Identification of conjugated metabolites of antipyrine in urine by pyrolysis-electron-impact mass spectrometry

Joumal of Ana&icarl and AppIied PymIjs&

Ekvier

Science Publishers B.V.. Amsterdam

6 (1984) 1-18

- Printed in The Netherlands

IDENTIFICATION OF CONJUGATED METABOLITES OF ANTIPYRINE IN URINE BY PYROLYSIS-ELEmON-IMPACI’MASS SPECmOMErRY J. B&ITCHER

and H. BASSMANN

InsfiKutrw Pharmakologie wad Toxikoiogie, TechnLsche. Unicetsiiriir Braunschweig, Mende&sohn.wmsse I, D - 33m Braunschweig (ERG.)

I. ERXLEBEN F~Mereich /ERG.)

BioIogie/Chemie,

H.-M. SCHIEBEL

Unicrrsiirar Bmmen,

Bibliorhekss~rase,

Bremen

33

l

iturirw fi%r Orgtanische Chemie. Technische Unicersiriir Bmwzschwei, Bmwuchweig

D -2&W

Schleini~zs~rasw D - 3300

(F. R G.)

(Received July 10th. 1983: accepted August 30th. 1983)

SUMMARY Pyrolysis-electron-impact mass spectrometry can be employed as a fast and sensitive method for the identification of unconjugatcd and conjugated metabolites of antipyrinc at different stages of the work-up procedure_ T5e method avoids derivatization of the highly poIar conjugates. generally a basic requirement for electron-impact investigation. Using crude extracts of human and rat urine. endogeneous products of the metabolic process did not interfere! with the studied metabolitts. It is demonstrated that all phase1 metabolites so far described in the literature can be thermally Iiierated from their uxresponding conjugates and subscqucntly identified by electron-impact mass spectrometry. The isomeric main metabolites 3-hydro~methylantipyn’nc and Qhydroxyantipyrinc can be distinguished by specific pyrolytic reactions.The different tanpcraturcs of decomposition of the conjugated sulphatcs and ghxuronides give additional information on the nature of the conjugates. Thus, the characteristic difference in the metabolic patterns in man and rat is reflected in the mass thermograms of the aglycones obtained by thermal decomposition of the conjugate mixtures_ IN-IRODUCI-ION

Lipophilic xenobiotics such as drugs are enzymatically converted in man and animals to more polar metabolites that can be easily excreted. The main biotransformation reactions for this conversion are oxidation to give phase-1 016!5-2370/84/So3.00

*E 1984 Elsevier Science Publishers B.V.

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1.

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phase-11 nctabotilc R-0-coqugate

Biotransformation of xenobiotics into phase-1 and phase-11 mctabolitcs.

metabdites and conjugation to give phase-11 metaboiites. For conjugation by. for instance. activation by ghxcuronic or sulphuric acid a suitable reaction center is required_ This has to be primarily available or has to be formed by phase-1 metabohsm. These steps of metabolic conversion are iIIustrated in Fig- 1. The first step. oxidation of the xenobiotic R. is catalysed by monooxygenases to produce the phase-1 metabolite R-OH. The second step. conjugation of the primarily formed hydroxyl group. is performed by specific transferases. By this step the phase-11 metabolite R-O-conjugate is obtained. Antipyrine I (Fig 2). one of the classic antipy-retic drugs. is a widely used model compound for pharmacokinetic studies in man and animals [1.2]. The antipyrine test gives evidence about the individual hydroxylation and conjugation capacity of the‘liver in normal and diseased states. To date this test has been !imited mostly to the elimination kinetics of the drug in plasma or saIiva [3]. Thus. only indirect conclusions about the metabolic capacity of the liver can be drawn. LMuch more deta.iIed information is expected to be obtained if the phase-1 and phase-11 metabolites. excreted in urine. are taken into account for diagnostic purposes. So far. such an analysis could not be performed because of fra&mentary knon-ledge of the pattern of both phase-1 and phase-11 metabolites. Only very recently have investigations been reported that result in B nearly complete anaIysis of the complex pattern [4-g]_ Antipyrinc undergoes extensive enzymatic oxidation at four different positions of the molecule. thus forming four hydroxylated phase-1 metaboIites (Figs. 2 and 3)_ These are subsequently conjugated by glucuronic acid and sulphuric acid to give glucuronides and sulphates as phase-II metaboIites. The complete metabolic pattern of antipyrine. so far described in the literature. and its typical distribution found in human and rat urine are shown in Fig. 3 and Table 1, As shoun in TabIe 1. the most of the

