Monitoring the behaviour of 4-ketocyclophosphamide versus cyclophosphamide during capillary gas chromatography by mass spectrometry

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Monitoring the behaviour of 4-ketocyclophosphamide versus

cyclophosphamide during capillary gas chromatography by

mass spectrometry

Citation for published version (APA):

Bruijn, de, E. A., Oosterom, van, A. T., Leclercq, P. A., Haan, de, J. W., Ven, van de, L. J. M., & Tjaden, U. R. (1987). Monitoring the behaviour of 4-ketocyclophosphamide versus cyclophosphamide during capillary gas chromatography by mass spectrometry. Biomedical & Environmental Mass Spectrometry, 14(11), 643-647. https://doi.org/10.1002/bms.1200141113

DOI:

10.1002/bms.1200141113 Document status and date: Published: 01/01/1987

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BIOMEDICAL A N D ENVIRONMENTAL MASS SPECTROMETRY, VOL. 14, 643-647 (1987)

-

Monitoring the Behaviour of

4-Ketocyclophosphamide uersus Cyclophosphamide

during Capillary Gas Chromatography by Mass

Spectrometry

E. A. de Bruijnt and A. T. van Oosterom

Leiden University Medical Centre, Department of Clinical Oncology, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands

P. A. Leclercq, J. W. de Haan and L. J. M. van de Ven

Eindhoven University of Technology, Laboratory of Instrumental Analysis, Department of Chemical Engineering, PO Box 513, 5600 MB Eindhoven, The Netherlands

U. R. Tjaden

Center for Bio-Pharmaceutical Sciences, Division of Analytical Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands

Capillary Gas Chromatography (CGC) is capable of determining underivatized cyclophosphamide (CPA) using SCOT OV 275 columns. Then CPA is subjected to in situ degradation resulting in formation of a cyclization product which can be determined selectively in biological fluids. In routine bioanalysis however cyclization products of CPA metabolites might interfere, e.g. 4-keto CPA. In the present study possible formation of cyclization products of 4-keto CPA similar to CPA was monitored by Mass Spectrometry. Cyclization of 4-keto CPA in siru was demonstrated to occur, resulting in a product similar to that of CPA. Both cyclization products could be determined selectively and it appeared that in situ cyclization of 4-keto CPA was negligible (<5’/0), probably owing to extra stabilization of the CPA metabolite by keto-enol tautomerism as has been demonstrated by NMR.

INTRODUCTION

Cyclophosphamide (CPA) (Fig. 1; 2-(bis-2-chloroethyl- amino)-tetrahydro-2H- 1,3,2-oxazophosphorin-2-oxide)

is a drug frequently used in cancer chemotherapy and as an immunosuppressant.122 CPA is metabolically acti- vated in vivo by the cytochrome P450 system to form 4-hydroxycyclophosphamide (4-OHCPA), iminocyc- lophosphamide (i-CPA), aldophosphamide (A-CPA), alcophosphamide (a-CPA) and N-dechloroethylcyc- lophosphamide ( N-dC1Et-CPA). Subsequent metabolism results in 4-ketocyclophosphamide (4-kCPA), chloroacetaldehyde, carboxyphosphamide (CPE), phosphoramide mustard (PM), acrolein, hydracrylic acid, nor-HN,, 3-(2-chloroethyl)-l,3-oxazolidin-2-one

and N-2-chloroethylaziridine (Fig. 1).

4-OHCPA is believed by most investigators of the mechanisms of action of CPA to be the primary extracel- lular mediator of antitumour a ~ t i v i t y . ~ - ~ Recently an important role for i-CPA has been demonstrated.6 Phar- macokinetics and metabolism of CPA have been found to vary widely;’.’ measurements of CPA and metabolites might be important in optimization of treatments with the alkylating agent.’.’’ Sensitive and selective assays are required then; to date methods of analysis include radioactivity measurements of l4C-labe1led CPA,” mass spectrometry,’2 gas chromatography/mass spectrometry (GC/MS),’3*14 gas-liquid chromatography with and without derivatization of CPA,”-’’ high performance liauid chromatography ( HPLC)20 and HPLC/field

t Author to whom correspondence should be addressed. 0887-6134/87/ 110643-06 $05.00

