Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 48: 1715-1719, 2002;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toennes, S. W.
Right arrow Articles by Kauert, G. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toennes, S. W.
Right arrow Articles by Kauert, G. F.
Related Collections
Right arrow Drug Monitoring and Toxicology
Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2002;48:1715-1719.)
© 2002 American Association for Clinical Chemistry, Inc.

Excretion and Detection of Cathinone, Cathine, and Phenylpropanolamine in Urine after Kath Chewing

Stefan W. Toennes1a and Gerold F. Kauert1

1 Institute of Forensic Toxicology, Center of Legal Medicine, University of Frankfurt/Main, Kennedyallee 104, D-60596 Frankfurt/Main, Germany.

aAuthor for correspondence. Fax 49-69-6301-7531; e-mail toennes{at}em.uni-frankfurt.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Introduction: The stimulating herbal drug kath is uncommon in most countries, and information on its detection and interpretation of analytical results is limited. Therefore, a study with kath was carried out to compare the efficiencies of different analytical techniques used to detect drug use.

Methods: Four volunteers chewed kath leaves for 1 h; urine samples were collected up to 80 h afterward and analyzed by the Abbott fluorescence polarization immunoassay (FPIA), the Mahsan-AMP300 on-site immunoassay, the Bio-Rad Remedi HS HPLC system with photodiode array detection (DAD), and gas chromatography–mass spectrometry (GC-MS).

Results: FPIA gave negative results, whereas positive results were obtained with the Mahsan test during the first day. With HPLC, one peak could be observed up to 50 h, but its DAD spectrum could not be identified by the system. Further investigations indicated that the kath alkaloids coeluted and produced a mixed DAD spectrum. With GC-MS, the specific kath ingredient cathinone was detected up to 26 h, whereas cathine and norephedrine were still detectable in the last samples. Maximum concentrations of cathinone, cathine, and norephedrine in urine samples from the study were 2.5, 20, and 30 mg/L, respectively, whereas in authentic cases the concentrations were much higher.

Conclusion: GC-MS is superior to the screening techniques Mahsan-AMP300 and Remedi with respect to specificity and sensitivity for the detection of kath use in urine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The psychostimulating herbal drug kath (Catha edulis Forsk.) is cultivated and used as a recreational drug predominantly in East Africa and the Arabian Peninsula. Emigrants from these countries try to maintain this habit (1)(2), and large quantities of fresh kath are imported into other world areas (3)(4)(5)(6). For example, >2300 kg of kath were confiscated at the Frankfurt airport (Germany) in 1998 (personal communication from German customs authorities).

The main psychoactive alkaloid of kath is the phenylpropylamine derivative S-(-)-{alpha}-aminopropiophenone (cathinone), which is accompanied by the less psychoactive phenylpropanolamine diastereomers S,S-(+)-norpseudoephedrine (cathine) and R,S-(-)-norephedrine (7)(8). The detection of these alkaloids in urine has been performed by thin-layer chromatography, HPLC, gas chromatography, and gas chromatography–mass spectrometry (GC-MS)1 (9)(10)(11)(12)(13)(14), but the usability of the results in routine toxicologic analysis is limited. Furthermore, in only one study from 1975 (13) was authentic kath material used at a time when cathinone, the principle active component of kath, was not known.

Because kath is not common outside of Africa and Arabia, this report examined which analytical methods would be most appropriate for screening and confirmation testing of urine samples from individuals with suspected kath use. This was accomplished by analyzing urine specimens from four volunteers, who chewed authentic kath leaves, by immunoassay, HPLC, and GC-MS. The developed methodology was subsequently used to investigate forensic specimens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
reagents, calibrators, and instrumentation
The reference standards, S-(-)-cathinone and (±)-phenylpropanolamine (norephedrine), and the internal standard, (±)-3,4-methylenedioxyamphetamine-d5 (MDA-d5), were purchased from Radian (Promochem), and D-norpseudoephedrine (cathine) was from Heinrich Mack Nachf. GmbH. The derivatization reagent N-methyl-bis(heptafluorobutyramide) (MBHFBA) was from Macherey & Nagel. All other reagents and organic solvents were of analytical grade and were from Merck.

