DETERMINATION OF EPHEDRINE, PSEUDOEPHEDRINE, AND NOREPHEDRINE IN HUMAN URINE USING GC-MS OF THE METHYLBORONIC ACID DERIVATIVES

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1 THE MANUSCRIPT FOR 2005 TIAFT PROCEEDINGS DETERMINATION OF EPHEDRINE, PSEUDOEPHEDRINE, AND NOREPHEDRINE IN HUMAN URINE USING GC-MS OF THE METHYLBORONIC ACID DERIVATIVES Jin Young Kim a, *, Moon Kyo In a, Bong Chul Chung b a Drug Analysis Laboratory, Supreme Prosecutors Office, Seoul , Korea b Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, P.O.Box 131, Cheongryang, Seoul , Korea *Corresponding author. Tel: ; Fax: address: paxus@spo.go.kr

2 Abstract A method using methylboronic acid derivatization and gas chromatography-mass spectrometry (GC-MS) was developed for the determination of ephedrine, pseudoephedrine, and norephedrine in human urine. Urine samples containing a deuterated internal standard were extracted with diethyl ether under an alkaline condition. The extracts were evaporated, and then the residue was derivatized by the ring-formation reaction of ephedrines with methylboronic acid (MBA). The derivatized extract (1 μl) was injected into the GC-MS. A good separation by GC was only achieved after derivatization of the amine and hydroxy groups, which also increased sensitivity to a limit of detection (LOD) of 5.0 ng/ml for both ephedrine and pseudoephedrine and 20.0 ng/ml for norephedrine. The recoveries were in the range %. The responses were linear with correlation coefficients (r 2 > ) for all analytes. The method was successfully applied to the selective determination of ephedrines from biological background or interference in positive control samples. Keywords: Ephedrines; GC-MS; Methylboronic acid; Selectivity - 2 -

3 1. Introduction Ephedrines (ephedrine, pseudoephedrine, and norephedrine) are sympathomimetic amines with central nervous system stimulating properties [1,2]. These substances are ingredients of many medicines commonly used in the treatment of flu, rhinitis, colds, and allergies [3]. Ephedrines share a chemical structure similar to amphetamines. Their structures differ from that of methamphetamine only by the substitution of a hydroxyl group for the hydrogen on the alpha-carbon atom. Because of their structural similarity, the misidentification as methamphetamine or amphetamine by immunoassays may be caused by the presence of ephedrines in the urine specimens [4,5]. The TDx/TDxFLx Amphetamine/Methamphetamine Ⅱ Assay fluorescence-polarization immunoassay (FPIA) used as an initial test also displays the cross-reactivity with ephedrines and other phenylisopropylamine derivatives that may cause false-positive results [6,7]. Several methods have been reported for the qualification or quantification of ephedrines, including high-performance liquid chromatography (HPLC) [8-10], gas chromatography (GC) [11,12], and gas chromatography-mass spectrometry (GC-MS) [5,13-15]. Due to specificity and sensitivity, most of the procedures devoted to drug detection in biological matrices are based on GC-MS. However the determination of ephedrines is not easy, because of the similar retention time of their derivatives in the gas chromatograph, as well as a similar mass fragmentation pattern. Therefore, a fast and reliable method is needed for the simultaneous quantification and confirmation of ephedrines. In this study, a selective, rapid, accurate GC-MS method with derivatization (O,Nmethylborated derivatives) was developed to confirm and quantify ephedrines in human - 3 -

4 urine. It is important to emphasize the following: (a) only one cyclic derivative was employed to form all the ephedrines; and (b) both a quadrupole (MS) and an ion-trap mass (MS-MS) spectrometer were applied to analyze the urine samples. 2. Experimental procedures 2.1. Chemicals Ephedrine, pseudoephedrine, and norephedrine were purchased from Cerilliant (Austin, TX, USA) in vials at a concentration of 1.0 mg/ml in methanol. Ephedrine-d 3 as their deuterated internal standard (IS) was purchased from Aldrich (Milwaukee, WI, USA). Diethyl ether and ethyl acetate were purchased from J. T. Baker (Phillipsburg, NJ, USA). All of the solvents were of HPLC grade. The derivatizing agent, methylboronic acid (MBA), was purchased from Fluka (Buchs, Switzerland) Preparation of solutions Working standard solutions (1 and 10 μg/ml) of ephedrine, pseudoephedrine, and norephedrine were prepared by appropriate dilution with methanol. A stock solution of ephedrine-d 3 was prepared by accurately weighing ~10 mg and dissolving in 10 ml. Subsequently, ephedrine-d 3 solutions were diluted with concentrations of 1, 10, and 100 μg/ml. All of these solutions were stored at -20 C in the absence of light until required Sample preparation - 4 -