@ 1987 by John Wiley & Sons Ltd

desorption mass spectrometry.2’ GC is the most wide-ly used technique in clinical pharmacological studies, both with and without derivatization. Introduction of unchanged CPA into G C results in a derivative as depicted in Fig. 2; conditions for the reaction can be optimized in such a way that CPA can be determined in body fluids of CPA-treated patients properly without derivatization. 1 3 7 1 8 ~ 1 9

The selectivity of the assays with respect to other drugs regularly combined with CPA has been demonstrated. Taking metabolities of CPA into account, however, products like the ‘on-column’ product of CPA, the fundamental of the assay, might be formed and hinder selective analysis of CPA. Introduction of metabolities of CPA in capillary GC assays as described earlier by us1’ revealed that only 4-kCPA might interfere. Injection of 4-kCPA in a gas chromatographic system consisting of SCOT OV-275 capillary columns and electron capture detection resulted in two peaks if 4-LCPA solutions were prepared just before introduction into the analytical system (Fig. 3). The peaks were thought to be related to unchanged 4-kCPA and an ‘on-column’ product as presented in Fig. 4.

Besides formation of ‘on-column’ products, another point needs further investigation: the peak shape of peak 2, which was considerably aberrant of peaks with comparable retention times in SCOT OV 275 capillary columns under similar conditions.”

The data of the present investigations place particular emphasis on the behaviour of 4-kCPA during elution on SCOT OV-275 columns and implications for the selectivity of CPA analysis by capillary G C without derivatization.

Received 4 September 1986 Accepted 27 January 1987

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644 E. A. De BRUIJN ET AL. r 1 spon toneous

-

co~P(N,CH7CH,Cl [ ' O 'CH H CH,CI ] l l v e r mtcrosomes 0, NAOPH CyclophosphamldelCPAl' hver mlcrosomes

O,\NAOPH

9WOg supernotant o f laver homogenate

/

y, HOICY), CPNICH2CH2CIIz 1 0 mediated by cytochrome

P

Alcophosphomide

/1-1/1-11

-hjdr[y CPA

P,,

, - I m k e d 0-CHCH, C H, OPNlC H,C H2 C I I,

soluble froction of Mixed - f u n c t i o n oxidose k H z

llver cells 0, Aldophospham!de H C ) ~ ~ c ~ , C H , c i N-Oechloroethyl CPA CICH,C=O H Chloroocetoldehyde

e

o

-

HOCCH,CH,OPNICH,CH, CII, N", Corboxy,phorphamade

rpontoneous Phosphorod~amme esterose

or rpontoneour I , t o #i Aldehyde enzymatic' NH, hydrose I spontaneous $'

L-ketoCPA lmlno CPA

HNICH,CH,CII, - HO-~NICH,CH,CII, + CH,=CHC=O oxl$se '

.

HOCH,CH,?OH

Nor -HN Phosphoramide mustord. Acrolem

"'

Hydrocryhc actd

CH, CH,CI I

3-V -Chloroethyll-1,3 - N - 2 - Chloroethyloziridine

oxozoi~din-2-one

Figure 1. Metabolism of cyclophosphamide.

EXPERIMENTAL Chemicals

CPA and metabolites of interest were kindly donated by ASTA-Werke (Bielefeld, FRG). Solvents used were of analytical grade and obtained from Baker (Deventer, The Netherlands) and Merck (Darmstadt, FRG). Freshly prepared ethyl acetate samples were introduced into the column at 250 "C (falling needle) and 83 "C (on column).

Columns

A fused silica WCOT CP SIL 5CB column (25x

0.21 mm i d . ) was used for elution of 4-kCPA.

