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Current Medical Research and Opinion
Vol. 4, No. 1, 1976
The determination of azapropazone in blood plasma
H. Leach, M.Sc.
Curr. med. Res. Opin., (1976), 4, 35.
Department of Biochemistry, University College of North Wales, and The Caernarvon and Anglesey General Hospital, Bangor, Wales
Received: 16th September 1975
Summary Three methods for the determination of azapropazone were studied for the analysis of blood plasma samples taken after oraladministration of azapropazone. A gas chromatographic method was compared with two simple spectrophotoinetric assay methods and the efSect of metabolites of the drug on each method is described. It is concluded that nzetabolites are unlikely to cause signiJicant interference with the two simple methods and that either is suitable for determination of plasma levels of the drug. Some problems associated with a gas chromatographic method are described and the comparative results of the examination of plasma samples using these methods are given. Keywords: Azapropazone -plasma levels - dosage - assay
Introduction For the determination of the concentration of a drug in body fluids there is, in general, considerable merit in choosing the simplest method of analysis which can give the required accuracy and reproducibility. Not only can large numbers of samples be handled expeditiously, analytical simplicity allows for replicate analysis with a consequent improvement in precision. The major objection which can often be levelled against simple methods is lack of specificity, and much of the elaboration introduced into analytical procedures is an attempt to improve this situation. Spectrophotometric methods in the ultra-violet or visible spectrum may include metabolites in the result if metabolites with the intact chromophore of the parent drug are present, and this may also happen when labelled compounds are used with radio-isotopic methods of analysis. To ensure specificity it may be necessary to introduce a separation stage in the analytical procedure or to use a method employing gas chromatography where separation and quantification are combined ; in either event there will be some loss in simplicity. If, however, chromatography can be used to demonstrate that metabolites are unlikely to cause serious interference, either because levels are insignificant or the metabolites do not appear in solvent extracts, much simpler methods may be applicable. In the course of studies on the plasma concentration of the antirheumatic drug azapropazone this approach was the choice. Initially, the method described by Schatz et aL3 was selected and compared with a gas chromatographic method of
The determination of azapropawne in blood plasma
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analysis and, in later studies, the simple colourimetricmethod describedby Farrier’ was also used.
Materials and methods Subjects All the subjects engaged in these experiments were young male and female volunteers aged between 19 and 26 years. They were in good health with no history of any gastric disorders. With the exception of one experiment when the dose of drug was taken before food all were instructed to take the dose of azapropazone after food, and they were asked to abstain from very fatty meals during the appropriate times during the experiment.
Drug Azapropazone in the form of 300 mg. capsules of azapropazone dihydrate was used. Within an experiment a single batch of capsules was used. Sampling Blood was taken from the antecubital vein and collected in lithium heparin anticoagulant. It was immediately centrifuged and the plasma removed to a siliconed glass vial. If the analysis could not be performed immediately the sample was stored at -20°C. Analytical methods (a) Gas chromatography. 2 ml. of plasma was added to 2 ml. of 0.1 M phosphoric acid and extracted by shaking vigorously with 20 ml. of dichloroethane. The solvent was removed to a clean tube and a second extraction with 20 ml. of solvent performed. The combined solvent extracts were then extracted with 10 ml. of 0.5 M sodium hydroxide in two 5 ml. portions. The combined alkali extracts were acidified with 5 N phosphoric acid and the azapropazone extracted into 10 ml. of dichloroethane. The solvent extract was evaporated to dryness in a fine pointed centrifuge tube or Dreyer’s tube in a heating block at 60°C using a stream of nitrogen to assist evaporation and exclude air. The residue was dissolved in 20 PI. of chloroform containing 1 mg. per ml. of C22hydrocarbon as an internal standard. Gas chromatography was done immediately and the concentration calculated from the relative peak areas of azapropazone and internal standard. A calibration curve was constructed by analysing plasma samples containing added azapropazone. The standards contained 20 pg., 50 pg. and 80 pg. azapropazone dihydrate per ml. and all were subjected to exactly the same analytical procedure as the test samples. Gas chromatography was done on 2.5% SE-30 on acid washed and silanised Chromosorb W. Columns were glass, 5 ft. (1.5 m.) in length and 0.25 inches (6 mm.) O.D. A Pye 104 instrument was used with flame ionisation detector. Gas flows were: carrier (nitrogen) 50 ml. per min; hydrogen 50 ml. per min; air 300 mi. per minute. 36
H.Leach
