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ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i891 2001 © The Japan Society for Analytical Chemistry

Analytical Examination of ß2-Agonists by Gas Chromatography-Mass Spectrometry

Rika NAKAJIMA, 1† Tatsuo SHINOZUKA, 1 Setsuko TAKEI,1 Osamu OHUE,1 Tatsuya MURAI, 1 and Masaru TERADA 2

1† Department of Legal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160- 8582, Japan (E-mail: [email protected]) 2 Department of Legal Medicine, Course of Social Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

This study presents a simple and fast method for the analysis of ß2- agonists in authentic samples using gas chromatography-mass spectrometry. It also includes the suggestion of the most suitable condition for the sample pre- treatment and the best derivatizing reagents. We established that five ß2-adrenergic agonists showed the best performance of separation was achieved when the residue were pre-treated by solid-phase extraction with Oasis® MCX, to which BSTFA (1% TMCS) and ethyl acetate were added for the derivatization at 70 ˚C for 15 min. We believe that this newly developed method may simplify the analysis of ß2-adrenergic agonists and will be of great value in the field of forensic toxicology.

(Received on August 9, Accepted on September 13, 2001)

Salbutamol, , , and cation exchange) and Oasis® HLB (hydrophilic-lipophilic are classified into ß2-adrenergic agonists. ß2- balance) cartridges were obtained from Waters (Milford, MA, adrenergic agonists are the potent and are widely USA). All other chemicals were of analytical grade. We used used in the treatment of bronchial . Meanwhile the the plasma samples collected from the young and healthy adults. number of poisoning cases that can be lethal has been recently on the increase in Japan. Although numerous studies for the Gas Chromatography-mass spectrometry (GC/MS) quantitative analysis of these agonists have been reported from GC/MS analyses were performed by using a M-9000 overseas1-9, none of them has been established in Japan. GC/3DQMS system (Hitachi, Tokyo, Japan). A capillary In this study, we searched for the most suitable measure for column (TC-1, 30 m×0.25 mm I.D., 0.25 µm film thickness) the pre-treatment of ß2-adrenergic agonists using solid-phase was used for separation. The column temperature was initially ® ® extraction by Oasis MCX (for the basic substances) and Oasis maintained at 120 ˚C for 2 min, then raised at 15 ˚C/min to 300 HLB (for both acidic and basic substances), and for the best ˚C to be held for 10 min. Carrier gas was helium at the flow-rate condition applied in the process of derivatization. Gas of 1.5 ml/min. The injector port and transfer line temperatures chromatography-mass spectrometry (GC/MS) was used for were kept at 230 ˚C and that of the ion source at 250 ˚C. Mass identification and quantification. spectra were obtained in the mass range 40 - 600 amu.

Sample preparation Experimental Plasma samples (0.5 ml) were spiked with 0.5–1 µg of ß2- adrenergic agonists, and added to 0.5 µg of clenbuterol as internal standard (i.s.), 1 ml of 10 mM phosphate buffer (pH 7.0) Chemicals and materials and 1.5 ml of distilled water. , terbutaline, fenoterol, orciprenaline (metaproterenol) and clenbuterol were purchased from Sigma (St. Extraction details Louis, MO, USA). Isoprenaline from Aldrich Chemical Solid-phase extractions of ß2-adrenergic agonists were Company (Milwaukee, WI, USA). N,O-bis (trimethylsilyl) performed on Oasis® MCX and HLB cartridges. The cartridges trifluoroacetamide (BSTFA) obtained from Pierce (Rockford, IL, were pre-conditioned with 3 ml of methanol and 3 ml of water. USA). Trimethylchlorosilane (TMCS) from Sigma (St.Louis, The spiked sample was then applied to the cartridge. The MO, USA). N-methyl-N-trimethylsilyl-trifluoroacetamide cartridge was washed with 3 ml of 0.1N hydrochloric acid and (MSTFA) and phenyltrimethylammonium hydroxide (PTAH) methanol, and eluted with 3 ml of 5% ammonium hydroxide in from Tokyo Kasei Kogyo (Tokyo, Japan). methanol (Oasis® MCX cartridge), and was washed with 3 ml of Pentafluoropropionic anhydride (PFPA) from Nacalai Tesque 5% methanol in water and eluted with 3 ml of methanol (Oasis® (Kyoto, Japan). Sep-Pak® C18, Oasis® MCX (mixed-mode

i892 ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT

HLB cartridge). The eluant was evaporated to dryness under a no reaction was observed when any other reagents but BSTFA stream of nitrogen at 40 ˚C. was used for the derivatization of ß2-adrenergic agonists.