5 Urine (1 ml) was submitted to a liquid liquid extraction 5ml of diethyl ether (twice), after adding 50 μl of methanolic solution of ephedrine-d 3 10 μg/ml internal standard solution, 1.0 ml of distilled water, and 0.2 ml of NaOH solution (2 M). Samples were mixed on a shaker for 15 min and centrifuged for 5 min (2800 g). The organic phase was transferred to a test tube ( mm) and evaporated until dry under a fine stream of nitrogen at 45 C. To the residue, a 50 μl mixture of MBA in ethyl acetate (1.0 mg/ml) was added and heated at 70 C for 15 min in a dry heating block. A 1 μl aliquot was injected into the GC-MS system Gas chromatography-mass spectrometry In the quantification study, the GC-MS system consisting of an Agilent Technologies (Palo Alto, CA, USA) 6890N GC coupled to a 5973 Network mass-selective detector (MSD) was used. The system was controlled by Drug Analysis Chemstation G1701CA software (C.00.00, Agilent Technologies). The gas chromatograph was equipped with a capillary column (DB-5MS, 25 m 0.25 mm i.d., 0.25 μm film thickness, J&W scientific, Folsom, CA, USA). Helium was used as the carrier gas at a flow rate of 0.8 ml/min. The GC temperature program was as follows: initial temperature of 120 C, was held for 1 min, increased to 255 C at a rate of 15 C/min, then to 290 C at a rate of 35 C/min, and then held for 1 min. The split ratio was 1:6, the injection temperature 250 C, and transfer line temperature was 280 C. The mass spectrometer was operated at 70 ev in the electron impact mode with selected ion monitoring (SIM) for the quantification. The selected ion groups of the derivatized analytes were monitored as follow: m/z 174, 189, and 132 (ephedrine and pseudoephedrine); m/z 160, 175, and 118 (norephedrine); and m/z 177 (ephedrine-d 3 )

6 The conditions of electron impact (EI) ionization and tandem mass spectrometer (MS-MS) with a ThermoFinnigan (San Jose, CA, USA) TRACE GC 2000 coupled to a ThermoFinnigan Polaris Q MS was employed to confirm the results. The gas chromatograph was equipped with a capillary column (DB-5MS, 25 m 0.25 mm i.d., 0.25 μm film thickness, J&W scientific, Folsom, CA, USA). The same GC temperature program above-noted was applied, and a split-injection mode with the split ratio (1:10) was used. The conditions for the MS-MS product ion mode applied are summarized in Table Validation of the analytical method The linearity of the method was checked in the concentration range ng/ml for ephedrine and pseudoephedrine and ng/ml for norephedrine. Using 1.0 ml samples of human urine spiked with methanolic solutions of ephedrine, pseudoephedrine, and norephedrine, a six-point calibration curve was established. The linear regression analysis was performed on the peak area ratios of analyte to the internal standard versus the analyte concentrations. For inter-day and intra-day validation, precision and accuracy were calculated for three concentrations corresponding to quality control (QC) samples of the method. To verify the method limit-of-detection (LOD) the signal-to-noise (S/N) ratio of the quantification ions was greater than the S/N ratio 3:1. Extraction recoveries of each analyte were quantified at three different concentrations (200, 500, and 1000 ng/ml) in five replicates each. 3. Results and discussion - 6 -

7 3.1. Chromatography and mass spectrometry Ephedrine, pseudoephedrine, and norephedrine were separated using a DB-5MS capillary column and a temperature-programmed GC. The methylborated ephedrines were identified by comparing the retention time with reference standards (Fig. 1). The SIM chromatograms for these drugs clearly indicated the order of elution. The methylborated norephedrine, pseudoephedrine, and ephedrine appear to have sharp and well-defined peaks at retention time of 4.50, 4.80, and 4.84 min, respectively. EI mass spectra obtained for the derivatives of (a) ephedrine and pseudoephedrine, (b) norephedrine, and (c) ephedrine-d 3 are shown in Fig. 2. The molecular ions, m/z 189 for ephedrine and pseudoephedrine and m/z 175 for norephedrine, were formed abundantly and also used as qualification ions Evaluation of validation data The described method was validated by evaluating selectivity, linearity, precision and accuracy, LOD, and recovery. Ephedrine, pseudoephedrine, and norephedrine are welldefined chromatographically after derivatization. Comparison of GC-MS SIM chromatograms for the blank urine sample and for a urine sample spiked with analyte and internal standard, no chemical interferences were observed from the biological matrix (Fig. 3). Table 2 presents the summarized quantitative parameters. Calibration standards at six concentration levels for each analyte were used to construct the calibration curves. Correlation coefficients were between and , indicating good linear regression. The limit of detection (LOD) was defined as the signal-to-noise ratio of 3 for - 7 -