Fi_e 2 Antipvrinc 1 and positions of cnqmatic cransfornufon into phase-1 met&o&s_ _

3 TABLE

1

Distribution pattern of ancipyrine and antipyrinc mecab@ites in human and rat obtained with [3-‘4CJancipyrine O-48 h and O-24 h. respectively. after administration NO.

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13

Antipyrine Norantipyrine 3-Hydroxymcchylantipyrine 3Karboxq;mtipyrine 4-Hydroxyantipyrine 4.4’-Dihydroxyantipyriie Norantipyrine glucuronidc Norantipyrinc sulphate 3-Hydroxymcthylantipyrine glucuronide 4-Hydroxyantipyrine glucuronide 4-Hydroxyantipyrinc sulphate 4.4’JXiydroxyantipyrine gkuronide 4.4’-Dihydroxyantipyrine sulphate

Man (%I PI

Rat cw

3.0

3.8

5.8 4.8 0.4 13 25.9 l-6 13.7 34.3

21.7 25

UIiIlC

WI

20.0 12.8 9.2

5.0

14.0

0.5 1.8

15.0

metabolites are eIiminated in the form of conjugated phase-11 metabolitcs. Only a small amount wili be left unchanged or unconjugated. Table 1

additionally shows a distinct difference in the phase-1 and phaseI reactions for man and rat. Thus, e.g., a characteristic feature of the metabolism of antip,yrine in rat is the preferred elimination of phase-1 metabolites as strIphates [9]Metabolites of antipyrine have been detected by gas chromatography-mass spectrometry (GC-MS) and by direct introduction ekctron-impact mass spectrometry (EI-MS) of isolated metabolites [lo-151. Identification of isolated phase-1 metabohtes by EI-MS presents few difficulties. This also applies to the isomeric main metabolites 3-hydroxymethylantipyrine 3 and LGhydroxyantipyrine 5. Both metabolites display a different fragmentation pattern [12]. In contrast to the unconjugated phase1 metabolites, the identification of the conjugated ph-II antipyrine metabolites by MS was to a Iarge content incomplete. This was due to the low volatility of the conjugates and the necessary preparation of derivatives of sufficient volatility for EI-MS. Identification by EI was limited to only three isolated and derivatized glucuronides [12-141. For the corresponding conjugated sulphates no derivatization procedure has been described so far. Recently, the first results have been obtained using field desorption (FD) [16] and fast atom bombardment (FAB) [17,18] for the molecular weight determination of underivatized antipyrine conjugates [19,20]. However, these results were obtained with carefully purified samples whereas with impure samples, the determination can be complicated by loss of sensitivity or completely prevented. This paper reports on the identification of undexivatized glucuronides and sulphates in antipyrine metabolism using pyrolysis EI-MS. The aim was to develop a rapid and sensitive method for the det_ermination of unconjugated

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Fig 4. Work-up procedure for isolation and mass spcctrometric identification of unconjugatcd and conjugated antipyrine metabolitcs (s. = sulphate; g. = ghcuronide).

and conjugated metabolites in crude extracts at different steps of the separation procedure, as illustrated in Fig. 4.