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The oven temperature was 225°C and the detector oven was maintained at 300°C. No injection point heater was used and samples were injected directly into the column packing. Under these conditions the retention time of azapropazone was 7.3 min. and the internal standard 3.4 min. (b) Spectrophotometric assay. The spectrophotometric procedure of Schatz et was used with minor modifications. 0.5 ml. of plasma was added to 4.5 ml. of methanol and, after shaking, the tube was closed with a screw cap and placed in a heating block at 100°C for 2 minutes with occasional shaking. After centrifuging until the supernatant was clear, the solvent was removed and scanned between 450 nm. and 225 nm. in a Unicam S.P.1800 spectrophotometer. An extract prepared from the zero time plasma sample was used as the blank. The concentration of azapropazone was determined from the absorbance reading at about 255 nm. A calibration curve was prepared from blank plasma samples containing added azapropazone from 10 to 100 pg. per ml. All results were the mean of parallel duplicates. Ethanol was also used as the protein precipitant and extractant with no significant change. Schatz et al. read the samples against methanol and subtracted the blank value from the test readings. It was found that more satisfactory precision was obtained if the blank plasma extract (zero time sample) was used as the reference solution. (c) Colourimetric assay method. This was the method of Farrier' with slight modification. To 6 ml. of methanol, 2 ml. of plasma was added and after vigorous mechanical shaking for 3 min. the mixture was centrifuged and the supernatant decanted into a clean centrifuge tube. The protein precipitate was extracted with a further 5 ml. of methanol and centrifuged. The combined extracts were evaporated to dryness in a heating block at 70°C with the aid of a stream of nitrogen and finally dried over phosphorus pentoxide in a dessicator for 1 hour. The surfactant BBS-3 (Beckman Instruments Company) was diluted 1 in 3 with methanol: 2 ml. of this solution was added to the residue and solution assisted by heating to 70°C; I ml. of sodium hypochlorite solution (6 % available chlorine) was added and after gentle mixing and centrifuging the supernatant was removed for photometry. The solutions were read at 450 nm. against a blank prepared from the zero time sample. A calibration curve of azapropazone dihydrate showed a straight line relationship between absorbance and concentration to above 200 pg. per ml. (d) Thin-layer chromatography for metabolites. Plasma samples taken after the administration of azapropazone for several days were pooled and 5 volumes of methanol added. After heating to boiling and frequent shaking the mixture was Centrifuged and the clear supernatant removed. The precipitate was extracted with a further 5 volumes of methanol and the combined extracts evaporated to a small volume in a rotary vacuum evaporator. The solution was transferred to a small tube and evaporation completed in a stream of nitrogen. 37
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The determination of azapropazone in blood plasma
The residue was dissolved in 50 pg, of methanol and centrifuged. The whole of the extract was applied to a Merck Kieselgel F254plate and reference spots of azar ropazone and 6-hydroxyazapropazone were applied either side of the sample streak. The plate was developed in a solvent mixture of ethyl acetate: ethanol: methylchloride: formic acid (40:5:45:10) until the solvent had run about 10 cm. The dried plates were then examined with short wave ultra-violet light and then sprayed with a location reagent of ferric chloride-potassium ferricyanide.
Results and discussions In the course of some early studies, 6 volunteers ingested 600 mg. azapropazone and blood samples were collected at zero time for the blank and after 2.5 hrs., 5 hrs., 8 hrs. and 48 hrs. Plasma levels of azapropazone were determined in duplicate by the ultra-violet spectrophotometric method described. The mean plasma levels found are shown in Figure 1. Figure 1. Mean plasma concentration of azapropazone from 6 subjects after the administration of 600 mg. of the drug, (mean dose 8.4 mg./kg.): ultra-violet spectrophotometry 50
1
Hour.\ after close
Samples of pooled plasma from this experiment were also examined by the gas chromatographic method and no significant difference was found between the two methods. There was no evidence in the gas chromatogram of any substance other than azapropazone and it appeared unlikely that any metabolites were contributing to the results obtained by spectrophotometry. However, it was observed that although azapropazone in plasma appeared stable when the samples were kept at
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H. Leach
-2O"C, the extracts showed a rapid loss of azapropazone when kept at room temperature while awaiting chromatography and it was necessary to prepare the extracts immediately prior to chromatography. This conversion of azapropazone to compounds with a shorter retention time was investigated further during later experiments. A similar study was conducted with the ingestion of 600 mg. azapropazone before and after food. Blood samples were taken after 2 hrs., 5 hrs. and 24 hrs. In view of the instability of extracts prepared for gas chromatography, these were prepared in batches of four just beforechromatographywas done. The results by gas chromatography were compared with those obtained by ultra-violet spectrophotometry, (Table I). Table I. Comparison of results obtained by gas chrornotography and by ultra-violet spectrophotometry:600 mg. azapropazone
Time
Gas chromatography
Ultra-violet spectrophotometry
Pg./ml.
Mean
Wml.
Mean
Before Food 2 hrs. 5 hrs. 24 hrs.