8 each compound. The extraction recoveries of ephedrine, pseudoephedrine, and norephedrine were determined at three concentration levels in replicates of five. The peak-area ratios for each analyte were compared between those for the samples that had been spiked with analytes prior to extraction and samples to which the sample levels of analytes were added after extraction. The recoveries of the analytes were %. Table 3 presents the intra- and inter-day precision and accuracy of the method. Intraday precision and accuracy were obtained by analyzing five replicates using three different spiked-urine samples (200, 500, and 1000 ng/ml) of ephedrine, pseudoephedrine, and norephedrine added with 500 ng of ephedrine-d 3. For intra-day assays, the precision ranged from 0.9 to 8.6% for all analytes. To determine the precision, the coefficients of variations (% C.V.) were calculated for the replicate measurements. To obtain the inter-day precision and accuracy, the same spiked humanurine samples were analyzed on each of 3 days for human urine. For inter-day assays, the overall precision ranged from 1.6 to 13.7% for all analytes. Considering the complexity of the biological matrix, these results were regarded as satisfactory Separation of ephedrines from compounds that have a similar chemical structure The fluorescence-polarization immunoassay as a screening test was used for the rapid determination of amphetamine type stimulants in urine samples. The fluorescencepolarization immunoassay method displays cross-reactivities with ephedrines and phenylisopropylamine derivatives (methoxyphenamine, mexiletine, methylephedrine, phenmetrazine, phendimetrazine) that may cause false-positive results. False-positive cases have been reviewed from the urine samples of drug abusers in our laboratory. Among these cases, ephedrines cross-reacted most frequently with the immunoassay

9 Thus the described method was applied for the selective determination of ephedrines to the methanolic solution of eight drugs containing norephedrine, ephedrine, pseudoephedrine, methoxyphenamine, mexiletine, methylephedrine, phenmetrazine, and phendimetrazine. The methylborated derivatives of ephedrines demonstrated the followings: (a) a better separation due to less peak-tailing, especially in the case of norephedrine; (b) a shift of retention time in the case of ephedrine, pseudoephedrine, and norephedrine; and (c) a clear separation of methoxyphenamine. Fig. 4 shows representative GC-MS total ion chromatograms of before- and after-derivatization with MBA in ethyl acetate Application to urine of a ephedrine user The method was applied to analysis of a positive control urine sample obtained from a ephedrines user. Fig. 3 shows the SIM-chromatogram for detecting ephedrine, pseudoephedrine, and norephedrine in this case. The concentrations measured in the urine sample are 3040 ng/ml (ephedrine), 8800 ng/ml (pseudoephedrine), and 405 ng/ml (norephedrine]. When analyzing the MS-MS (ion trap) of the parent ion in the product ion-scan mode, one can effectively check for the presence of the above-mentioned analytes found in the GC-MS-MS chromatogram. 4. Conclusions The mass spectral identification of ephedrines using methylboronic acid in ethyl acetate as a derivatizing mixture has many advantages. It is a convenient, quick, - 9 -

10 selective, and unmistakable way to confirm ephedrines in human urine. Methylboronate derivatives can clearly separate ephedrines from adjacent and other sympathomimetic amines on the GC chromatogram using cyclic derivatives. The method has been validated and effectively applied to urine sample collected from ephedrine user