EXPERIMENTAL

Chemicals

Antipyrine (1)(2,3-dimethyI-1-phenyl-3-pyrazolin-S-one) was purchased from Sigma (Munich, F.R.G.). Urcase (Jack bean) was obtained from Boehringer (Mannheim, F-R-G.). Solvents (reagent grade) were from Merck (Darmstadt, F.KG.). Experiments in man and rat

Four healthy male volunteers, aged 28-35 years, received qrally a single dose of 1200 mg of antipyrine and total urine was collected for the next 48 h after drug intake. AU volunteers were Caucasians, non-smokers and had not taken any medication for 1 month prior to the study. MaIe Sprague-Dawley rats (Lippische VcrsuchstieranstaIt, External,

6

F.R.G.). body weight 350 f 20 g. were kept on a standard diet (Altromin Xo. 1324. Laage. F.R.G.) and had free access to tap water. Antipyrine was given intraperitoneally with a single dose of 40 mg/kg. Urine was collected from all-glass metabolic cages for the next 24 h. Wbrk-up

proceciiues

Urine samples were incubated with ureas= for 6 h at 25°C. evaporated under reduced pressure and the residue was extracted with methanol After concentration. this extract was layered on a silica gel column and separated into three fractions. Fraction I contained unchanged antipyrine and phase-I metabolites. fraction II sulphates of phase-1 metabolites and fraction III glucuronides of phase1 metabolites. in a subsequent chromatographic step. the sulphate and the glucuronide fractions were separated into their individual phase-11 metabolites (Fig. 4). Details of the work-up procedure have been published elsewhere [4.9]. For MS analysis of crude extracts. 1 ml of urine u-as incubated with urease (6 h. 25OC). evaporated to dryness and extracted twice with 20.0 ~1 of anhydrous methanol. The methanolic solution was finally evaporated to dqness and the residue analysed.

The mass spectra were measured on an AEI MS 902 double-focusing mass spectrometer at 70 eV. High-resolution data were obtained by peak matching at a resolution of appro.ximately 8000 (10% valley definition) using an AEI hfS 30 mass spectrometer at 70 eV. Mass thermograms were produced on a Varian-,CiAT CH-7A single-focusing mass spectrometer in combination with a Varian SS 100 data system. The electron energy was 25 eV. The probe temperature ~3s increased in steps of IO”C/min and spectra were recorded 30 s after each step. An exponential scan of 3 s per decade was employed; 32.760 counts correspond to a signal of 10 V. The collisional activation (CA) spectra were obtained using a MAT 8230 double-focusing mass spectrometer. A few micrograms of the mixture and of the isolated conjugates were used for 52s analysis.

RFSULTS

Idmiificurion of isolarerlconjugates In principle. the molecular ions of underivatized phase-11 metabolites cannot be detected by EI-MS because of their high polarity and consequentIy low volatility. However, at increasing sample temperatures these conjugates are clearly decomposed by release of the basic phase-1 metabo-

7

lites, thus allowing an indirect determination of the original metabolites. This behaviour was found to be characteristic of all conjugated antipyrine metabolites so far described, as we11as for ghrcuronides and sulphates. Noranripynke gkuronide

and sulphate

A low temperature of decomposition (about 100 and 200°C respectively) and pronounced volatility of the released aglycone are characteristic features of the two-phase-II metabolites 7 and 8 of norantipyrine 2. The spectra of both conjugates are characterized by the high relative abundance of the molecular ion of norantipyrine, base peak at m/z 174, and its EI fragmentation pattern m/z 132, 105,91,77 and 51 [ll]. Interfering ions of degradation products of the sulphuric acid or glucuronic acid residue are of minor importance. This is shown by the spectrum of norantipyrine sulphate 8 and synthetic norantipyrine 2 for comparison (Fig. 5). 34Yydroxymethylantipyn~ne gkuronide The only phase-II metabolite of 3-hydroxymethylantipyrine

3 described so far in the literature is the giucuronide 9. In contrast to the conjugates of norantipyrine 2, decomposition of the glucuronide 9 and release of the

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Fig. 5. (a) EI mass spectrum of norantipyrine sulphatc 8 obtained from rat urine at a sample

temperature of 9oOC. l = Impurities from the work-up procedure. (b) EI mass spectrum of synthetic norantipy-rinc 2

Fis 6, El mss spectrum of Ih~droxvmcth~l~tip~~ne ghxuronide 9 obtained from human urine. Mws region of the mokcuiar ion of the &-cone 3 at different sample temperatures. 4~) 26O’C-ztb) 350°C.

