Archives of
Arch. Toxicol. 35, 137--139 (1976)
TOXICOLOGY 9 by Springer-Verlag 1976
Quinine-N-Oxide -- A Urinary Component after the Consumption of Quinine Beverages J. Jovanovi6*, G. Remberg, M. Ende and G. Spiteller Organisch-Chemisches Institut der Universitw GOttingen
Abstract. After the consumption of quinine-containing beverages, quinine-Noxide could be identified in the urine mass spectrometrically. Consequently in the analysis of physiological fluids for drugs not only the drugs themselves must be considered but also their metabolites. Key words: Tonic water -- Quinine-N-oxide - Drug and Doping Controls. Zusammenfassung. Nach dem Genu/3 von chininhaltigen Getr/inken konnte im Urin von Versuchspersonen Chinin-N-oxid festgestellt und massenspektrometrisch identifiziert werden. Es sind daher beim analytischen Nachweis von Arzneimitteln neben den unver~inderten Wirkstoffen auch deren Metabolite zu beachten. Schliisselwiirter: Tonic Water -- Chinin-N-oxid -- Sucht- und Dopingmittelnachweis.
Recently we received two sealed urine samples from The Yugoslav Water Polo Association for examination for doping drugs. After work-up a light blue fluorescent spot (R: = 0.27; acetone: methanol = 1 : 1) under UV light A = 254 nm) was found in the thin-layer chromatogram (TLC) (silica gel with fluorescence indicator). The mass spectrum of the eluted spot is shown in Fig. la. By means of high resolution mass spectrometry the molecular ion was identified as C20H24N203. The intensive m/e 136 fragment (Spiteller et al., 1963) suggested that the compound could be a metabolite of quinine which is also characterized by an intensive m/e 136 (Fig. lb). Since the determined molecular composition is one oxygen atom more than that of quinine, it was probable that a quinine oxidation product was involved. This additional oxygen can only be located in the quinoline part of the * Tehnolow167 fakultet, 11000 Beograd, Jugoslawien. Send offprint requests to: Prof. Dr. Gerhard Spiteller, Organisch-ChemischesInstitut der Universit~it, D-3400 G6ttingen, Tammannstr. 2, Federal Republic of Germany.
138
Jovanovi6 et
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molecule as the base fragment m/e 136 corresponds to the quinuclidine portion of the molecule. The prominent loss of an OH-fragment (Fig. la) indicated a N-oxide. It is well known that many nitrogen-containing compounds are transformed into N-oxides under physiological conditions (Bickel, 1969). Therefore quinine was oxidized for 4 days in an H202 solution. The mass spectrum of the isolated oxidation product was
E
Quinine-N-Oxide after the Consumption of Tonic Water
139
identical to the spectrum of the u n k n o w n compound. The I R spectrum (PerkinElmer PE 621) shows intensive absorption at 1240 cm -1 confirming the presence of an N-oxide. F o r confirmation of the above results two volunteers each drank 1.1 1 of quininecontaining tonic w a t e r (Schweppes). The 24-hrs urines were collected for 3 days and worked up in the same m a n n e r as the submitted urine sample. Even after 3 days easily detectable quantities of quinine-N-oxide could be found in addition to quinine itself (Sch/itz et al., 1974). As no indication is given as to the quantities of quinine present we have not attempted to carry out any quantitative work. According to a local producer of beverage concentrates (D~ning & Krausse, Braunschweig) commercial tonic waters usually contain between 30 and 70 ppm quinine. These results demonstrate that the chromatographic detection of drugs m a y not be sufficient if only the parent drugs are taken for comparison. As chromatographic data have yet to be determined for m a n y drug metabolites, both false positives and false negatives m a y be obtained, false positives when the u n k n o w n metabolites have the same Rz or R t as a k n o w n drug or metabolite and false negatives when only metabolites with u n k n o w n R r or R t are present. Without confirmation of T L C or G L C results by means of mass spectrometry these should be treated with the utmost reserve. The occurrence of quinine -N-oxide in the urine of persons who have consumed no drugs indicates the problems occurring from u n k n o w n or unexpected components of foods and beverages.
