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. 2006 Nov 22;8(4):E709–E717. doi: 10.1208/aapsj080480

Quantitative determination of total methamphetamine and active metabolites in rat tissue by liquid chromatography with tandem mass spectrometric detection

Howard Hendrickson 1,✉, Elizabeth Laurenzana 2, S Michael Owens 2
PMCID: PMC2751367  PMID: 17233534

Abstract

High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng·mL−1 using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3–1000 ng·mL−1). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg−1 day−1) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.

Keywords: methamphetamine, rats, LC-MS/MS, matrix ion effects

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References

  • 1.Rawson RA, Marinelli-Casey P, Anglin MD, et al. A multi-site comparison of psychosocial approaches for the treatment of methamphetamine dependence. Addiction. 2004;99:708–717. doi: 10.1111/j.1360-0443.2004.00707.x. [DOI] [PubMed] [Google Scholar]
  • 2.Greenwald MK, Schuh KJ, Hopper JA, Schuster CR, Johanson CE. Effects of buprenorphine sublingual tablet maintenance on opioid drugseeking behavior by humans. Psychopharmacology (Berl) 2002;160:344–352. doi: 10.1007/s00213-001-0975-0. [DOI] [PubMed] [Google Scholar]
  • 3.Harrod SB, Dwoskin LP, Crooks PA, Klebaur JE, Bardo MT. Lobeline attenuates d-methamphetamine self-administration in rats. J Pharmacol Exp Ther. 2001;298:172–179. [PubMed] [Google Scholar]
  • 4.Ginawi OT, Al-Majed AA, Al-Suwailem AK. NAN-190, a possible specific antagonist for methamphetamine. Regul Toxicol Pharmacol. 2005;41:122–127. doi: 10.1016/j.yrtph.2004.10.005. [DOI] [PubMed] [Google Scholar]
  • 5.Sattar SP, Bhatia SC, Petty F. Potential benefits of quetiapine in the treatment of substance dependence disorders. J Psychiatry Neurosci. 2004;29:452–457. [PMC free article] [PubMed] [Google Scholar]
  • 6.Johnson BA, Roache JD, Ait-Daoud N, Wallace C, Wells LT, Wang Y. Effects of isradipine on methamphetamine-induced changes in attentional and perceptual-motor skills of cognition. Psychopharmacology (Berl) 2005;178:296–302. doi: 10.1007/s00213-004-1998-0. [DOI] [PubMed] [Google Scholar]
  • 7.Laurenzana EM, Byrnes-Blake KA, Milesi-Halle A, Gentry WB, Williams DK, Owens SM. Use of anti-(+)-methamphetamine monoclonal antibody to significantly alter (+)-methamphetamine and (+)-amphetamine disposition in rats. Drug Metab Dispos. 2003;31:1320–1326. doi: 10.1124/dmd.31.11.1320. [DOI] [PubMed] [Google Scholar]
  • 8.Byrnes-Blake KA, Laurenzana EM, Carroll FI, et al. Phamacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats. Eur J Pharmacol. 2003;461:119–128. doi: 10.1016/S0014-2999(03)01313-X. [DOI] [PubMed] [Google Scholar]
  • 9.Byrnes-Blake KA, Carroll FI, Abraham P, Owens SM. Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats. Int Immunopharmacol. 2001;1:329–338. doi: 10.1016/S1567-5769(00)00019-9. [DOI] [PubMed] [Google Scholar]
  • 10.Byrnes-Blake KA, Laurenzana EM, Landes RD, Gentry WB, Owens SM. Monoclonal IgG affinity and treatment time alters antagonism of (+)-methamphetamine effects in rats. Eur J Pharmacol. 2005;521:86–94. doi: 10.1016/j.ejphar.2005.08.016. [DOI] [PubMed] [Google Scholar]
  • 11.Kosten T, Owens SM. Immunotherapy for the treatment of drug abuse. Pharmacol Ther. 2005;108:76–85. doi: 10.1016/j.pharmthera.2005.06.009. [DOI] [PubMed] [Google Scholar]