correspondins a&-cone 3 (n:/- - 204) starts at a markedIy higher temperature (220°C) and has its maximum at 260°C (Fig. 6a). Above 300°C the formaticm

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molecular ion of the a&cone at m/z 204 to its pyrolytic degradation product at nr/z 188 is about 1: 1 (Fig_ 6b)- Its formation seems to be conjugate-specific. The-exact mass of this ion corresponds to antipyrine 1 (Fig. 7). Its identity was confirmed by collisional activation (CA) [21]. The formation of an ion at m/z IS8 was first observed by Zietz and SpiteIIer [12] in the EI mass spectrum of a trimethylsilyl derivative of 3-hydroxymethylactipyrinc glucuronide 9. We also assume that, in this instance. the occurrence of the ion at m/z 188 was caused by thermaliy induced degradation and not by EI fragmentation-

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224 zs

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-#-IQ-droxwntip_r7irre ghcuronide and sulphare

The range of the thermol_ytic fission of the glucuronide 10 is about 50°C lower than that of the isomeric ghrcuronide of 3-hydroxymethylantipyrine 9. In comparison with the ghtcuronide 10 the maximum of decomposition of the 4-hydroqantipyrine sulphate II can be obtained at distinctIy lower

9 202

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I 204 11 ‘II ii

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Fig. 8. EI mass spectrum of 4-hydroxyantipyrine glucuronide 10 obtained from human urine_ Mass region of the molecular ion of the aglycone 5 at different sample tcmpa-aturcs. (a) 250°c: (b) 310°C_

temperatures. as is the case for the corresponding conjugate of norantipyrine 8. In contrast to 3-hydroxymethyl antipyrine 3, at higher source temperatures pyrolytic degradation of the aglycone 4-hydroxyantipyrine 5 results in a product obtained by dehydrogenation and formation of an ion at m/z 202 (Fig. 8). The formation of an ion at m/z 202 was first observed by Momose and Tsuji [lo] during their GC-MS investigation of antipyrine phase-1 metabolites. This specific degradation of 4-hydroxyantipyrine makes possibIe a differentiation of the isomeric antipyrine metabolites 3 and 5 without an analysis of their fragmentation pattern which is. in general, not practicable for the investigation of complex biological mixtures without the use of

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Fig. IO. EI mass spectrum of 4.4’-dih_vdroxyantipyine glucuronidc 12 obtained from rat urine [23]. Mass region of the mokcukr ion of the aglyconc 6. Sample temperature 180°C.

mass spectrometric techniques [21]. The pyrolytic product at m/z 202 has the elemental composition C,,H,,N,O+ and may be 2-N-methyl-3-meth_vIcne-l-N-phenylpyrazolidine-4.5-dione 14 (Fig. 9). Its formation can be observed not only for the isolated conjugates 10 and II but also for the synthetic aglycone 5 at approprizte sample temperatures.

special

q~‘-oi~~dro.~anrip~rine gkuronide The temperatures of decomposition

and sulphate

of the phase-11 metabolites 12 and 13 of the recently discovered 44’-dihydroxyantipyrine 6 [22] exhibit onIy slight differences in comparison with the conjumtes 10 and 11 of the 4-hydroxyantipyrine. Fig. 10 shows the mass range of the molecular ion of the agIycone of 4.4’-dihydroxyantipyrine glucuronide 12. As can be seen from the intensive signa at m/z 204. this compound still contained considerable amounts of 4-hydroxyantipyrine gIucuronide 10. At the start of the decomposition of the conjugate. degradation of the released aglycone and formation of 2 didehydro compound at m/z 218 can be observed. This reaction is similar to the pyrolytic degradation of 4hydroxyantipyrine 5 and is characteristic of antipyrine mctabolites hydroxylated in position C-4. A na&.sis of conjqate

mixtures

Giucuronide fraction from human urine A characteristic feature of the EI mass spectra of the crude and purified

mixtures of antipyrine and its phase-1 metabolites is 2 considerable change in the molecular ion intensities with increasing temperature. as a resuIt of the different volatilities and molecular ion stabilities of the individual phase-1 metabolites and of the stabilities of the conjugated metabolites towards thermai cieavage. The temperature-dependent change in intensities is shown