study design
Four healthy, non-drug-using volunteers chewed a total of 0.6 g of authentic kath leaves per kg of body weight continuously for 1 h. The kath material had been confiscated at Frankfurt airport and was stored at -20 °C until use. According to the method of Widler et al. (15), the cathinone, cathine, and norephedrine content in the leaves was 1.14, 0.83, and 0.44 mg/g of kath, respectively. After chewing, each volunteer spit out the residual material, which was analyzed for its alkaloid content. A blank urine sample was obtained from each study participant before the chewing, and spontaneous urine was collected up to 80 h after ingestion; for each sample the total volume was determined and an aliquot was stored at -20 °C for toxicologic analysis. The study protocol was approved by the Ethical Committee of the University of Frankfurt/Main (Germany), and written informed consent was obtained from the participants.

immunologic screening for kath alkaloids in urine
Immunologic screening of the urine samples was performed with two different immunoassays: the automated AxSYM analyzer (Abbott) with the Abbott fluorescence polarization immunoassay (FPIA) Amphetamine/Methamphetamine II and the Mahsan-AMP300 on-site test (Mahsan Diagnostika). The tests were carried out according to the recommendations of the manufacturers.

screening of urine with the Remedi HS HPLC SYSTEM
All urine samples were screened using the Remedi HS automated HPLC analyzer (Bio-Rad). According to the recommendations of the manufacturer, 1 mL of urine was mixed with 0.2 mL of "internal standard combination" (a defined solution of ethyldiazepam and chlorpheniramine). The fully automated analysis consisted of a column-switching system that provided extraction and chromatographic separation, photodiode array detection (DAD), and a computer library search report of the recorded spectra.

determination of cathinone, cathine, and norephedrine in urine by gc-ms
Urine samples were prepared using a standard procedure for basic drugs (16). An aliquot of 0.2 mL or less, if necessary, was diluted with 4 mL of 0.1 mol/L phosphate buffer, pH 6.0, and 100 µL of internal standard solution (1 mg/L MDA-d5 in methanol) was added and vortex-mixed. The diluted samples were extracted using 3-mL Bond Elut Certify HF 300-mg solid-phase extraction cartridges (Varian) with the extraction robot RapidTrace (Zymark). The extraction protocol was as follows: conditioning with 2 mL of methanol and 3 mL of phosphate buffer, application of the sample on the column at 1 mL/min, rinsing with 2 mL of 0.1 mol/L acetic acid and 3 mL of methanol at 1.5 mL/min, elution of the analytes with 3 mL of a freshly prepared solution of methylene chloride–2-propanol–ammonia (80:20:2 by volume) at 1 mL/min.

The extracts were evaporated to dryness and derivatized with 40 µL of MBHFBA for 30 min at 60 °C. Analysis of 1-µL aliquots of these solutions was performed on a Hewlett Packard (HP) GC-MS system (HP 5890 Series II GC, HP 6890 ALS, HP 5972 MSD) with a HP-1 MS capillary column [30 m x 0.25 mm (i.d.); 0.25-µm film thickness]. The carrier gas was helium with a flow rate of 1.0 mL/min. The GC conditions were as follows: injection port temperature, 260 °C; temperature program, 120 °C for 0.5 min, increase of 10 °C/min to 170 °C, increase of 30 °C/min to 310 °C, and hold for 5 min. The MS conditions were as follows: transfer line temperature, 280 °C; ionization energy, 70 eV. Data analysis was performed on a Windows computer with HP ChemStation software (Rev. C.03.00).

Qualitative analysis was performed in the full scan mode (m/z range, 50–550), and quantification was in the in single-ion monitoring (SIM) mode with MDA-d5 as internal standard. The following fragment ions were used in SIM mode (quantifier ion underlined): MDA-d5 heptafluorobutyramide (HFBA), 136, 166, 167; cathinone HFBA, 105, 77, 240; cathine bis-HFBA, 330, 303, 240; norephedrine bis-HFBA 330, 303, 240. Analytical recoveries (mean ± SD) for cathinone, cathine, and norephedrine were 82% ± 3%, 73% ± 4%, and 77% ± 2%, respectively (0.5 mg/L; n = 6). The limits of detection were <0.01 mg/L for the three analytes, and calibration curves were linear from 0.01 to 10 mg/L (calibrator concentrations, 0, 0.01, 0.025, 0.05, 0.25, 0.50, 1.25, 2.50, 5.00, and 10.00 mg/L) with regression coefficients >0.999. The regression equation for cathinone was: y = 0.51x (Sy|x = 0.7 mg/L); for cathine it was y = 0.62x (Sy|x = 0.04 mg/L); and for norephedrine it was: y = 0.52 (Sy|x = 0.06 mg/L). In-series reproducibility for cathinone, cathine, and norephedrine was 7%, 2%, and 7%, respectively (1.00 mg/L; n = 6).