  • 12.Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324:14–30. doi: 10.1097/00000441-200207000-00004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Haney M, Kosten TR. Therapeutic vaccines for substance dependence. Expert Rev Vaccines. 2004;3:11–18. doi: 10.1586/14760584.3.1.11. [DOI] [PubMed] [Google Scholar]
  • 14.Zernig G, Giacomuzzi S, Riemer Y, Wakonigg G, Sturm K, Saria A. Intravenous drug injection habits: drug users' self-reports versus researchers' perception. Pharmacology. 2003;68:49–56. doi: 10.1159/000068731. [DOI] [PubMed] [Google Scholar]
  • 15.Schiffer WK, Lee DE, Brodie JD, Dewey SL. Imaging addiction with PET: is insight in sight? Drug Discov Today. 2005;10:547–562. doi: 10.1016/S1359-6446(05)03412-4. [DOI] [PubMed] [Google Scholar]
  • 16.Nakamura H, Hishinuma T, Tomioka Y, et al. Positron emission tomography study of the alterations in brain distribution of [11C]methamphetamine in methamphetamine-sensitized dog. Ann N Y Acad Sci. 1996;801:401–408. doi: 10.1111/j.1749-6632.1996.tb17462.x. [DOI] [PubMed] [Google Scholar]
  • 17.Volkow ND, Fowler JS, Ding YS, Wang GJ, Gatley SJ. Imaging the neurochemistry of nicotine actions: studies with positron emission tomography. Nicotine Tob Res. 1999;1:127–128. doi: 10.1080/14622299050011941. [DOI] [PubMed] [Google Scholar]
  • 18.Volkow ND, Fowler JS, Wang GJ. Imaging studies on the role of dopamine in cocaine reinforcement and addiction in humans. J Psychopharmacol. 1999;13:337–345. doi: 10.1177/026988119901300406. [DOI] [PubMed] [Google Scholar]
  • 19.Riviere GJ, Gentry WB, Owens SM. Disposition of methamphetamine and its metabolite amphetamine in brain and other tissues in rats after intravenous administration. J Pharmacol Exp Ther. 2000;292:1042–1047. [PubMed] [Google Scholar]
  • 20.Wessinger WD, Owens SM. Chronic administration of phencyclidine: pharmacokinetic comparison of intravenous and subcutaneous infusions in Sprague-Dawley rats. Drug Metab Dispos. 1991;19:719–721. [PubMed] [Google Scholar]
  • 21.Papac DI, Shahrokh Z. Mass spectrometry innovations in drug discovery and development. Pharm Res. 2001;18:131–145. doi: 10.1023/A:1011049231231. [DOI] [PubMed] [Google Scholar]
  • 22.Mallet CR, Lu Z, Mazzeo JR. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts. Rapid Commun Mass Spectrom. 2004;18:49–58. doi: 10.1002/rcm.1276. [DOI] [PubMed] [Google Scholar]
  • 23.Fiori M, Civitareale C, Mirante S, Magaro E, Brambilla G. Evaluation of two different clean-up steps, to minimize ion suppression phenomena in ion trap liquid chromatography-tandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine. Anal Chim Acta. 2005;529:207–210. doi: 10.1016/j.aca.2004.08.068. [DOI] [Google Scholar]
  • 24.Dunn-Meynell KW, Wainhaus S, Korfnacher WA. Optimizing an ultrafast generic high-performance liquid chromatography/tandem mass spectrometry method for faster discovery pharmacokinetic sample throughput. Rapid Commun Mass Spectrom. 2005;19:2905–2910. doi: 10.1002/rcm.2147. [DOI] [PubMed] [Google Scholar]
  • 25.Villa JS, Cass RT, Karr DE, Adams SM, Shaw JP, Schmidt DE. Increasing the efficiency of pharmacokinetic sample procurement, preparation and analysis by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2004;18:1066–1072. doi: 10.1002/rcm.1447. [DOI] [PubMed] [Google Scholar]