Mass Spectrometry

A Finnigan model 4000 quadrupole mass spectrometer (Finnigan, Sunnyvale, California, USA) was used both in the electron impact (EI) and chemical ionization (CI) mode. Mass spectral data were obtained under the following conditions: ionizing electron energy, 70 eV;

Figure 2. Cyclization process of CPA following injection on capillary GC.

electron current, 0.30 mA; scan time, 1 s; source temperature, 250 "C (EI) and 200 "C (CI). For the CI mode, NH3 reactant gas was introduced via the make-up gas line. The ion source was maintained at 0.15 Torr gauge

jll

_I T . . , . C PA

5

R (min) t

Figure 3. A chromatogram of 4-kCPA ( 1 ) and an 'on-column' prod- uct (2) and compounds not related to CPA and/or4-kCPA. 5-FUraH,: 5,6 dihydro-5-fluorouracil; 5-FUra: 5-fluorouracil; 5-CUra: 5- chlorouracil. H H H ,,0 c,H2 ,,o CICLC,2 N-C, c y c I i z o t i o n H 2 C , N-C, ,N-yS, ,CHz - W:=C CH,+ HCI CLC-c 0-c 01 CIC-c' 0-c' HZHZ H2 Hz Hz H2 4 - k e t o CPA

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SELECTIVITY OF CAPILLARY GAS CHROMATOGRAPHY OF CKETOCYCLOPHOSPHAMIDE * I 645

I

2 1 0

j

i o

f (min)

Figure 5. Chromatographic profile of 4-kCPA following on-column injection at 83 "C. Temperature programme: 83 "C to 225 "C (1.5 deg. C/S).

reading. The WCOT fused silica column was operated isothermally in MS experiments.

-

RESULTS AND DISCUSSION

A chromatogram obtained upon on-column injection at 83°C using a fused silica WCOT C P SIL-5 column followed by programmed temperature increase from 83 "C to 225 "C at 1.5 deg./s is presented in Fig. 5. Two peaks can be noted, with retention times ( tR) of 6.1 and 8.3 min, respectively. The peak area of peak 1 was less than 5% of peak 2. A slightly raised baseline between peaks 1 and 2 could be distinguished, indicating that on-column reactions as well as reactions in the injection system of 4-kCPA take place.

When a total ion current chromatogram of 4-kCPA was recorded using split injection at 250 "C under other- wise similar conditions, again two peaks were observed, with tR = 4.5 min and f R = 7.0 min (Fig. 6). The peak area

of peak 1 was 8% of peak 2. Furthermore, asymmetric peaks were observed in contrast to peaks of Fig. 5.

EI mass spectral fragmentation

The EI mass spectrum of peak 1 is given in Fig. 7(a). Fragment ions m / z 189, 161 and 117 were produced. The ions are degradation products of the M+ ion at

c l o o 0 800

t

c

0 100 200 300 LOO 500

f (s)

Figure 6. Total ion current chromatogram of 4-kCPA using solid injection at 250°C. A fused silica WCOT CP Sil-5 column (25x 0.21 mm i.d.) at 230 "C was used.

m / z 238, which was not observed owing to rapid degra- dation to the ion at m / z 189 by loss of CH2Cl. The EI mass spectrum of peak 2 is given in Fig. 7(b). Ions at m / z 225, 189, 163, 161, 134, 117 and 92 were formed. The fragmentation processes, together with their percentages as obtained from EI mass spectrometry, of peaks 1 and 2 are presented in Fig. 8.

CI mass spectral fragmentation

The CI mass spectrum of peak 1 is given in Fig. 9(a). Two ions were formed: MH+ ( m / z 239) and MNH: ( m / z 256). The CI mass spectrum of peak 2 is presented in Fig. 9(b). MH+ ( m / z 275) and MNH,' ( m / z 2 9 2 ) were formed, as well as the fragment ion MH'-HCI ( m / z 239). Pathways of degradation during capillary G C and C I mass spectrometry with percentages of products formed are depicted in Fig. 10.