Derivatization The residue was added to 15 µl of BSTFA (1% TMCS) and 60 µl of ethyl acetate. The mixture to react was vortexed and heated at 70 ˚C for 15 min. After cooling, the solution was evaporated to dryness under a nitrogen stream at 40 ˚C. The residue was dissolved in 100 µl of ethyl acetate and 1 µl was injected into the GC/MS.

Results and Discussion

Recovery from extraction The recoveries of several extraction procedures were compared; liquid-liquid extraction with ethyl acetate and chloroform and solid-phase extraction with Sep-Pak® C18 cartridge both showed poor recovery of ß2-adrenergic agonists. Relatively high and stable recovery was obtained by solid-phase extraction using Oasis® MCX and HLB cartridges (Tables 1 and 2). We, therefore, used the ß2-adrenergic agonists extracted Fig. 1 Derivative scheme for the silylation of salbutamol to by Oasis® MCX and HLB for the selection of the most suitable TMS-salbutamol. reagent for the derivatization. The detection limits of salbutamol, terbutaline, orciprenaline and isoprenaline were 0.5 ng, that of fenoterol was 5 ng. Recovery from extraction was GC/MS analysis calculated and shown in the ratio of concentration of ß2- Interference peaks that influence on total ion chromatogram of to that of internal standard, clenbuterol. GC/MS were more ofen seen when the residue to be derivatized was the extract from Oasis® HLB than it was from Oasis® MCX. ® Table 1 Recovery of ß2-adrenergic agonists by Oasis In Fig. 2 and 3 respectively, the total ion chromatograms and MCX cartridges (n=5) full-scan mass spectra for five ß2-adrenergic agonists and internal standard after extracted from spiked plasma sample by Oasis®MCX Oasis® MCX and derivatized by BSTFA were illustrated. Drug added Mean RSD (%) Retention time of isoprenaline, orciprenaline, terbutaline, (µg/ml) internal standard (clenbuterol), salbutamol and fenoterol was Salbutamol 1.0 83.87 3.33 approximately 8.75, 8.85, 8.98, 9.09, 9.45 and 13.36 min, Terbutaline 1.0 104.01 8.94 respectively. The peak regularly appearing at 12.20 min is the Fenoterol 5.0 75.30 11.51 peak derived from the solid-phase cartridge. Orciprenaline 1.0 93.75 10.60 Isoprenaline 1.0 79.18 8.89

® Table 2 Recovery of ß2-adrenergic agonists by Oasis HLB cartridges (n=5)

® Oasis HLB Drug added Mean RSD (%) (µg/ml) Salbutamol 1.0 87.95 11.77 Terbutaline 1.0 77.62 10.55 Fenoterol 5.0 87.77 7.05 Orciprenaline 1.0 83.27 12.61

Isoprenaline 1.0 107.17 10.11

Derivatization To select the best condition for the derivatization of ß2- adrenergic agonists, we conducted experiments using various derivatizing reagents (silylation with BSTFA and MSTFA, acylation with PFPA, and methylation with PTAH) under different environment: (1) concentration (1% and 50% for TMCS, 50mM and 100mM for PTAH), (2) temperature (the room temperature, 70˚C, 80˚C), and (3) reaction time (15min, 30min). ß2-adrenergic agonists showed the best performance of separation when the residue was added to BSTFA (1% TMCS) Fig. 2 Total ion chromatograms of the TMS derivatives of and ethyl acetate at 70 ˚C for 15 min (Fig. 1). Except for isoprenaline (peak1), orciprenaline (peak2), terbutaline (peak3), clenbuterol, which was derivatized by any reagent listed above, salbutamol (peak4) and fenoterol (peak5) along with clenbuterol as internal standard (i.s.).

ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT i893

Numerous analytical studies for several extraction methods and derivatizing reagents of ß2-adrenergic agonists have been reported.1-9 For those studies, we examined the methods to use ® the ß2-adrenergic agonists extracted from plasma by Oasis MCX and HLB as the pre-treatment, for the selection of the most suitable reagent for the derivatization. In this experiment, different five kinds of ß2-adrenergic agonists showed that the best performance of separation was achieved when the residues were pre-treated by solid-phase extraction with Oasis® MCX, to which BSTFA (1% TMCS) and ethyl acetate were added for the derivatization at 70˚C for 15 min. We established the GC/MS method to analyze five kinds of ® ß2-adrenergic agonists by using Oasis MCX based on this result.

References

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Fig. 3 Full-scan mass spectra of salbutamol (A), terbutaline (B), fenoterol (C), orciprenaline (D), isoprenaline (E) and clenbuterol (F) as internal standard (all as TMS derivatives).