  • 26.Tang L, Kebarle P. Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution. Anal Chem. 1993;65:3654–3668. doi: 10.1021/ac00072a020. [DOI] [Google Scholar]
  • 27.Kebarle P, Tang L. From ions in solution to ions in the gas phase— the mechanism of electrospray mass spectrometry. Anal Chem. 1993;65:972A–986A. doi: 10.1021/ac00070a001. [DOI] [Google Scholar]
  • 28.King R, Bonfiglio R, Fernandez-Metzler C, Miller-Stein C, Olah T. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom. 2000;11:942–950. doi: 10.1016/S1044-0305(00)00163-X. [DOI] [PubMed] [Google Scholar]
  • 29.Hayen H, Karst U. Strategies for the liquid chromatographic-mass spectrometric analysis of non-polar compounds. J Chromatogr A. 2003;1000:549–565. doi: 10.1016/S0021-9673(03)00505-3. [DOI] [PubMed] [Google Scholar]
  • 30.Hori Y, Fujisawa M, Shimada K, Hirose Y, Yoshioka T. Method for screening and quantitative determination of serum levels of salicylic acid, acetaminophen, theophylline, phenobarbital, brom valerylurea, pentobarbital, and amobarbital using liquid chromatography/ electrospray mass spectrometry. Biol Pharm Bull. 2006;29:7–13. doi: 10.1248/bpb.29.7. [DOI] [PubMed] [Google Scholar]
  • 31.Kahlich R, Gleiter CH, Laufer S, Kammerer B. Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:275–283. doi: 10.1002/rcm.2257. [DOI] [PubMed] [Google Scholar]
  • 32.Bhatt J, Jangid A, Shetty R, et al. Rapid and sensitive liquid chromatography-mass spectrometry method for determination of ropinirole in human plasma. J Pharm Biomed Anal. 2006;40:1202–1208. doi: 10.1016/j.jpba.2005.09.024. [DOI] [PubMed] [Google Scholar]
  • 33.Yulan S, Fang F. Sensitive liquid chromatography-tamdem mass spectrometry method for the determination of scutellarin in human plasma: application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830:1–5. doi: 10.1016/j.jchromb.2005.10.016. [DOI] [PubMed] [Google Scholar]
  • 34.Zang X, Luo R, Song N, Chen TK, Bozigian H. A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites. Rapid Commun Mass Spectrom. 2005;19:3259–3268. doi: 10.1002/rcm.2188. [DOI] [PubMed] [Google Scholar]
  • 35.Rook EJ, Hillebrand MJ, Rosing H, van Ree JM, Beijnen JH. The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;824:213–221. doi: 10.1016/j.jchromb.2005.05.048. [DOI] [PubMed] [Google Scholar]
  • 36.Nordgren HK, Holmgren P, Liljeberg P, Eriksson N, Beck O. Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers. J Anal Toxicol. 2005;29:234–239. doi: 10.1093/jat/29.4.234. [DOI] [PubMed] [Google Scholar]
  • 37.Wood M, Laloup M, Ramirez Fernandez Mdel M, et al. Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography-tandem mass spectrometry. Forensic Sci Int. 2005;150:227–238. doi: 10.1016/j.forsciint.2004.11.027. [DOI] [PubMed] [Google Scholar]
  • 38.Li M, Alnouti Y, Leverence R, Bi H, Gusev AI. Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;825:152–160. doi: 10.1016/j.jchromb.2005.03.047. [DOI] [PubMed] [Google Scholar]
  • 39.Rodriguez-Rosas ME, Medrano JG, Epstein DH, Moolchan ET, Preston KL, Wainer IW. Determination of total and free concentrations of the enantiomers of methadone and its metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine) in human plasma by enantioselective liquid chromatography with mass spectrom etric detection. J Chromatogr A. 2005;1073:237–248. doi: 10.1016/j.chroma.2004.08.153. [DOI] [PubMed] [Google Scholar]