11 References [1] P.J. van der Merwe, S.E. Hendrikz, J. Chromatogr. B. 663 (1995) 160. [2] J. Dickerson, D. Perrier, M. Mayersohn, R. Bressler, Eur. J. Clin. Pharmacol. 14 (1978) 253. [3] G. Joseph, J. Chromatogr. 307 (1984) 220. [4] C.L. Hornbeck, J.E. Carrig, R.J. Czarny, J. Anal. Toxicol. 17 (1993) 257. [5] A. Dasgupta, C. Gardner, J. Forensic Sci. 40 (1995) [6] J. D'Nicuola, R. Jones, B. Levine, M. L. Smith, J. Anal. Toxicol. 16 (1992) 211. [7] Amphetamine/Methamphetamine Ⅱ. In: List No. 1A99, /R2, Abbott Park, IL: Abbott Laboratories, 2001, p. 1. [8] D. J. Hood, H. Y. Cheung, J. Pharm. Biomed. Anal. 30 (2003) [9] G. Gmeiner, T. Geisendorfer, J. Kainzbauer, M. Nikolajevic, H. Tausch, J. Chromatogr. B. 768 (2002) 215. [10] Y. Makino, Y. Urano, T. Nagano, J. Chromatogr. A. 947 (2002) 151. [11] P. Van Eenoo, F. T. Delbeke, K. Roels, P. De Backer, J. Chromatogr. B. 760 (2001) 255. [12] J. Jonsson, R. Kronstrand, M. Hatanpaa, J. Forensic Sci. 41 (1996) 148. [13] M. E. Spyridaki, C. J. Tsitsimpikou, P. A. Siskos, C. G. Georgakopoulos, J. Chromatogr. B. 758 (2001) 311. [14] G. Aymard, B. Labarthe, D. Warot, I. Berlin, B. Diquet, J. Chromatogr. B. 744 (2000) 25. [15] B. M. El-Haj, A. M. Al-Amri, M. H. Hassan, H. S. Ali, R. K. Bin Khadem, Forensic Sci. Int. 135 (2003)

12 Table 1 Mass spectrometric conditions of the GC-MS-MS method Source temperature ( C) 150 Start time (min) 4.0 Scan mode MS-MS; product ion mode Precursor ion (m/z) 174 (ephedrine and pseudoephedrine) 160 (norephedrine) 177 (ephedrine-d 3 ) Width (m/z) 1 Excitation voltage 1 Product ions (m/z) (ephedrine and pseudoephedrine) (norephedrine) (ephedrine-d 3 ) - 12-

13 Table 2 Validation data for the analysis of ephedrines in human urine Ephedrine Pseudoephedrine Norephedrine Concentration range (ng/ml) Linearity a LOD b (ng/ml) Recovery (% C.V., n = 5) 200 ng/ml 87.7 ± ± ± ng/ml 92.3 ± ± ± ng/ml 95.4 ± ± ± 7.2 a Linearity is described by the correlation coefficients for the calibration curve b Limit of detection (LOD) was defined as the concentration at which the characteristic ion was detectable on the corresponding mass chromatogram at signal-to-noise (S/N) =3 or greater - 13-

14 Table 3 Precision a and accuracy b for determining ephedrine and related compounds Drug Added amount (ng/ml) Mean (ng/ml) Intra-day (n=3) Accuracy (% bias) Precision (% C.V.) Mean (ng/ml) Inter-day (n=3) Accuracy (% bias) Precision (% C.V.) Epedrine Pseudoephedrine Norephedrine a Expressed as coefficient of variance of the peak area ratios of analyte/internal standard. b Calculated as [(mean calculated concentration nominal concentration)/nominal concentration]

15 Figure legends Fig. 1. Chemical structure of ephedrines: Ephedrine (A), pseudoephedrine (B), and norephedrine (C). Fig. 2. EI spectra of ephedrines derivatized with methaneboronate: Ephedrine and pseudoephedrine (A), norephedrine (B), and ephedrine-d 3 (C). Fig. 3. SIM GC-MS determination of ephedrines in human urine: Extract from blank urine (A) and Extract from amphetamine/methamphetamine false-positive human urine (B). Fig. 4. SIM GC-MS chromatograms of eight compounds (norephedrine, ephedrine, pseudoephedrine, methoxyphenamine, mexiletine, methylephedrine, phenmetrazine, and phendimetrazine) before (A) and after derivatization with MBA in ethyl acetate (B). The acquisition was made by using 50 μl of a standard mixture (100 ppm) containing eight compounds in Full-Scan mode (m/z ). The arrow in the panel (B) indicates norephedrine elutes before the derivatization

16 H OH H CH OH CH N CH CH N CH OH CH NH 2 A B C Fig

17 Abundance (A) 95 O B 90 N m/z (B) O B 95 NH m/z (C) O B N CD m/z Fig

18 (A) 1 Norephedrine 2 Pseudoephedrine 3 Ephedrine IS Relative Abundance m/z 174 m/z IS 30 Relative Abundance (B) & IS NL: m/z Relative Abundance m/z 177 IS Time (min) Time (min) Fig

19 (A) (a) 1 Norephedrine 2,3,4 2 Ephedrine Pseudoephedrine 4 Methoxyphenamine Mexiletine 6 Methylephedrine 7 Phenmetrazine 8 Phendimetrazine Abundance (B) (b) 1 2, Methylborated norephedrine 2 Methylborated pseudoephedrine 3 Methylborated ephedrine 4 Methoxyphenamine 5 Mexiletine 6 Methylephedrine 7 Phenmetrazine 8 Phendimetrazine Time (min) Fig