The analytical procedure was tested for interference with the following phenylpropylamine derivatives and common sympathomimetic amines: amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, ephedrine, pseudoephedrine, N-methylephedrine, amphetaminil, etilefrin, fencamfamine, fenethylline, fenfluramine, fenproporex, mefenorex, norfenefrine, phenylephrine, methylphenidate, diethylpropion, propylhexedrine, and prolintane.

authentic forensic cases
In six forensic cases of suspected driving under the influence of drugs, the suspects admitted the ingestion of kath and urine samples were collected. The samples were analyzed for kath alkaloids by the Remedi HS HPLC and the GC-MS methods. For the quantitative assays, only 0.1 mL or less of urine was analyzed, and a calibration up to 30 mg/L was used.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
In the study, 36.1–59.2 g of kath material was chewed, which is equivalent to one-fourth of the amount chewed during a typical kath session. The alkaloid composition of the kath used was almost identical to the material used in a controlled study on pharmacokinetics (15) and corresponded to the average composition of kath (17)(18). During the chewing of kath, the leaves were not swallowed, and analysis of the residues indicated that 10.2% ± 4.5% cathinone, 10.9% ± 5.0% cathine, and 6.1% ± 1.0% norephedrine remained. The difference from the initial alkaloid content was considered to be the dose ingested (Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Ingested and excreted amounts of cathinone, cathine, and norephedrine of the four participants in the kath study.1

immunologic screening
Negative results for all urine samples were obtained with the Abbott FPIA, whereas the Mahsan-AMP300 on-site test gave positive results for urine samples from ~2 h up to 10 h after ingestion. In one case the 22-h urine was also positive (Fig. 1 ). To confirm whether cathinone, cathine, or norephedrine exhibited cross-reactivity with the antibodies of the Mahsan immunoassay, we added one of the three alkaloids in a concentration of 50 mg/L to three portions of a drug-free urine. Cathine and norephedrine yielded positive results, but not cathinone. These findings suggest that this immunoassay designed for the detection of amphetamine is insensitive to cathinone but may exhibit a low sensitivity for phenylpropanolamines.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Time range of kath alkaloid detection in urine.

The minimum ({blacksquare}) and maximum ({square}) times of the four participants in the kath study are given for the different analytical techniques. The last urine samples were collected 80 h after kath ingestion.

hplc screening
With the automated HPLC screening system Remedi HS, one peak, which was not present before kath ingestion, was detected in all urine samples up to 34 h after chewing and was still found in urine samples of one participant up to 50 h. The DAD spectrum of this peak is shown in Fig. 2C . In 35.8% of these cases, the Remedi library search algorithm found no candidates at all and reported "No Candidates, No Match", whereas in 41.8% of the cases the following library entries were reported as candidates (cited as printed), but a match was not obtained: "Fenfluramine, N-desmet", "Phenylpropanolamine/(Norpseudoephedrine)", "Pseudoephedrine, N-de", "Prolintane Metab.#2", "Isometheptene Metab.#", "AMPHETAMINE/(Phentermine)/(Phenethylamine)", "Mexiletine". Only in 22.4% of the cases (15 of 67 positive urine samples) did the candidate "phenylpropanolamine" qualify for a match.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. HPLC chromatogram of an authentic urine sample and DAD spectrum of the peak at 4.1 min (inset C), which results from a combination of the coeluting cathinone (inset A) and cathine/norephedrine (inset B).

To investigate the nature of the unknown peak, we added 10 mg/L cathinone or norephedrine to drug-free urines, and both were analyzed with the Remedi HS system. We found that the DAD spectra of cathinone (Fig. 2AUp ) and norephedrine (Fig. 2BUp ) were correctly matched by the computer library, but both compounds exhibited identical retention times. A urine sample to which both cathinone and norephedrine were added showed one single peak with a DAD spectrum similar to that found in urine samples from the study. From these results we concluded that the unknown peak in the urine samples results from the coeluting alkaloids cathinone, cathine, and norephedrine and that the Remedi HS system is therefore not suitable for reliable and unambiguous identification of ingested kath phenylpropylamine alkaloids.

In the Remedi analyses of urine samples from six forensic cases (Table 2 ) in which kath ingestion was later confirmed by GC-MS, a peak with the same retention time and DAD spectrum as in the samples from our study was found. This suggests that the detection of this peak in a Remedi HS analysis may be an indicator for the presence of both cathinone and phenylpropanolamines and that an analysis using GC-MS should be applied to confirm this.


