Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection I. Bartosek, M. Corada, S. Dallarda, C. Lucas, P. Deloffre & A. Guaitani To cite this article: I. Bartosek, M. Corada, S. Dallarda, C. Lucas, P. Deloffre & A. Guaitani (1991) Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection, Xenobiotica, 21:2, 235-242 To link to this article: http://dx.doi.org/10.3109/00498259109039465
Published online: 27 Aug 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California, San Diego]
Date: 07 November 2015, At: 04:04
XENOBIOTICA,
1991, VOL. 21,
NO.
2, 235-242
Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection I. BARTOSEKt, M. CORADAT, S. DALLARDAt C. LUCASS, P. DELOFFRES and A. GUAITANItO
t Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy Downloaded by [University of California, San Diego] at 04:04 07 November 2015
$ Institut de Recherches Internationales Servier, NeuilIy-sur-Seine Cedex, France Received 26 March 1990; accepted 12 August 1990
1. A differential pulse polarographic (DDP) assay of diethyl-l-[3-(2-~hloroethy1)-3nitrosoureido] ethylphosphonate (fotemustine) was developed to determine the kinetics of this nitrosourea in plasma, brain, liver, lung and kidney. The optimized polarographic determination, previously applied to BCNU and CCNU, attained a limit of detection of 0.3 pg fotemustine/ml plasma and 1pg/g in other tissues; the calibration curve in electrolyte or in plasma was linear between 0 5 and 100pg/mI.
2. The choice of electrolyte, the effects of pH, temperature, light, and the stability of fotemustine in samples were investigated. Recovery of fotemustine was 7690% from lung > kidney > plasma > brain >liver; the variability coefficients were low (4~67.3%). Tissue samples could be stored for 20 days at -20°C without loss of the compound. 3. Plasma kinetics of fotemustine and BCNU given to male rats at therapeutic doses (20mg/kg i.v.) fitted a bi-exponential equation. Two minutes after injection plasma, levels of unchanged nitrosoureas were 15 and 11pg/ml respectively. Fotemustine could be measured (092pg/ml) for 3 h, while BCNU could not be detected after 60 min. Unchanged fotemustine was cleared from the blood stream 3-5 times more slowly than BCNU.
Introduction Fotemustine (diethyl- 1-[3-(2-chloroethyl)-3 -nitrosoureido]ethylphosphonate; S10036) is a new nitrosourea which has been synthesized in order to achieve better penetration of the cell membrane and better passage through the blood/brain barrier (Foster and Pardee 1969, Montgomery and Mayo 1974). Fotemustine is more active than other nitroureas against several experimental tumour models (Reed 1984, Cudennec et al. 1987, Alvarez et al. 1988, Filippeschi el al. 1988). Clinical phase 1-11 studies (Khayat et al. 1987, 1988) have shown high response rates in patients with disseminated malignant melanoma, primary and metastatic brain tumours, and haematological malignancies; the clinical activity has been confirmed by large multicentre studies (Jacquillat et al. 1988). As expected for a nitrosourea, the chemotherapeutic activity is accompaned by delayed, dose-related, cumulative toxic effects. Therefore the choice of doses and treatment schedules must be based on accurate pharmacokinetic studies to establish the safety and efficacy of maintenance therapy. Most chloroethylnitrosoureas are rapidly cleared from plasma, the rate of total clearance being related to their tissue distribution and chemical reactivity. Current assays of chloroethylnitrosoureas are based on the use of radiolabelled compounds (Levin et al. 1978, Freed et al. 1982), mass spectrometric techniques (Smith et al. 1981, 1983, Smith and Cheung 1982), normal or reverse-phase high-performance liquid chromatography (Caddy et al. 1982, Yeager et al. 1984, Labarre et al. 1987, Polacek et al. 1988).
8 Address correspondence to: A. Guaitani, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62 201 57 Milan, Italy.
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
236
J. Bartosek
et al.
Recently a new h.p.1.c. procedure was developed to measure fotemustine (Gordon et al. 1989). A differential pulse polarographic assay, previously proposed for BCNU (1,3bis(2-chloroethyl)-l -nitrosourea) and CCNU (1-(2-chloroethyl)-3-cyclohexyl-lnitrosourea) determinations (Bartoseket al. 1978,1984,Russo et al. 1981,1984),has now been optimized and used for fotemustine assay in biological samples. This analytical technique offers high specificity for measuring the novel nitrosourea, fotemustine, in various tissues within a wide range of linearity, with good recovery and accuracy. Although the method described in this paper is slightly less sensitive than other analytical procedures (Gordon et al. 1989),it is suitable for routine assays, and is less expensive. T h e method is proposed both for preclinical studies in animals and for clinical pharmacokinetics in patients.
Materials and methods Instrumentation Measurements were made using a modular polarograph Bruker Spectrospin E 3 10 (Karlsruhe, Germany) with standard polarographic vessels (Metrohm E 607 Herisau, Switzerland) and a threeelectrode arrangement (dropping mercury, platinum auxiliary and saturated calomel electrode). An X-Y recorder (Hewlett-Packard 7010A) was connected with the polarograph for plotting current-potential curves. A particular module was employed for the 'three-point' reading of polarographic currents at preset potentials. A rotating extractor (60r.p.m.) was used for plasma and tissue homogenate extraction to prevent formation of water-solvent emulsions. Chemicals Fotemustine was supplied by Servier (Neuilly-sur-Seine, France) and BCNU was provided by Simes Milan, Italy). Both compounds were stored at - 20°C in the dark. Fotemustine was provided as a solution in ethanol at 50mg/ml. All reagents (methylene chloride, ethanol, n-pentane, citric acid and sodium citrate) were analytical grade. All solutions were prepared in bi-distilled water. Tri-distilled, commercially supplied mercury was used for the dropping-mercury electrode, and oxygen was removed from polarography samples by highpurity nitrogen. Animals Male Cr1:CD (SD) BR rats (Charles River Italy, Calco), 6-7 weeks old, 18&200g, were housed under standard conditions (22? l"C, 55-65% relative humidity, 12/12h light/dark cycle) and fed an 'open formula' diet (Altromin, Rieper, Italy) with water ad libitum. Procedures Sample preparation. Blood was collected from animals under light ether anaesthesia, mixed with 3.8% sodium citrlite (10 : 1 v/v) adjusted to pH 5.5 with HCl, cooled in ice and centrifuged (IOmin, 300g, 4°C). T h e organs (liver, kidney, lung, and brain) were rapidly removed, weighed, minced and homogenized with 0.1 M citric acid, p H 2 (1 : 5). T h e plasma samples or tissue homogenates were frozen and stored in the dark at -20°C. Fofemustine extroction. Samples (1 ml) were rapidly thawed (37°C) under shaking and extracted three times with 5 ml of methylene chloride on a rotating extractor (60 r.p.m., 5 min). T h e combined extracts were evaporated to dryness in a water bath at 40°C under a nitrogen stream then 10 ml of electrolyte ( 0 1 M citric acid pH 2) were added, keeping the samples in darkness all the time. BCNU was extracted from plasma with n-pentane (three times) as described previously (Bartosek et ol. 1984). Polorographzc ussay. The optimum instrumental parameters were: differential pulse 100mV, drop time 1.5 sec, mercury column height 80cm. T h e sweep interval for the measurement of fotemustine was between -250 and -650mV; the potentials for 'three-point' reading of the polarographic current were Imi,= - 350 mV, Ipepk = - 478 mV and I,,, = -605 mV. Polarographic assay of BCNU was as described (Imin= -350mV,ICesk= -500mV, I,,,= -650mV)(Bartoseket al. 1978,1984, Russoetal. 1981,1984). Calculation of nitrosourea concentration. The height of the differential pulse polarographic peak is proportional to the drug concentration. T h e total height of the peak of the sample (T&,reference (T,) or
Polarographic determination of fotemustine
237
blank (Tb) is calculated from the polarographic current (I) measured at three potentials: ( T s ,T , or Tb) - Ipear- 0 5 (Imin Imax).The net height corresponds to the difference between sample and blank total heights H , (or H,) = T,(or Tr)- Tb.Comparison of the net height of the sample (H,) with the net height of the reference (H,)having a known concentration of nitrosourea (C,) gives the concentration of the sample C, = H , x C,/h.
+
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
Pharmacokinetics. T h e dose, 20 mg/kg body wt, was injected in a rapid bolus (10-20 s) in the rat tail vein. Blood for fotemustine and BCNU analysis was collected at pre-set intervals (2, 5, 10, 15, 20, 30, 60, 120 and 180min) as described above. Blood levels of fotemustine and BCNU were fitted by a bi-exponential equation (see details in legend to table 3). The NL-Fit computer program (Sacchi-Landriani et al. 1983) was used for calculating the kinetic constants.
Results and discussion Efiect of p H and basic electrolyte A solution of 0.1 M KCl with 0 0 1 M citric acid was used initially as the electrolyte. T h e pH of this solution was adjusted to different values between 1.4 and 11.2. T h e concentrated solution of fotemustine was added to the electrolyte and a differential pulse polarogram was recorded between 0.0 and - 1500 mV. T h e peak was highest at pHl.46. With increasing pH, the peak became lower and was slightly shifted towards more negative potentials (figure 1). Starting from p H values higher than 3.0 a secondary peak appeared at about -950mV. T h e second polarographic wave indicates the intrinsic instability common to the nitrosoureas (Reed 1984) and also described for fotemustine (Gordon et al. 1989). Thus the p H of all samples was immediately adjusted to 2.0, before freezing, in order to avoid decomposition of the drug in the biological matrix. T h e secondary reduction step might be due to the presence of another electrochemically active form of the nitrosourea at higher pH values. In the light of our previous experience with citric acid, the heights of the fotemustine peaks were recorded at different concentrations (0.025-0.5 M) of citric acid buffered to p H 2.0. No polarographic signal could be detected when the possible products of decomposition (e.g . 2-chloroethylamine and 2-chloroethyl-isocyanate) (Bartosek et al. 1984) were measured at the conditions set up for fotemustine assay.
p H 1.46
0
POTENTIAL, v
- 1.500
Figure 1. Differential pulse polarograms of fotemustine showing dependence on p H from 1.46 to 5.35. The pH of the solution of 0.1 M KCI with 0.01 M citric acid was adjusted to different values.
J . Bartosek et al.
238
T
t-0.025M
0.05M 0.1 M
3uunH t
+- O.2M
Z w
+- 0.5M
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
a CK 3 V
0
-0.35 -0.50
-0.70
POTENTIAL, v Figure 2 .
Effect of citric acid concentration on the height of the polarographic peak of foternustine.
The concentrations of basic electrolyte, buffered to pI'I 2.0, for the curves (from top to bottom) werc 0425, 0.05, 0.1, 0.2 and 0 . 5 ~ .
T h e height of the nitrosourea peak decreased with increasing concentrations of citric acid (figure 2). Thus, 0.1 M citric acid was chosen as a compromise solution to maintain stable p H and to reach good sensitivity.
Optimizution of polarogrupphic analysis of fotemustine Initially fotemustine was added to rat plasma (10, 50, lOOpg/ml) and samples were analysed at room temperature in the light. In such conditions the total recovery decreased with increasing concentrations of fotemustine in the sample: recovery was 98-1 10%at 10pg/ml but dropped to 81%and 73% at 50 and 100pg/ml, respectively. When fotemustine was added directly to the electrolyte, the detection limit was close to the range measured by the h.p.1.c. technique (Gordon et ul. 1989) (i.e. 300ng). However, at concentrations lower than 1 pg/ml and higher than 100pg/ml plasma, the calibration curve was not linear because complete drug extraction was hampered by formation of a solvent-protein emulsion. Very few authors have considered the possible effect of light on nitrosourea stability. O ur experience indicates that the photosensitivity of fotemustine must be borne in mind, together with temperature and pH of the solution. Both light and temperature seemed to influence recovery of fotemustine (table 1). Statistical analysis o f the results supports the evidence that these factors have additive effects. Rccovery was higher when the samples were kept in darkness during extraction at room temperature. When the extraction and evaporation were done at O°C, however, the reproducibility and recovery were both lower, probably becaupe of lower solubility of the drug. Recovery of fotemustine from various tissues (lOOpg of nitrosourea were added per ml of homogenate) was: 76% in liver, 78% in brain, 83%
Polarographic determination of fotemustine Table 1.
Effect of temperature and darkness on the polarographic assay of fotemustine
Room temperature +room lighta
Experimental conditions Fotemustine 100pg extracted from citrate buffer
Ice bath +room lightb
Room temperature +darkness
77 If: 4.7 p g
Ice bath +darkness
li
7*-,
90 k 2.6 p g
95 f 2.4 p g
98 & 2.9 p g
,
#
Foternustine 10Opg extracted from rat plasma
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
239
#
69 & 3 9 p g
48
5.0 pcg
84 1.9 p g
6 7 f 6 1 pg
\-
-##A
MeanfSEM, n=6. T h e same amount of fotemustine was either assayed directly (and considered reference for the other conditions) or added to citrate buffer o r to rat plasma (acidified to p H 3.8) and extracted. a Extraction was made at room temperature and methylene chloride was evaporated under nitrogen at 40°C. Extraction and evaporation procedures were performed at 0°C. Statistical analysis of the effects that influence fotemustine assay was done by two-way ANOVA. Differences between means were tcsted by Tukey's procedure (*P
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection I. Bartosek, M. Corada, S. Dallarda, C. Lucas, P. Deloffre & A. Guaitani To cite this article: I. Bartosek, M. Corada, S. Dallarda, C. Lucas, P. Deloffre & A. Guaitani (1991) Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection, Xenobiotica, 21:2, 235-242 To link to this article: http://dx.doi.org/10.3109/00498259109039465
Published online: 27 Aug 2009.
Submit your article to this journal
Article views: 9
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California, San Diego]
Date: 07 November 2015, At: 04:04
XENOBIOTICA,
1991, VOL. 21,
NO.
2, 235-242
Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection I. BARTOSEKt, M. CORADAT, S. DALLARDAt C. LUCASS, P. DELOFFRES and A. GUAITANItO
t Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy Downloaded by [University of California, San Diego] at 04:04 07 November 2015
$ Institut de Recherches Internationales Servier, NeuilIy-sur-Seine Cedex, France Received 26 March 1990; accepted 12 August 1990
1. A differential pulse polarographic (DDP) assay of diethyl-l-[3-(2-~hloroethy1)-3nitrosoureido] ethylphosphonate (fotemustine) was developed to determine the kinetics of this nitrosourea in plasma, brain, liver, lung and kidney. The optimized polarographic determination, previously applied to BCNU and CCNU, attained a limit of detection of 0.3 pg fotemustine/ml plasma and 1pg/g in other tissues; the calibration curve in electrolyte or in plasma was linear between 0 5 and 100pg/mI.
2. The choice of electrolyte, the effects of pH, temperature, light, and the stability of fotemustine in samples were investigated. Recovery of fotemustine was 7690% from lung > kidney > plasma > brain >liver; the variability coefficients were low (4~67.3%). Tissue samples could be stored for 20 days at -20°C without loss of the compound. 3. Plasma kinetics of fotemustine and BCNU given to male rats at therapeutic doses (20mg/kg i.v.) fitted a bi-exponential equation. Two minutes after injection plasma, levels of unchanged nitrosoureas were 15 and 11pg/ml respectively. Fotemustine could be measured (092pg/ml) for 3 h, while BCNU could not be detected after 60 min. Unchanged fotemustine was cleared from the blood stream 3-5 times more slowly than BCNU.
Introduction Fotemustine (diethyl- 1-[3-(2-chloroethyl)-3 -nitrosoureido]ethylphosphonate; S10036) is a new nitrosourea which has been synthesized in order to achieve better penetration of the cell membrane and better passage through the blood/brain barrier (Foster and Pardee 1969, Montgomery and Mayo 1974). Fotemustine is more active than other nitroureas against several experimental tumour models (Reed 1984, Cudennec et al. 1987, Alvarez et al. 1988, Filippeschi el al. 1988). Clinical phase 1-11 studies (Khayat et al. 1987, 1988) have shown high response rates in patients with disseminated malignant melanoma, primary and metastatic brain tumours, and haematological malignancies; the clinical activity has been confirmed by large multicentre studies (Jacquillat et al. 1988). As expected for a nitrosourea, the chemotherapeutic activity is accompaned by delayed, dose-related, cumulative toxic effects. Therefore the choice of doses and treatment schedules must be based on accurate pharmacokinetic studies to establish the safety and efficacy of maintenance therapy. Most chloroethylnitrosoureas are rapidly cleared from plasma, the rate of total clearance being related to their tissue distribution and chemical reactivity. Current assays of chloroethylnitrosoureas are based on the use of radiolabelled compounds (Levin et al. 1978, Freed et al. 1982), mass spectrometric techniques (Smith et al. 1981, 1983, Smith and Cheung 1982), normal or reverse-phase high-performance liquid chromatography (Caddy et al. 1982, Yeager et al. 1984, Labarre et al. 1987, Polacek et al. 1988).
8 Address correspondence to: A. Guaitani, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62 201 57 Milan, Italy.
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
236
J. Bartosek
et al.
Recently a new h.p.1.c. procedure was developed to measure fotemustine (Gordon et al. 1989). A differential pulse polarographic assay, previously proposed for BCNU (1,3bis(2-chloroethyl)-l -nitrosourea) and CCNU (1-(2-chloroethyl)-3-cyclohexyl-lnitrosourea) determinations (Bartoseket al. 1978,1984,Russo et al. 1981,1984),has now been optimized and used for fotemustine assay in biological samples. This analytical technique offers high specificity for measuring the novel nitrosourea, fotemustine, in various tissues within a wide range of linearity, with good recovery and accuracy. Although the method described in this paper is slightly less sensitive than other analytical procedures (Gordon et al. 1989),it is suitable for routine assays, and is less expensive. T h e method is proposed both for preclinical studies in animals and for clinical pharmacokinetics in patients.
Materials and methods Instrumentation Measurements were made using a modular polarograph Bruker Spectrospin E 3 10 (Karlsruhe, Germany) with standard polarographic vessels (Metrohm E 607 Herisau, Switzerland) and a threeelectrode arrangement (dropping mercury, platinum auxiliary and saturated calomel electrode). An X-Y recorder (Hewlett-Packard 7010A) was connected with the polarograph for plotting current-potential curves. A particular module was employed for the 'three-point' reading of polarographic currents at preset potentials. A rotating extractor (60r.p.m.) was used for plasma and tissue homogenate extraction to prevent formation of water-solvent emulsions. Chemicals Fotemustine was supplied by Servier (Neuilly-sur-Seine, France) and BCNU was provided by Simes Milan, Italy). Both compounds were stored at - 20°C in the dark. Fotemustine was provided as a solution in ethanol at 50mg/ml. All reagents (methylene chloride, ethanol, n-pentane, citric acid and sodium citrate) were analytical grade. All solutions were prepared in bi-distilled water. Tri-distilled, commercially supplied mercury was used for the dropping-mercury electrode, and oxygen was removed from polarography samples by highpurity nitrogen. Animals Male Cr1:CD (SD) BR rats (Charles River Italy, Calco), 6-7 weeks old, 18&200g, were housed under standard conditions (22? l"C, 55-65% relative humidity, 12/12h light/dark cycle) and fed an 'open formula' diet (Altromin, Rieper, Italy) with water ad libitum. Procedures Sample preparation. Blood was collected from animals under light ether anaesthesia, mixed with 3.8% sodium citrlite (10 : 1 v/v) adjusted to pH 5.5 with HCl, cooled in ice and centrifuged (IOmin, 300g, 4°C). T h e organs (liver, kidney, lung, and brain) were rapidly removed, weighed, minced and homogenized with 0.1 M citric acid, p H 2 (1 : 5). T h e plasma samples or tissue homogenates were frozen and stored in the dark at -20°C. Fofemustine extroction. Samples (1 ml) were rapidly thawed (37°C) under shaking and extracted three times with 5 ml of methylene chloride on a rotating extractor (60 r.p.m., 5 min). T h e combined extracts were evaporated to dryness in a water bath at 40°C under a nitrogen stream then 10 ml of electrolyte ( 0 1 M citric acid pH 2) were added, keeping the samples in darkness all the time. BCNU was extracted from plasma with n-pentane (three times) as described previously (Bartosek et ol. 1984). Polorographzc ussay. The optimum instrumental parameters were: differential pulse 100mV, drop time 1.5 sec, mercury column height 80cm. T h e sweep interval for the measurement of fotemustine was between -250 and -650mV; the potentials for 'three-point' reading of the polarographic current were Imi,= - 350 mV, Ipepk = - 478 mV and I,,, = -605 mV. Polarographic assay of BCNU was as described (Imin= -350mV,ICesk= -500mV, I,,,= -650mV)(Bartoseket al. 1978,1984, Russoetal. 1981,1984). Calculation of nitrosourea concentration. The height of the differential pulse polarographic peak is proportional to the drug concentration. T h e total height of the peak of the sample (T&,reference (T,) or
Polarographic determination of fotemustine
237
blank (Tb) is calculated from the polarographic current (I) measured at three potentials: ( T s ,T , or Tb) - Ipear- 0 5 (Imin Imax).The net height corresponds to the difference between sample and blank total heights H , (or H,) = T,(or Tr)- Tb.Comparison of the net height of the sample (H,) with the net height of the reference (H,)having a known concentration of nitrosourea (C,) gives the concentration of the sample C, = H , x C,/h.
+
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
Pharmacokinetics. T h e dose, 20 mg/kg body wt, was injected in a rapid bolus (10-20 s) in the rat tail vein. Blood for fotemustine and BCNU analysis was collected at pre-set intervals (2, 5, 10, 15, 20, 30, 60, 120 and 180min) as described above. Blood levels of fotemustine and BCNU were fitted by a bi-exponential equation (see details in legend to table 3). The NL-Fit computer program (Sacchi-Landriani et al. 1983) was used for calculating the kinetic constants.
Results and discussion Efiect of p H and basic electrolyte A solution of 0.1 M KCl with 0 0 1 M citric acid was used initially as the electrolyte. T h e pH of this solution was adjusted to different values between 1.4 and 11.2. T h e concentrated solution of fotemustine was added to the electrolyte and a differential pulse polarogram was recorded between 0.0 and - 1500 mV. T h e peak was highest at pHl.46. With increasing pH, the peak became lower and was slightly shifted towards more negative potentials (figure 1). Starting from p H values higher than 3.0 a secondary peak appeared at about -950mV. T h e second polarographic wave indicates the intrinsic instability common to the nitrosoureas (Reed 1984) and also described for fotemustine (Gordon et al. 1989). Thus the p H of all samples was immediately adjusted to 2.0, before freezing, in order to avoid decomposition of the drug in the biological matrix. T h e secondary reduction step might be due to the presence of another electrochemically active form of the nitrosourea at higher pH values. In the light of our previous experience with citric acid, the heights of the fotemustine peaks were recorded at different concentrations (0.025-0.5 M) of citric acid buffered to p H 2.0. No polarographic signal could be detected when the possible products of decomposition (e.g . 2-chloroethylamine and 2-chloroethyl-isocyanate) (Bartosek et al. 1984) were measured at the conditions set up for fotemustine assay.