23-70 41-74 15-23
46 51 20
21-73 40-17 18-26
41 59 21
After Food 2 hrs. 5 hrs. 24 hrs.
22-71 52-73 10-22
50 60 15
26-73 51-78 9-24
53 61 16
The close agreement between the two methods suggests that the spectrophotometric method determines azapropazone alone and that metabolites cause no significant interference. The remaining plasma samples were pooled and examined for the presence of the major metabolite - 6-hydroxyazapropazone - by thin-layer chromatography. No band corresponding to this substance was detected and the only substance giving the same reaction as azapropazone had a Rf value identical with authentic material. On standing in air, the extracts and the material on the thin-layer plate developed a yellow colour. The material was eluted and re-chromatographed. The second plate showed some unchanged azapropazone and a spot with a Rf corresponding to the compound described by Mixich2 as the product of alkaline oxidation of azapropazone, 3-dimethylamino-7-methyl-1,2,4-benzotriazine. Repeated dose study Six subjects ingested 300 mg. azapropazone 3-times daily. Blood samples were taken at 24 hr. intervals and 12 hrs., 36 hrs. and 60 hrs. after the last dose. In addition to ultra-violet spectrophotometry and gas chromatography, the samples were also examined using the colourimetric procedure described by Farrier.' The results of this experiment are shown in Table 11. It will be seen that a plateau is reached after 4 to 5 days and the half-life of azapropazone is approximately 12 hrs. 39
The determinationof azapropazone in blood plasma
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Table II. Comparison of results obtained by ultra-violetspectrophotometry,gas chromatography, and colourimetric assay: 1200 mg. azapropazone daily. Mean figures (pg./ml.) for 6 subjects Day
Ultra-violet spectrophotometry
Gas chromatography
Colourimetric method
1 2 3 4
36.8 79.0 84.6 89.3 94.6 96.5 48.0 15.7 5.2
34.0 74.5 75.0 84.5 81.0 89.0 41.O 14.5 5.0
34.0 75.6 82.8 86.2 89.5 91.5 44.2 16.0 below 10.0
5
6 12 hrs. after last dose 36 hrs. after last dose 60 hrs. after last dose
There was close agreement between the results obtained by the three methods of analysis and although the spectrophotometric method gives rather higher results than the other two assays the differences are not significant. The plasma samples from this experiment were examined for the presence of metabolites by gas and thin-layer chromatography. No detectable amounts of any metabolite were found.
Recoveries andprecision of methods The recovery of azapropazone added to plasma at a concentration of 50 pg. per ml. was 92 % with gas chromatography, 107 % with ultra-violet spectrophotometry, and 96% with the colourimetric method. Results of the analysis of plasma samples containing 50 pg. and 100 pg. per ml. and calculated from a series of standards taken through the entire analytical procedure are given in Table 111. Table Ill. Recoveries and precision of the three different assay methods Method
Gas chromatography Ultra-violet spectrophotometry Colourimetricmethod
Mean values f standard deviation
50 pg. per ml.
100 pg. per ml.
46.6&2.3 53.9 +3.05 48.0k4.05
98.2k3.9 107+5.15 97.2k6.1
Comparison of assay methods As can be seen from Figure 2 showing a gas chromatogram of the 6-hydroxy metabolite of azapropazone, this is well removed from azapropazone and its detection should present no problem if it is present in solvent extracts of plasma. Although the specificity of the gas chromatographic assay method is better than can be obtained with either of the two photometric methods, it is technically far more demanding and prone to error. If extracts are not processed soon after preparation or stored in the dark in the absence of air while awaiting chromatography the azapropazone will be low or even absent. Figure 3 shows two gas chromato-
H. Leach
Figure 2. Gas chromatogram of azapropazone reference material and internal standard and of 6-hydroxy metabolite of azapropazone
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100
01 /O
90 80
70 60 50
40
30 20 10 0
1
L
grams of the same plasma extract containing 40 pg per ml. azapropazone. Figure 3a is the extract immediately after preparation and the chromatogram is unencumbered by any significant peaks apart from azapropazone and the internal standard. In the second chromatogram, (Figure 3b) the sample has been allowed to stand at room temperature for I hour. The azapropazone peak has almost disappeared and has been replaced by 3 more rapidly eluted products. One of the peaks corresponds to the oxidation product described by MLxich2 and studies are now in progress on a new method based on the conversion of azapropazone to the oxidation product prior to gas chromatography. Preliminary work suggests that this procedure will give some increase in sensitivity with no loss of specificity or precision. Results obtained with the ultra-violet spectrophotometric method are higher than those obtained with the colourimetric assay or by gas chromatography. This cannot be associated with the presence of contributing metabolites since, even after several days administration of the drug, attempts to detect metabolites have been unsuccessful. While metabolites may not appear in relatively non-polar solvent 41
The determination of azapropazone in blood plasma
Figure 3. Gas chromatograms of azapropazone extracted from plasma showiiig appearance of extract immediately after preparation (a) and after standing at room temperature (b) for 1 hour n
100
L D
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3 90 80
70
3 !