View this table:
[in this window]
[in a new window]
  		Table 2. Concentrations of cathinone, cathine, and norephedrine determined by GC-MS-SIM in urine samples from six forensic cases of suspected driving under the influence of drugs.

gc-ms analysis
For derivatization, we selected heptafluorobutyrylation using MBHFBA because the derivatives of the diastereomers cathine and norephedrine are well separated by GC. The derivatization procedure using MBHFBA is very convenient compared with the more common heptafluorobutyrylation method (11)(19) because the analytes are dissolved in the reagent, no reactive species are produced during the derivatization reaction, and therefore, no evaporation step is necessary; hydrolysis of the derivatives is prevented by an excess of the reagent. The mass spectral characteristics of the HFBA derivatives have been described by Thurman et al. (19) and Valentine and Middleton (11). During GC-MS analyses, no interferences were observed with matrix compounds or with other phenylpropylamine derivatives and common sympathomimetic substances (see Materials and Methods for list of tested compounds).

In the urine samples from the study, the phenylpropanolamines cathine and norephedrine could be detected in the MS scan mode up to 50–70 h after kath ingestion; in the MS-SIM mode, the compounds were still detectable at concentrations up to 0.05 mg/L in the last urine sample collected at ~80 h after ingestion. Both phenylpropanolamines could be assayed much longer than the characteristic kath ingredient cathinone, which was detectable in MS-SIM mode only up to 22–26 h after ingestion.

Total amounts of the three analytes excreted were calculated and compared with the ingested doses (Table 1Up ). We found that <=7% of the applied cathinone doses were excreted unchanged in the urine, which is in agreement with the results of Brenneisen et al. (9). On the other hand, the amount of norephedrine excreted was much higher than the amount ingested, which is consistent with the finding of Mathys and Brenneisen (14), indicating that S-(-)-cathinone is metabolized to R,S-(-)-norephedrine. The maximum concentrations of cathinone, cathine, and norephedrine in urine samples from the study were 2.5, 20, and 30 mg/L, respectively, and were 28.8, >300, and >300 mg/L, respectively, in urine samples from the forensic cases.

In other fields, such as clinical toxicology or doping control, screening for phenylpropylamine stimulants plays an important role and the question arises whether an analytical distinction is possible between kath use and the intake of related substances. During the excretion phase, 26–80 h after the last kath use, only the phenylpropanolamines cathine and norephedrine were detected in urine. But because cathine is also available as an anorectic and norephedrine as a cold medication [(±)-phenylpropanolamine] or as metabolite of ephedrine, these compounds are not characteristic for kath. After the single intake of cathine or norephedrine, only one peak is to be expected, whereas after kath use both diastereomers are present. However, because drug abusers may use both stimulants, only the alkaloid cathinone can be considered characteristic for kath. Unfortunately, cathinone could be expected to be a desalkyl metabolite of the homologous 2-aminopropiophenones methcathinone, dimethylpropion, and diethylpropion. Although this has been reported for diethylpropion (20), no cathinone has been found in urine samples from methcathinone users (10) or after the intake of dimethylpropion (21). Therefore, kath use can be confirmed only by the detection of cathinone in the absence of N-alkylated homologs.