Experimental Urine Work-Up. The entire 24-hrs urine (pH in each case 6.1) was brought to pH of about 2 with 0.1 N HC1 and extracted 3 times with chloroform. The water phase was then brought to pH 10 with 1 N NaOH and extracted 3 times with ether. After drying over anhydrous Na2SO4 the ether was carefully evaporated on a rotary evaporator and the residue analyzed by TLC (Merck Fertig-Platten, Kieselgel 60 F2~4, methanol/acetone = 1 : 1). The light blue fluorescent spot (Rj,= 0.27) was removed and eluted with methanol. Preparation of Quinine-N-Oxide. Quinine (10 mg) was stirred for 4 days at ambient temperature with approx. 5 ml 5% H202 in water and 0.3 ml acetone as cosolvent. A spatula tip of paUadium black was added to destroy excess peroxide and the solution was filtered after 1 hr. The water solution was saturated with NaC1 and extracted with ether. The ether solution was dried over anhydrous Na2SO4, filtered, concentrated, and submitted to TLC and mass spectrometry. Thin-Layer-Chromatography. Merck Fertig-Platten, Kieselgel60 F254,0.25 mm thickness, eluantmethanol/acetone = 1 : 1. Zone A: Ry = 0.27; quinine-N-oxide, mass spectrum Fig. la IR(KBr): 1240 cm-1 (N-O). Zone B: Ry = 0:67; quinine, mass spectrum Fig. lb. Mass Spectrometry. Mass spectra were taken on a Varian MAT CH-7 (low resolution) and a Varian MAT 731 (high resolution) on the solid sample probe, probe temperature: 140~ C. All spectra were taken at 70 eV.
References Bickel, M. H.: The pharmacology and biochemistry of N-oxides. Pharmacol. Rev. 21, 325-355 (1969) Sch/Jtz, H., Hempel, J.: Renale Ausscheidungsprofilenach der Einnahme chininhaltiger Tonic-Water. Arch. Toxikol. 31, 1--6 (1974) Spiteller, G., Spiteller-Friedmann, M.: Schl/isselbruchstiickein den Massenspektren von Alkaloiden. Tetrahedron Letters 3, 153--158 (1963) Received September 24, 1975
Arch. Toxicol. 35, 137--139 (1976)
TOXICOLOGY 9 by Springer-Verlag 1976
Quinine-N-Oxide -- A Urinary Component after the Consumption of Quinine Beverages J. Jovanovi6*, G. Remberg, M. Ende and G. Spiteller Organisch-Chemisches Institut der Universitw GOttingen
Abstract. After the consumption of quinine-containing beverages, quinine-Noxide could be identified in the urine mass spectrometrically. Consequently in the analysis of physiological fluids for drugs not only the drugs themselves must be considered but also their metabolites. Key words: Tonic water -- Quinine-N-oxide - Drug and Doping Controls. Zusammenfassung. Nach dem Genu/3 von chininhaltigen Getr/inken konnte im Urin von Versuchspersonen Chinin-N-oxid festgestellt und massenspektrometrisch identifiziert werden. Es sind daher beim analytischen Nachweis von Arzneimitteln neben den unver~inderten Wirkstoffen auch deren Metabolite zu beachten. Schliisselwiirter: Tonic Water -- Chinin-N-oxid -- Sucht- und Dopingmittelnachweis.
Recently we received two sealed urine samples from The Yugoslav Water Polo Association for examination for doping drugs. After work-up a light blue fluorescent spot (R: = 0.27; acetone: methanol = 1 : 1) under UV light A = 254 nm) was found in the thin-layer chromatogram (TLC) (silica gel with fluorescence indicator). The mass spectrum of the eluted spot is shown in Fig. la. By means of high resolution mass spectrometry the molecular ion was identified as C20H24N203. The intensive m/e 136 fragment (Spiteller et al., 1963) suggested that the compound could be a metabolite of quinine which is also characterized by an intensive m/e 136 (Fig. lb). Since the determined molecular composition is one oxygen atom more than that of quinine, it was probable that a quinine oxidation product was involved. This additional oxygen can only be located in the quinoline part of the * Tehnolow167 fakultet, 11000 Beograd, Jugoslawien. Send offprint requests to: Prof. Dr. Gerhard Spiteller, Organisch-ChemischesInstitut der Universit~it, D-3400 G6ttingen, Tammannstr. 2, Federal Republic of Germany.
138
Jovanovi6 et
al.
J 0 r tN "N
E o i
i
6
:2
q~
az
,~
to
t~ .Q
rJ
~o--=_-
tt~ : tt3-~_-
oo
to
-~
tN
~
~o
to
,,~
tx4
molecule as the base fragment m/e 136 corresponds to the quinuclidine portion of the molecule. The prominent loss of an OH-fragment (Fig. la) indicated a N-oxide. It is well known that many nitrogen-containing compounds are transformed into N-oxides under physiological conditions (Bickel, 1969). Therefore quinine was oxidized for 4 days in an H202 solution. The mass spectrum of the isolated oxidation product was
E
Quinine-N-Oxide after the Consumption of Tonic Water
139
identical to the spectrum of the u n k n o w n compound. The I R spectrum (PerkinElmer PE 621) shows intensive absorption at 1240 cm -1 confirming the presence of an N-oxide. F o r confirmation of the above results two volunteers each drank 1.1 1 of quininecontaining tonic w a t e r (Schweppes). The 24-hrs urines were collected for 3 days and worked up in the same m a n n e r as the submitted urine sample. Even after 3 days easily detectable quantities of quinine-N-oxide could be found in addition to quinine itself (Sch/itz et al., 1974). As no indication is given as to the quantities of quinine present we have not attempted to carry out any quantitative work. According to a local producer of beverage concentrates (D~ning & Krausse, Braunschweig) commercial tonic waters usually contain between 30 and 70 ppm quinine. These results demonstrate that the chromatographic detection of drugs m a y not be sufficient if only the parent drugs are taken for comparison. As chromatographic data have yet to be determined for m a n y drug metabolites, both false positives and false negatives m a y be obtained, false positives when the u n k n o w n metabolites have the same Rz or R t as a k n o w n drug or metabolite and false negatives when only metabolites with u n k n o w n R r or R t are present. Without confirmation of T L C or G L C results by means of mass spectrometry these should be treated with the utmost reserve. The occurrence of quinine -N-oxide in the urine of persons who have consumed no drugs indicates the problems occurring from u n k n o w n or unexpected components of foods and beverages.
Experimental Urine Work-Up. The entire 24-hrs urine (pH in each case 6.1) was brought to pH of about 2 with 0.1 N HC1 and extracted 3 times with chloroform. The water phase was then brought to pH 10 with 1 N NaOH and extracted 3 times with ether. After drying over anhydrous Na2SO4 the ether was carefully evaporated on a rotary evaporator and the residue analyzed by TLC (Merck Fertig-Platten, Kieselgel 60 F2~4, methanol/acetone = 1 : 1). The light blue fluorescent spot (Rj,= 0.27) was removed and eluted with methanol. Preparation of Quinine-N-Oxide. Quinine (10 mg) was stirred for 4 days at ambient temperature with approx. 5 ml 5% H202 in water and 0.3 ml acetone as cosolvent. A spatula tip of paUadium black was added to destroy excess peroxide and the solution was filtered after 1 hr. The water solution was saturated with NaC1 and extracted with ether. The ether solution was dried over anhydrous Na2SO4, filtered, concentrated, and submitted to TLC and mass spectrometry. Thin-Layer-Chromatography. Merck Fertig-Platten, Kieselgel60 F254,0.25 mm thickness, eluantmethanol/acetone = 1 : 1. Zone A: Ry = 0.27; quinine-N-oxide, mass spectrum Fig. la IR(KBr): 1240 cm-1 (N-O). Zone B: Ry = 0:67; quinine, mass spectrum Fig. lb. Mass Spectrometry. Mass spectra were taken on a Varian MAT CH-7 (low resolution) and a Varian MAT 731 (high resolution) on the solid sample probe, probe temperature: 140~ C. All spectra were taken at 70 eV.
References Bickel, M. H.: The pharmacology and biochemistry of N-oxides. Pharmacol. Rev. 21, 325-355 (1969) Sch/Jtz, H., Hempel, J.: Renale Ausscheidungsprofilenach der Einnahme chininhaltiger Tonic-Water. Arch. Toxikol. 31, 1--6 (1974) Spiteller, G., Spiteller-Friedmann, M.: Schl/isselbruchstiickein den Massenspektren von Alkaloiden. Tetrahedron Letters 3, 153--158 (1963) Received September 24, 1975