11

Fig. 11. JZJmass spectrum of ghacuronide fraction from human urine. Single scan at different sample temperatures. (a) 200°C; (b) 38OOC.

as an example in Fig. 11 for the gkuronide fraction which is typical of man. The stability of the conjugates towards acidic hydrolysis [4] correlates well with the stability towards thermal cleavage. As demonstrated in Fig. lla. the agIycone of norantipyrine glucuronide 7 at nz/z 174 is detected almost exclusively at a sample temperature of 200°C. However, at higher temperatures, the contribution of this ion to the total ion current decreases (Fig. 11 b). Simultaneously, the agIycone of 3-hydroxymethylantipyrine ghtcuronide 9 and of 4hydroxyantipyrine glucuronide 10 at m/r 204 becomes evident. The ion at m/z 202 is a typical dehydrogenation product of 4-hydroxyantipyrine 5. The high relative abundance of this ion is an indication of the large amount of 4hydroxyantipyrine glucuronide 10 in human urine [5.7]. Sulphatefraction from rat urine

In contrast to man, in rat phase-1 metabolites are predominantly bound to sulphate [4.9.23.24]. The stability of the sulphates towards acidic hydrolysis is Iower than the stability of the appropriate ghtcuronides [4]. This difference .,-

-7

.r.._-c’

b

Fig. 12 EI mass spectrum of sulphate fraction from rat urine Single scan at diffa.cnt sample temperatures. (a) 1OOT; (b) 380°C.

in stability is also refkcted in the EI mass spectra. Sulphates are decomposed at considerable lower source temperatures. This behaviour is especially pronounced in the case of norantipyrine sulphate 8. Norantipyrine 2 (Mi’ 174) appears with a high relative abundance at a sample temperature of 100°C (Fig. 12a). In contrast.. thermal cleavage of 4hydroxyantipyrine suIphate 11 starts at considerably higher temperatures. At a sample temperature of 3SOOC the sulphate fraction of rat is characterized by three metabolites. norantipyrine sulphate 8 (agIycone nz/z 174). 4hydroxyantipyrine suiphare 11 (agiycone m/z 204) and its dehydrogenation product 14 at m/z 202. and 4.4-dihydroxyantipyrine strIphate 13 (aglycone m/z 220) (Fig. 12b). The signal at nr/z 218 represents the dehydrogenation product of 3.4’-dihydroxyantipyrinc suIphatc 13.

Concentrated methanolic extracts of urine can be investigated directly without any pre-treatment. However. the amount of urea results in a strongIy fluctuating ion curren; and consequently in a more complicated determination. This difficuhy can be avoided by an initial enzymatic degradation of urea (Fig. 4). As is demonstrated for a crude extract of rat urine in Fig. 13. all phase-I metaboiites of antipyrine (Fig. 3) can be recorded by one magnetic scan at a sample temperature of 18OOC. Under these conditions the phase-I I metabolites. with the exception of 3-hydroxymethylantipyrine glucuronide 9. are decomposed to a large extent to the corresponding phase-1 metabolks. The signal at w/z 174 represents norantipyrine 2 from 8. m/z

_.; -7:

Fig sin&

13. EI mass spectrum .san.

of mcthanolic extract from rat urine_

%mple tempc~turc180°C.

13

188 unchanged antipyrine 1,m/z 204 the unconjugated 3-hydroxymethylantipyrine 3 (see Table 1) and the isomeric 4-hydroxyantipyrine 5 from 11, respectively. The ion at m/z 218 corresponds to 3-carboxyantipyrine 4 and the ion at m/r 220 to 4,4’-dihydroxyantipyrine 4 from 13. The characteristic temperature for pyrolytic dehydrogenaticn of the 4,4’-dihydroxyantipyrine 6 was found to be above 200°C. Therefore, the ion at m/z 218 displayed in Fig. 13 can be associated in part with 3-carboxyantipyrine 4 under the indicated experimental conditions_ The ion at m/z 202 is formed by pyroIytic degradation of the conjugated 4hydroxyantipyrine 11. Spectra of methanolic extracts of blank rat urine, which were recorded under the same experimental conditions. did not display interfering ions in the mass range of antipyrine and the aglycones of phase-11 metabolites (m/z 150-250). Differentiation between glucuronides and sut’phates by fractionated pyrolysis