   Footnotes
 
1 Nonstandard abbreviations: GC-MS, gas chromatography–mass spectrometry; MDA-d5, (±)-3,4-methylenedioxyamphetamine-d5; MBHFBA, N-methyl-bis(heptafluorobutyramide); FPIA, fluorescence polarization immunoassay; DAD, photodiode array detection; HP, Hewlett-Packard; SIM, single-ion monitoring; and HFBA, heptafluorobutyramide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Rousseau C, Said TM, Gagne MJ, Bibeau G. Between myth and madness: the premigration dream of leaving among young Somali refugees. Cult Med Psychiatry 1998;22:385-411.[ISI][Medline] [Order article via Infotrieve]
  2. Ahmed AG, Salib E. The khat users: a study of khat chewing in Liverpool’s Somali men. Med Sci Law 1998;38:165-169.[ISI][Medline] [Order article via Infotrieve]
  3. Mayberry J, Morgan G, Perkin E. Khat-induced schizophreniform psychosis in UK [Letter]. Lancet 1984;1:455.
  4. Dietschy PJ. Catha edulis und Ibogain. Schweiz Apoth Ztg 1992;130:447-449.
  5. Kalix P, Braenden O. Pharmacological aspects of the chewing of khat leaves. Pharmacol Rev 1985;37:149-164.[ISI][Medline] [Order article via Infotrieve]
  6. Kalix P. Khat, an amphetamine-like stimulant. J Psychoactive Drugs 1994;26:69-74.[ISI][Medline] [Order article via Infotrieve]
  7. Schorno X, Steinegger E. CNS-active phenylpropylamines of Catha edulis FORSK (Celastraceae) of Kenyan origin. Experientia 1979;35:572-574.
  8. Brenneisen R, Geisshüsler S. Psychotropic drugs. III. Analytical and chemical aspects of Catha edulis Forsk. Pharm Acta Helv 1985;60:290-301.[ISI][Medline] [Order article via Infotrieve]
  9. Brenneisen R, Geisshüsler S, Schorno X. Metabolism of cathinone to (-)-norephedrine and (-)-norpseudoephedrine. J Pharm Pharmacol 1986;38:298-300.[ISI][Medline] [Order article via Infotrieve]
  10. Paul BD, Cole KA. Cathinone (khat) and methcathinone (CAT) in urine specimens: a gas chromatographic-mass spectrometric detection procedure. J Anal Toxicol 2001;25:525-530.[ISI][Medline] [Order article via Infotrieve]
  11. Valentine JL, Middleton R. GC-MS identification of sympathomimetic amine drugs in urine: rapid methodology applicable for emergency clinical toxicology. J Anal Toxicol 2000;24:211-222.[ISI][Medline] [Order article via Infotrieve]
  12. Guantai AN, Maitai CK. Metabolism of cathinone to D-norpseudoephedrine in humans. J Pharm Sci 1983;72:1217-1218.[ISI][Medline] [Order article via Infotrieve]
  13. Maitai CK, Mugera GM. Excretion of the active principle of Catha edulis (Miraa) in human urine. J Pharm Sci 1975;64:702-703.[ISI][Medline] [Order article via Infotrieve]
  14. Mathys K, Brenneisen R. Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection. J Chromatogr 1992;593:79-85.[ISI][Medline] [Order article via Infotrieve]
  15. Widler P, Mathys K, Brenneisen R, Kalix P, Fisch H-U. Pharmacodynamics and pharmacokinetics of khat: a controlled study. Clin Pharmacol Ther 1994;55:556-562.[ISI][Medline] [Order article via Infotrieve]
  16. Toennes SW, Kauert GF. Importance of Vacutainer selection in forensic toxicological analysis of drugs of abuse. J Anal Toxicol 2001;25:339-343.[ISI][Medline] [Order article via Infotrieve]
  17. Geisshüsler S, Brenneisen R. The content of psychoactive phenylpropyl and phenylpentenyl khatamines in Catha edulis Forsk of different origin. J Ethnopharmacol 1987;19:269-277.[ISI][Medline] [Order article via Infotrieve]
  18. Baselt RC. Cathinone. Baselt RC eds. Disposition of toxic drugs and chemicals in man 2000:142-143 Chemical Toxicology Institute Foster City, CA. .
  19. Thurman EM, Pedersen MJ, Stout RL, Martin T. Distinguishing sympathomimetic amines from amphetamine and methamphetamine in urine by gas chromatography/mass spectrometry. J Anal Toxicol 1992;16:19-27.[ISI][Medline] [Order article via Infotrieve]
  20. Pokrajac M, Miljkovic B, Misailovic B. Mass spectrometric investigation of 2-aminopropiophenones and some of their metabolites. Rapid Commun Mass Spectrom 1991;5:59-61.[ISI][Medline] [Order article via Infotrieve]
  21. Markantonis SL, Kyroudis A, Beckett AH. The stereoselective metabolism of dimethylpropion and monomethylpropion. Biochem Pharmacol 1986;35:529-532.[ISI][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 Hum ReprodHome page
S. A. Adeoya-Osiguwa and L. R. Fraser
Cathine and norephedrine, both phenylpropanolamines, accelerate capacitation and then inhibit spontaneous acrosome loss
Hum. Reprod., January 1, 2005; 20(1): 198 - 207.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toennes, S. W.
Right arrow Articles by Kauert, G. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toennes, S. W.
Right arrow Articles by Kauert, G. F.
Related Collections
Right arrow Drug Monitoring and Toxicology
Right arrow Endocrinology and Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS