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QUANTITATIVE 252Cf-PDMS FOR ETOPOSIDE
H. Jungclas, K.-H. Pflüger, L. Schmidt, M. Hahn
To cite this version:
H. Jungclas, K.-H. Pflüger, L. Schmidt, M. Hahn. QUANTITATIVE 252Cf-PDMS FOR ETOPO- SIDE. Journal de Physique Colloques, 1989, 50 (C2), pp.C2-41-C2-46. �10.1051/jphyscol:1989208�.
�jpa-00229404�
QUANTITATIVE 2 5 2 ~ f - ~ ~ ~ S FOR ETOPOSIDE
H. J U N G C L A S , K.-H. PFLUGER, L. SCHMIDT* and M. HAHN
Fachbereich Humanmedizin PhiliDDs-Universit~t, 0-3550 Marburu, F.R.G.
Fachbereich Physikalische ~ h e i i e
philipps-~niversitdt .
0-3550 Marburg, F.R.G.RBsum6 - Les serum de patients traites par etoposide ont et6 marques par tbniposide, un compos4 analogue, puis extraits. . La purification des Bchantillons a 6tB obtenue par chromatographie en couches fines et l'analyse quantitative realisCe par 25ZCf-PDMS. L'addition de substances adequates en exces h la cible finale PDMS amkliore la sensibilite et la linearit6 de cette analyse quantitative.
Abstract - Serum samples from cancer patients, who were treated with etoposide, are labeled with the homologous compound teniposide as internal standard and extracted. Sample purification is achieved by thin layer chromatography and the quantitative analysis is performed by 2szCf-PDMS.
Adding suitable substances in excess to the f.inal PDMS target is an effective tool to improve the sensitivity and linearity of this quantitative analysis.
INTRODUCTION
The detection of pharmaceuticals in biological fluids like blood and urine requires a careful sample preparation and a sensitive detection system. A reasonable way for cleaning up the sample after extraction is thin layer chromatography (TLC). For the quantitative analysis a fluorescence, UV absorption or electrochemical detector is normally used in liquid chromatography, but mass spectrometry is a more specific method. Here TLC and 2 5 2 ~ f - ~ ~ ~ ~ (plasma desorption mass spectrometry) are operated in an off-line combination in order to perform a quantitative detection of pharmaceuticals in serum /1,2/.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1989208
C2-42 JOURNAL DE PHYSIQUE
Our research aims to investigate the pharma- cokinetics of some antitumor drugs in clinical use. Two of the common cytotoxic drugs are the epipodophyllotoxine derivatives etoposide (VP16-213) and teniposide (VM26), which are both active against a number of human malig- nant diseases /3/. Etoposide was introduced into clinical trials in the early 1970's and is now established in the treatment of several malig- nancies, including small cell lung cancer, germ cell tumors and lymphomas. However, the pharmacokinetic knowledge concerning both substances is still limited and the available information reveals marked interpatient variability. Both compounds show nearly the same physicochemical properties during the extraction (by chloroform) and detection process, thus one can be used as an internal standard for the analysis of the other /1,2/.
Etoposide Teniposide VP 16 - 213 VM 26
R -CH,
ANALYTICAL METHODS
The pharmacokinetic analysis is performed in a three-step procedure: sample extraction, sample purification by TLC and quantitative sample detection by time-of-flight mass spectrometry. The quantitative result is obtained by comparing the two mass lines corres- ponding to the two molecular ions at m/z 588 and m/z 656. The feasability of the method has been tested varying the concentration of etoposide from 1 to 100 pg cm-3 in a blank serum sample and observing a linear calibration curve /1,2/.
PHARMACOKINETIC RESULTS
Fifteen adult patients, thirteen males and two females, were included in the study after informed consent. Twentyeight 24h-kinetics were determined in these patients, who received parenterally administered etoposide in a dosis range of 80 - 120 mg m-2 (related to patient's surface in m2).
I n Table 3 the resulting mean values for some pharmacokinetic parameters are compared to earlier findings from other investigators. This comparison indicates similar individual deviations but shorter elimination half-lives with increasing specificity of the detection system /4/.
CIS: systemic clearance
determined by different methods. The given mean values are weighted by the number of patients, included in each study.
Method 3-~-labeled HPLC-UV HPLC-EC LC-MS
No. of patients 18 28 28 15
t?ha/h 0.71 1 .O 0.87 0.31
tl/,@/h 11.9 7.3 6.26 4.4
cls/cm3 min-I m-2 27.1 27.3 19.26 23.7
PROBLEMS OF THE QUANTITATIVE ANALYSIS
During the course of our quantitative kinetic measurements we run into serious problems, which affected the sensitivity and calibration. Both compounds ionize as M'. radicals and exhibit strong yield sensitivity on impurities in the prepared solid matrix. In spite of extraction and purification by TLC, the final samples can not be kept totally clear of such impurities. Another problem is the slightly different solubility of the internal standard teniposide, which causes a difference in the deposition profile. The resulting inhomo- genity of etoposide and teniposide influences the calibration curve.
In order to achieve more reliable and reproducible results in routine analysis, it became necessary to develop a sample preparation method, which allows to tolerate unclean conditions to a certain extend. According to our understanding the production of mole- cular ions is dominated by the decomposition of large metastable clusters and by a fast chemistry (= 1 ns) under highly excited conditions /5,6/. Thus we searched for additive substances, which are able to form clusters with the sample molecules, and which are volatile and fragile enough to support the decay and evaporation process of these clusters.
At the same time the additives might protect the sample molecules against the chemical attack of impurities in the excited cluster.
We concluded three criteria for the design of an optimal matrix for etoposide:
-
The additive should have a solubility in chloroform and ether comparable with etoposide and teniposide;- the additive should be added in excess without decreasing the molecular ion yield of the sample too much;
- the additive should not interfere with the sample in the mass spectrum.
C2-44 JOURNAL DE PHYSIQUE
NEW PREPARATION METHOD
Time -of - flight I n s
Fig.1 Three time-of-flight spectra of the same patient sample prepared in three different steps (see explanation in text).
Finally we found a combination of two substances, sucrose octaacetate and urotropin, which can be added in excess to pure etoposide/ teniposide samples without decreasing the molecular ion yield too much. Moreover, this mixture fulfills our requirements for an easy preparation and solubility in chloroform/ ether. Its protection ability against impuri- ties could also be proven by the successful analysis of numerous serum samples (some hundred) during the course of our pharmacokinetic studies.
600 j
- 400 1
a )
VP 16
M * VM 26
-
M +200 6
0 : .
C- 600 1
E -
9 - 400
U)
-
4-
C
= 200 '
0 0
0 1
600 <
- 400 1
-
200 4
678 + 23678 + 331
0
588 656
C )
sample after each of the following steps:
a) serum sample, separated by TLC, redissolved in chloroform : ether = 1: 1 and dried o n target foil;
b) 100 pg sucrose octaacetate (mol.wt. 678) with 50 pg urotropin (mol.wt. 140)
dissolved in chloroform : ether = 1: 1 and added to sample;
c) further treatment of the sample with 100 pg urotropin in chloroform.
After the third step the matrix contains the sample plus 250 pg additives.
Spectrum a) is noisy due to a variety of impurities in the sample. The yield of the two molecular cations at m/z 588 and 656 is rather low and Na-attachment is observed. After step b) the related signal-to-noise ratios are improved and the Na-attachment is totally suppressed. The yield of impurity cations is strongly reduced. The new line at m/z 701 is due to the added sucrose octaacetate attached by ~ a + , thus this additive adsorbs the Na- impurity. After step c) the important ratio of 588 to 656 intensities is significantly different from the initial value in a). The influence of the impurities is now minimized. The arising line at m/z 1009 corresponds to a composite ion of sucrose octaacetate and its own main fragment (m/z 331). The added urotropin does not interfere in the shown mass range, although it is the major matrix component and helps to mix the two interesting compounds.
CONCLUSION
In the etoposide analysis the matrix design is a n effective tool for the suppression of the uncontrolled influence of impurities on the molecular ion yield. From our experience with the optimization of the matrix we conclude the following generalized rules, how to select the proper additive substances.
The additive should:
- be more fragile than the sample molecules and have higher volatility;
- produce different ions, if possible opposite charge than the ions from sample compounds;
- be able to form metastable cluster ions, which decompose and emit molecular sample ions.
As sucrose octaacetate contributes to composite ions and urotropin shows a n extensive fragmentation pattern, these rules agree with our etoposide experiments. However, they are not yet proven in this generalized form.
JOURNAL DE PHYSIQUE
SUMMARY
The cluster desorption model /6/ showed us a way to solve the problems in the quantita- tive analysis of etoposide. The successful application of the matrix design confirms our conception of the desorption mechanism involved in the 2 5 2 ~ f - ~ ~ ~ ~ : - Fast chemical reactions within the desorbed metastable clusters influence the
production and release of heavy organic molecular ions.
- Each type of ion has its own special chemical formation history. Thus the chemical design of a matrix (additive) can increase certain ion yields while other ions may be suppressed.
The success in the quantitative analysis is also a success of the model. The cluster desorp- tion and decomposition must play an important role in the 2 5 2 ~ f - ~ ~ ~ ~ mechanism.
REFERENCES
H. Danigel, L. Schmidt, H. Jungclas: Biomed. Mass Spectrom. 12,542 (1985)
H. Danigel, K.-H. Pfluger, H. Jungclas, L. Schmidt, J. Dellbriigge: Cancer Chemother. Pharmacol. 15,121 (1985)
/3/ B. F. Issel: Cancer Chemother. Pharmacol. 2,73 (1982)
/4/ K.-H. Pfluger, L. Schmidt, M. Merkel, H. Jungclas, K. Havemann: Cancer Chemother. Pharmacol. 20.59 (1987)
/5/ L. Schmidt, H. Jungclas: in "PDMS and Clusters", E. R. Hilf, F. Kammer, K. Wien (eds.), Springer Lecture Notes in Physics 269,234 (1987)
/6/ L. Schmidt, H. Jungclas: in "Ion Formation from Organic Solids (IFOS IV)", A.
Benninghoven (ed.) J. Wiley, in press
Work supported by the Deutsche Forschungsgemeinschaft, Bonn.