p H 1.46
0
POTENTIAL, v
- 1.500
Figure 1. Differential pulse polarograms of fotemustine showing dependence on p H from 1.46 to 5.35. The pH of the solution of 0.1 M KCI with 0.01 M citric acid was adjusted to different values.
J . Bartosek et al.
238
T
t-0.025M
0.05M 0.1 M
3uunH t
+- O.2M
Z w
+- 0.5M
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
a CK 3 V
0
-0.35 -0.50
-0.70
POTENTIAL, v Figure 2 .
Effect of citric acid concentration on the height of the polarographic peak of foternustine.
The concentrations of basic electrolyte, buffered to pI'I 2.0, for the curves (from top to bottom) werc 0425, 0.05, 0.1, 0.2 and 0 . 5 ~ .
T h e height of the nitrosourea peak decreased with increasing concentrations of citric acid (figure 2). Thus, 0.1 M citric acid was chosen as a compromise solution to maintain stable p H and to reach good sensitivity.
Optimizution of polarogrupphic analysis of fotemustine Initially fotemustine was added to rat plasma (10, 50, lOOpg/ml) and samples were analysed at room temperature in the light. In such conditions the total recovery decreased with increasing concentrations of fotemustine in the sample: recovery was 98-1 10%at 10pg/ml but dropped to 81%and 73% at 50 and 100pg/ml, respectively. When fotemustine was added directly to the electrolyte, the detection limit was close to the range measured by the h.p.1.c. technique (Gordon et ul. 1989) (i.e. 300ng). However, at concentrations lower than 1 pg/ml and higher than 100pg/ml plasma, the calibration curve was not linear because complete drug extraction was hampered by formation of a solvent-protein emulsion. Very few authors have considered the possible effect of light on nitrosourea stability. O ur experience indicates that the photosensitivity of fotemustine must be borne in mind, together with temperature and pH of the solution. Both light and temperature seemed to influence recovery of fotemustine (table 1). Statistical analysis o f the results supports the evidence that these factors have additive effects. Rccovery was higher when the samples were kept in darkness during extraction at room temperature. When the extraction and evaporation were done at O°C, however, the reproducibility and recovery were both lower, probably becaupe of lower solubility of the drug. Recovery of fotemustine from various tissues (lOOpg of nitrosourea were added per ml of homogenate) was: 76% in liver, 78% in brain, 83%
Polarographic determination of fotemustine Table 1.
Effect of temperature and darkness on the polarographic assay of fotemustine
Room temperature +room lighta
Experimental conditions Fotemustine 100pg extracted from citrate buffer
Ice bath +room lightb
Room temperature +darkness
77 If: 4.7 p g
Ice bath +darkness
li
7*-,
90 k 2.6 p g
95 f 2.4 p g
98 & 2.9 p g
,
#
Foternustine 10Opg extracted from rat plasma
Downloaded by [University of California, San Diego] at 04:04 07 November 2015
239
#
69 & 3 9 p g
48
5.0 pcg
84 1.9 p g
6 7 f 6 1 pg
\-
-##A
MeanfSEM, n=6. T h e same amount of fotemustine was either assayed directly (and considered reference for the other conditions) or added to citrate buffer o r to rat plasma (acidified to p H 3.8) and extracted. a Extraction was made at room temperature and methylene chloride was evaporated under nitrogen at 40°C. Extraction and evaporation procedures were performed at 0°C. Statistical analysis of the effects that influence fotemustine assay was done by two-way ANOVA. Differences between means were tcsted by Tukey's procedure (*P
