposed in Scheme I. T o assist in the interpretation of the spectrum, fragmentation of several analogous etorphine derivatives was examined. T h e peak a t mle 396 in the spectrum of etorphine-TMS, resulting from the loss of stable tertiary radical (CH3-C(OH)-C3H7), as expected, is shifted to higher mass by 3 a m u in the spectrum of etorphine-d3-TMS (Figure 3); to lower mass by 14 a m u in t h e spectrum of etorphine-dimethylsilyl ether (not shown here) a n d appears a t the same m / e value in the spectrum of etorphine-(TMS)2(Figure 2). Again, in agreement, with the proposed fragmentation, the ions at mle 354, 272 in the spectrum of etorphine-TMS are shifted to higher mass by 4 amu in the spectrum of ditritiated etor~hine-TMS((15,16-~H)8) and to lower mass by 14 m u in
!!
8
486
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Figure 3. Mass spectrum of etorphine-d,-TMS
the spectrum of etorphine-dimethylsilyl ether. GC-MS-SIM Quantitation. The ion at mle 483 is specific for etrophine-TMS (mle 486 for etorphine-d3-TMS),is a convenient working mass for SIM assay and is not observed in the electron impact ionization spectra of other opiates (18, 19) and known metabolites of etorphine (20). Consequently, urinary etorphine extract along with the labeled etorphine-d3 was treated with BSTFA at 60 "C, excess reagents were evaporated, an aliquot of the reconstituted solution was injected into the GLC-mass spectrometer; etorphine was quantitated by measuring the ion intensities a t mle 483 a n d 486, respectively. Control urine samples subjected to the described procedure for etorphine analysis showed no significant background ions at mle 483 and 486. Known amounts of etorphine along with their isotopic analogue in "fixed" amount were added to control urine a n d processed as described above. A plot of ion intensity ratio (mle 4831486) vs. the amount of etorphine per fixed amount of etorphine-d3, was linear with a slope of 0.98 f 0.02 and an intercept of 0.04 k 0.02 ng. These d a t a affirm a simple linear relationship between t h e appropriate ion intensity ratios and t h e concentration of etorphine and exclude any isotopic exchange or any significant kinetic isotope effect in the fragmentation
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979 C
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Selective ion chromatograms for etorphine-TMS ((-) m l e and etorphine-d,-TMS (---) m/e 486) obtained from 4-mL aliquots of selected samples of rabbit urine and processed as described above. Part A, Urine at 1.25 h after the dose, 5 ng of etorphine-d, ( - - - ) was added as internal standard. Etorphine found was 5.3 ng. Part B, Urine at 3 h after the dose, 20 ng of etorphine-d, (---) was added as internal standard and etorphine found was 42 ng. Part C, Urine at 5 h after the dose, 20 ng of etorphine-d, (---) was added as internal standard and etorphine found was 21.6 ng. Part D, Urine at 52 h after the dose, 5 ng of etorphine-d, (---) was added as internal standard and etorphine found was 2.96 ng Figure 4. 483
process. Six control urine samples containing 5 ng/mL of etorphine were analyzed by the above method using 5 ng/mL of etorphine-d3 as internal standards. T h e results for these samples were 4.8 f 0.25 ng/mL of etorphine. Another set of six control urine samples containing 5 ng/mL of etorphine were processed as above, this time the internal standard etorphine-d3, 5 ng/mL was added in each sample after the extraction. T h e recoveries for these samples, based on comparison of the ion intensity ratios of the two sets were 68 f 1 2 % . T h e wide range of recoveries observed is expected in the field of trace analysis and is attributed to variable glassware and GLC column adsorption. T h e assay of etorphine, presented here, is sensitive, specific, and, with minor changes, is applicable to other body fluids and tissues. T h e sensitivity of the assay, being a function of extraction efficiencies, GLC column conditions, and the ion source, cannot be quoted in absolute terms. With near-zero leak current in t h e ion source, clean and freshly silanized GLC column, glassware, and better than 60% recoveries, an assay sensitivity of approximately 2 n g / m L is possible. A number of experiments were performed on rabbits to determine urinary excretion after the subcutaneous admin-
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istration of varying doses of etorphine. In a typical experiment, a 6-kg rabbit was given a single subcutaneous dose of 5 Fg of etorphine hydrochloride in water. Urine was collected by catheterization periodically at 2-h intervals for 7 h after the dose was given. T h e last sample was collected 52 h after the administration. Appropriate amounts of urine samples were taken and processed as described. Selected ion chromatograms (Figure 4) obtained from urine samples are clean symmetrical peaks and the amounts of endogenous etorphine were calculated from the ratio of ion intensities a t m / e 483 and 486, respectively. T h e cumulative urinary excretion of intact drug, indicating the half-life of excretion to be approximately 5 h, corroborates its reported pharmacological activity of short duration. Furthermore, the animal excretes less than 20% of the drug in its native form, evidently pointing to its extensive metabolism and or conjugation to biologically inactive conjugates. LITERATURE CITED (1) K. W. Bentley and D. G. Hardy, J . Am. Chem. Soc., 89, 3267 (1967). (2) K. W. Bentley, D. G. Hardy, and B. Meek, J . Am. Chem. Soc., 89, 3273 (1967). (3) K. W. Bentley and D. G. Hardy, J . Am. Chem. Soc., 89, 3281 (1967). (4) J. D. Robinson, B. A. Morris, and V. Marks, Res. Commun. Cbem. Pathol., Pharmacol., 10, 1 (1975). (5) J. M. King and 6.H. Carter, fast A h . Wild/. J . , 3 , 19 (1965). (6) D. R. Jasinski, J. D. Griffith, and C. B. Carter, Clin. Pharmacol. Ther., 17. 267 11975). (7) C. W. Gordetzky and M. P. Kullberg, Clin. Pharmacol. Ther., 17, 273 (1975). (8) S. P. Jindal and P. Vestergaard, J . Pharm. Sci., 67, 260 (1978). (9) C.-G. Hamrnar, 8. Holmstedt, and R. Ryhage. Anal. Biochem., 25, 53 (1968). (IO) 8. Holmstedt and L. Palmer, A&. Bkzhem. Psychopharmacoi..7, 1 (1973). (1 1) R. E. McMahon and F. J. Marshall, Adv. Tracer Methodol.. 4. 29 (1968). (12) K. Verebey, M. A. Chedekel, S.J. Mul6, and D. Rosenthal. Res. Commun. Chem. Pathol. Pharmacol., 12, 67 (1975). (13) R. S. Gohlke and R . W. McLafferty. Anal. Chem., 34, 1281 (1962). (14) D. M. S. Wheeler, T. H. Kinstie, and K. L. Rinehart, Jr., J . Am. Chem. Soc.. 89. 4494 (1967). (15) H. Aubier,'M. Fetizon, D.'Ginsburg, A. Mandelbaum, and T. Rull. T e t r a M o n Len., 13 (1965). (16) H. Nakata, Y. Hirata, A. Tatematsu, H. Tada, and Y. K. Sawar, Tetrahedron Lett., 829 (1965). (17) G. R. Waller, "Biochemical Applications of Mass Spectrometry", John Wilev and Sons. New York. 1972. D 655. (18) W. 0. R. Ebbighausen, J. H. Mowat, H. Stearns, and P. Vestergaard, Biomed. Mass Spectrom., 1, 305 (1974). (19) J. Chao, R. Saferstein. and J. Manura. Anal. Chem., 46, 296 (1974). (20) M. Gordon and J. A . Vida, Annu. Rep. Med. Chem., 12, 20 (1977).
RECEIVED for review July 21, 1978. Accepted November 20, 1978. Supported by the Office of Research of the Department of Mental Hygiene of the State of New York.