As seen above for the isolated phase-11 metabolites, thermal cleavage of the conjugates and release of the corresponding aglycones can be observed in temperature ranges that arc typical of the individual metabolite. The kinetics of the reIease of phase1 metabolites from their conjugated phase-11 metabolites and the formation of characteristic pyrolysis products can be clearly demonstrated by the mass thermograms of the corresponding molecular ions of the aglycones obtained with increasing sample temperature. These thermograms obviously display the different degradation temperatures between glucuronides and sulphates and, thus. yield fast and qualitative information about the ratio of both forms of conjugates within the mixture Conjugate fraction from rat urirre

A characteristic feature of the metabolism of antipyrine in rat is the formation of norantipyrine as main phase-1 metabolite and the preferred elimination of the phase-1 metabolites as suIphates. In this instance gIucuronidcs arc of minor importance (Table 1). Fig 14a shows the mass thermogram of the purified conjugate fraction of rat obtained after enzymatic degradation of urea. Unchanged antipyrine and unconjugated metabolites were removed by extraction with chloroform. With increasing temperature the molecular ions of norantipyrine 2 at m/z 174 and of the two isomeric metabolites 3-hydroxymethylantipyrine 3 and 4-hydroxyantipyrine 5 at m/z 204 are produced by fractionated pyrolysis of the conjugate mixture_ In addition, the characteristic degradation product of 4hydroxyantipyrine at m/z 202 is detected at a sample temperature of about 27OOC. The large amount of norantipyrine sulphate 8 in the mixture (about 30%), its low decomposition temperature and the stability of the released norantipyrine 2 towards EI excitation results in an intense and clearly separated signal in the mass thermogram of this conjugate mixture. Thermal fission of the 4-hy-

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14. (a) Conjugated antip?;r;nt metabolites (suiphatcs and gkuronidcs) from rat urine: mass thermogram obtained bv temperature-dependent liberation of phase-1 metabolica and generation of characteristic degradation products. Norantipyrine 2 (m/z 174). 3-hydroxymcthykmtipyrinc 3 ( zn /= 204) and 4-hvdroxwntipyrinc 5 (m/z 204/X2). (b) Extended section of the mass thtimogmm given in (ai rcprcsenting the ions pt tn/: 202 and 204 (3-h~droxymcth$mtipyrinc 3 and 4-h~droxyantipyine 5) and at m/z 220and 215 (4,4’-dihydros_vantipyrine 6). Fig

droxyantipyrine sulphate 11 starts at 160*C and is characterized by the appearance of the molecular ion- of the corresponding agIycone at m/z 204. This signal decreases at 220-23OOC and becomes a plateau produced by the decomposition of the two isomeric glucuronides of 4-hydroxyantipyrine 10 and 3-hydroxymethyIantipyrine 9. The release of 4hydroxyantipyrine 5 from its glucuronide is connected at higher temperatures with pyrolytic dehydrogenation of the original metabolite and formation of the ion detected at na/z 202. Evidence for the isomeric 3-hydroxymethylantipyrine ghrcuronide 10 is given by the conjugate-specific pyrolysis product of this metabolite formed at temperatures above 300°C at m/z 188 (not considered in this chromatogram). An expansion of the thermogram is seen in Fig. 14b. In addition to the ions at m/z 204 and 202. only the molecular ion of 4,4’-dihydroxyantipyrine