As Figs 8 and 10 demonstrate, peak 1 is associated with a cyclization product of 4-kCPA produced during capillary chromatography, while selectivity towards unchanged 4-kCPA is sufficient. The comparable cyclization product of CPA and unchanged CPA could be well separated from 4-kCPA and its 'on-column' product. Moreover, the amount of the 'on-column' product of CPA is negligible when capillary G C with a temperature programme is used. Keto-enol tautomerism of 4-kCPA was thought to be related to the relatively low amounts of 'on-column' products of 4-kCPA as well as some forms of peak asymmetry. This possibility was

m/z m /I

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646 >r

2

600- al C

-

- 400 - 200- 1 .. E. A. De BRUIJN E T A L . I GCmoi n1238,GC meoh 1 L - k e t o C P A . m o l w t 2 i 4 . G C p e a k 2 _..' m / z 189, 100% m / i 2 2 5 , 100% m / i 134. 10%

Figure 8. Fragmentation processes of El mass spectrometry of 4-kCPA. 200 300 m /z (b)

1

1000 000 600 ? Ul

..

c - 275 100 2 00 300 rn /I

Figure 9. CI mass spectra of (a) peak 1 and (b) peak 2, as presented in Fig. 6. GC moi wt 238. GC p e o k 1 4 - k e t o C P A . m o l w l 2 7 4 , G C peok 2 MS I I

y--;A,

, , , ~ 214 214 226 0 100 200

Figure 11. Thermal desorption of 4-kCPA.

239

'"ooII

800 (b)

m /I

Figure 12. Thermal desorption mass spectra of (a) peak A and (b) peak 6, as presented in Fig. 11.

CI OH No t h e r m o l "L-, d e g r o d o t i o n

*

flN-'% CI C I I - k C P A . m / r 2 7 4 ECP H ,p

1

NH;KII CI H O ,,

3

C N 8 0

>&&L

- ' l C H 2 C H 2 N q O = $4 -HCI y-P:o] 15 %I m , z 225,100% -HCI m / z 1 3 4 , 5 % MH', r , ~ 239. 100% _{MH',m/z 275,100 %} 140 %I

/

\

#-P-0

h

i

CH, ' 0 a n d mjz189, 5 % ond C I ""?O cil,

M N H f , m / i 256, 1 0 %

[

Adduct EM * NHI, ions I*] M N H l . m i > 292, 10%

/

o l s o f o r m e d Minor f r a g m e n t ions

l?H; 0'

m j z 1 6 3 , 8% m,z92. 2 5 %

Figure 10. Fragmentation processes of CI mass spectrometry of

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SELECTIVITY OF CAPILLARY GAS CHROMATOGRAPHY OF 4-KETOCYCLOPHOSPHAMIDE ₆₄₇

further investigated by thermal desorption mass spectrometry and nuclear magnetic resonance (NMR). Measurements by thermal desorption (EI) mass spec- trometry with a fast temperature programme (0.15 A/s) and N M R confirmed the existence of keto-enol tautomerism at temperatures below 150°C, as can be seen in Figs 11, 12, 13 and 14. Both peaks (A and B), as depicted in Fig. 11, show identical mass spectra (Fig. 12(a) and (b)).

Figure 13 shows pathways of degradation during thermal desorption mass spectrometry, together with percentages of formed products. NMR data of 4-kCPA solutions in CDCI, finally give confirmation of the existence of keto-enol tautomerism as an OH signal at 1.65 ppm.

Tautomerism might also be responsible for peak asymmetry. However, peak broadening can increase to such an extent'' that absorption of one or both of the compounds of the tautomeric misture cannot be excluded. Implications of the keto-enol tautomerism of 4-kCPA with respect to in uiuo metabolism of 4-kCPA are cur- rently being investigated; indications for reversible CPA metabolism have now been found in which the tautomerism might play a key role.22

- B

I-.-

+A+

ip

J

O H

Figure 14. NMR spectrum of 4-kCPA in CDCI,.

CONCLUSIONS

The experimental evidence presented here strongly sup- ports the selectivity of capillary GC of underivatized CPA and 4-kCPA with respect to their 'on-column' products as well as unchanged compounds. A keto-enol tautomerism of 4-kCPA has been demonstrated at temperatures below 150 OC; the tautomerism is assumed to

be a determining factor for the behaviour of 4-kCPA during capillary GC.

Acknowledgements

The authors are grateful to ASTA-Werke, de 'Saal van Zwanenberg Stichting' and 'Linea Science'.

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