  • 40.Heavner DL, Richardson JD, Morgan WT, Ogden MW. Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry. Biomed Chromatogr. 2005;19:312–328. doi: 10.1002/bmc.463. [DOI] [PubMed] [Google Scholar]
  • 41.Bi HC, Zhong GP, Zhou S, Chen X, Huang M. Determination of adefovir in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study. Rapid Commun Mass Spectrom. 2005;19:2911–2917. doi: 10.1002/rcm.2141. [DOI] [PubMed] [Google Scholar]
  • 42.Proenca P, Teixeira H, Castanheira F, et al. Two fatal intoxication cases with imidacloprid: LC/MS analysis. Forensic Sci Int. 2005;153:75–80. doi: 10.1016/j.forsciint.2005.04.015. [DOI] [PubMed] [Google Scholar]
  • 43.Yun JH, Myung JH, Kim HJ, et al. LC-MS determination and bioavailability study of imidapril hydrochloride after the oral administration of imidapril tablets in human volunteers. Arch Pharm Res. 2005;28:463–468. doi: 10.1007/BF02977677. [DOI] [PubMed] [Google Scholar]
  • 44.Li K, Chen X, Xu J, Li X, Zhong D. Liquid chromatography/tandem mass spectrometry for pharmacokinetic studies of 20(R)-ginsenoside Rg3 in dog. Rapid Commun Mass Spectrom. 2005;19:813–817. doi: 10.1002/rcm.1862. [DOI] [PubMed] [Google Scholar]
  • 45.Kousoulos C, Tsatsou G, Apostolon C, Dotsikas Y, Loukas YL. Development of a high-throughput method for the determination of itraconazole and its hydroxy metabolite in human plasma, employing automated liquid-liquid extraction based on 96-well format plates and LC/MS/MS. Anal Bioanal Chem. 2005;384:199–207. doi: 10.1007/s00216-005-0159-6. [DOI] [PubMed] [Google Scholar]
  • 46.Zhang N, Yang A, Rogers JD, Zhao JJ. Quantitative analysis of simvastatin and its β-hydroxy acid in human plasma using automated liquid-liquid extraction based on 96-well plate format and liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2004;34:175–187. doi: 10.1016/j.japna.2003.08.016. [DOI] [PubMed] [Google Scholar]
  • 47.Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R. Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Techmol Biomed Life Sci. 2003;785:263–275. doi: 10.1016/S1570-0232(02)00914-5. [DOI] [PubMed] [Google Scholar]
  • 48.Hendrickson HP, Milesi-Halle A, Laurenzana EM, Owens SM. Development of a liquid chromatography-tandem mass spectrometric method for the determination of methamphetamine and amphetamine using small volumes of rat serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;806:81–87. doi: 10.1016/j.jchromb.2004.03.038. [DOI] [PubMed] [Google Scholar]
  • 49.Matuszewski BK. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. J Chromatogr B. 2006;830:293–300. doi: 10.1016/j.jchromb.2005.11.009. [DOI] [PubMed] [Google Scholar]
  • 50.Shima N, Kamata HT, Katagi M, Tsuchihashi H. Urinary excretion of the main metabolites of methamphetamine, including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide, in humans and rats. Xenobiotica. 2006;36:259–267. doi: 10.1080/00498250600627475. [DOI] [PubMed] [Google Scholar]
  • 51.Fielding J, Ryall RG. Some characteristics of trichloroacetic acidprecipitated proteins and their effects on biochemical assay. Clin Chim Acta. 1971;33:235–240. doi: 10.1016/0009-8981(71)90272-5. [DOI] [PubMed] [Google Scholar]

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