15

6 and its pyrolysis product at m/z 220and 218, respectively, are considered. The amount of 4,4’-dihydroxyantipyrine conjugates in the metabolic pattern of antipyrine in rat is about 15%. It will be almost exclusively eliminated as sulphate. Therefore, a differentiation between the two conjugate forms is not necessary. Both signals are of low intensity owing to the small amount of the dihydroxy compound in the mixture and the instability of the corresponding aglVcone towards EI ionization. The release of the aglycone and the pyrolytic degradation are not resolved with time or temperature. In this instance both processes start simultaneously. Ghcuronide fraction from human wine

enhanced stability of glucuronides towards thermal fission in comparison with the analogous sulphates is reflected in the mass thermogram obtained by fractionated pyrolysis of a ghrcuronide fraction from human urine that contained the three main phase-II metabolites 7, 9 and 10 in different amounts (Fig 15). Again, the conjugate of norantipyrine 7 proves to be most sensitive towards thermal stress.The release of the aglycone (m/z 174) has its maximum at approximately 200°C and therefore about 50°C higher than the corresponding sulphate shown in Fig 14. The signal at m/z 204 represents the mo1ecuIarions of the released isomeric phase-1 metabolites 3 and 5. The intensity of this signal is determined to a large -tent by the amount of 4hydroxyantipyrine glucuronide 10 in the mixture (see Table 1). A reliable indication of this metabolite is given by the signal at m/z 202. Decomposition of the conjugate (maximum at 250°C) and pyrolytic dehydrogenation (maximum at 26OOC)are clearly separated processes. For the sulphate and glucuronide of 4-hydroxyantipyrine the same difference in degradation temperature can be observed as for the conjugate 7 and 8 of norantipyrine- The maximum of the signal at m/z 204 for the ghtcuronide is ca. 25OOC(Fig. 15), whereas it is ca. 2OOOCfor the sulphate (Fig. 14). The The

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human urine: mass thermogram obtained by phase-1 metabolitcs norantipyrinc 2 (m/z 174). 4hydroxyantipyrinc 5 (m/z 204), and generation of the 14 of compound 5 at m/z 202. of the

16

ion at m/z 188, a conjugate-specific degradation product of the glucuronide 9. was observed only with weak intensity and was therefore not considered in this plot.

The application of fractionated pyrolysis EI-LMS permits the fast and sensitive analysis of the complete metabohc pattern of antipyrine in rat and human urine. The analysis is feasible using isolated metabolites. purified mixtures and crude extracts. Both phase-1 and phase-11 metabolites can be identified. the latter after thermally induced decomposition of the conjugates and subsequent identification of the corresponding phase-1 metabolites. This approach avoids the derivatization of the conjugates or their hydrolysis to the corresponding aglycones. The “direct” analysis of the metabolic pattern prevents or limits the loss of biotransformation products during the work-up procedure and the possible formation of artefacts by derivatization of the conjugates. Registration can be limited to the mass range of the moIecuIar ions of the phase-1 nietaboIites (nz/= 150-250). An unequivocal identification of the conjugated and unconjugated isomeric main metabolites 3hydroxymethylantipyrine and 4-hydroxyantipyrine is obtained without the use of special mass spectrometric techniques by specific pyroIytic= degradation of these metaboiites prior to ionization. On the basis of the varying temperature of decomposition of the individual phase-11 metabolites. a clear distinction can be made between the two forms of conjugation. gIucuronid_ and sulphates. Analysis can be performed by recording a number of single spectra at different sample temperatures or. much faster and clearer. by repetitive magnetic scanning and a temperature-programmed rise using an MS data system. Thus. the pronounced different courses of the phase-1 and phase-11 reactions of man and rat are reflected in the thermograms of the conjugate mixtures. Unquestionabiy. FD and FAB will be the most suitable ionization methods for the unequivocal determination of the moIecuIar weights of the free. individua1 phase-II metabohtes. However. discrimination effects or a complete Ioss of sensitivity can be observed for both techniques with complex mixtures or fractions of Iow purity. Using antipyrine as a model compound for pharmacokinetic studies. fractionated By-EI-MS appears to be a valuabIe analytical tool for the rapid screening of crude or pre-purified mixtures to give a quick, initial survey of the complete metabolite pattern.

ACKXOWLEDGEME’Si

The authors thank Prof. Dr. R. Schiippel, Braunschwcig. for his support and interest in this work and H. Steinert for exceIIent technical assistance.

17

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