60
i I
>
50
>
P
1
L
W
40
0 0
P 30
z
J
~
m
20
O
J
extracts, it would be expected that they would be easily extractable by methanol if present in any significant amount and would be detected by chromatography. Farrier’ demonstrated that the oxidation product of azapropazone had an absorbance maximum at 450 nm. while the 6-hydroxy metabolite is oxidised to a substance with a maximum at 370 nm. and which faded rapidly. Consequently, the colourimetric method is not affected by the presence of the major metabolite of azapropazone. For the determination of plasma levels of the drug, there is little to choose between the two spectrophotometric methods although the ultra-violet assay is more sensitive at low concentrations. Both methods will give results of acceptable accuracy and have the virtue of analytical simplicity. The more demanding nature of the gas chromatographic method makes it more suitable for use as a reference method when the number of samples is small enough to permit rapid processing of the extracts and where the presence of metabolites must be considered. The results of the analyses following oral dosage with azapropazone indicate that peak plasma levels are reached 2 to 6 hours after ingestion and that significant 42
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H. Leach
amounts remain in the blood after 24 hours. On repeated doses of 300 mg. 3-times daily, a plateau is reached after approximately 4 days with plasma levels between 61 and 144 pg. per ml. and a mean of approximately 90 pg. in the 6 subjects involved. The half-life of azapropazone is about 12 hours. The results obtained when plasma levels of the drug taken after food are compared with those after ingestion on an empty stomach are interesting in that little difference is discernible between the two sets of results. The individual variation within each experiment was considerably greater than the slight difference between the two sets of results.
Acknowledgement
I am indebted to Dr. Stewart Templeton, Mr. Francis Walker and Mr. Christopher Jones of A. H. Robins Company Limited for providing material for these studies and for helpful discussions.
References
1. Farrier, D. S.,(1974).Spectroscopic methods for the determination of azapropazone in serum developed and evaluated using radio-isotopic techniques. Avzneini.-fovsch., 24,747. 2. Mixich, G.,(1968). Zum chemischen Verhalten des Antiphlogisticums “Azapropazon” (Mi 85)=3-Dimethylamino-7-methyl-l,2-(n-propylmalonyl)-1,2-dihydro-l,2,4-benzotriazin. Helv. chinz. Acra, 51,532. 3. Schatz, F.,Adrian, R. W., Mixich, G., Molnarova, M., Reller, J., and Jahn U.,(1970). Pharmakokinetische Untersuchungen mit dem Antiphlogistikum Azapropazon (Prolixan 300) am Menschen. Therupiewoche, 20,39.
43
Current Medical Research and Opinion
Vol. 4, No. 1, 1976
The determination of azapropazone in blood plasma
H. Leach, M.Sc.
Curr. med. Res. Opin., (1976), 4, 35.
Department of Biochemistry, University College of North Wales, and The Caernarvon and Anglesey General Hospital, Bangor, Wales
Received: 16th September 1975
Summary Three methods for the determination of azapropazone were studied for the analysis of blood plasma samples taken after oraladministration of azapropazone. A gas chromatographic method was compared with two simple spectrophotoinetric assay methods and the efSect of metabolites of the drug on each method is described. It is concluded that nzetabolites are unlikely to cause signiJicant interference with the two simple methods and that either is suitable for determination of plasma levels of the drug. Some problems associated with a gas chromatographic method are described and the comparative results of the examination of plasma samples using these methods are given. Keywords: Azapropazone -plasma levels - dosage - assay
Introduction For the determination of the concentration of a drug in body fluids there is, in general, considerable merit in choosing the simplest method of analysis which can give the required accuracy and reproducibility. Not only can large numbers of samples be handled expeditiously, analytical simplicity allows for replicate analysis with a consequent improvement in precision. The major objection which can often be levelled against simple methods is lack of specificity, and much of the elaboration introduced into analytical procedures is an attempt to improve this situation. Spectrophotometric methods in the ultra-violet or visible spectrum may include metabolites in the result if metabolites with the intact chromophore of the parent drug are present, and this may also happen when labelled compounds are used with radio-isotopic methods of analysis. To ensure specificity it may be necessary to introduce a separation stage in the analytical procedure or to use a method employing gas chromatography where separation and quantification are combined ; in either event there will be some loss in simplicity. If, however, chromatography can be used to demonstrate that metabolites are unlikely to cause serious interference, either because levels are insignificant or the metabolites do not appear in solvent extracts, much simpler methods may be applicable. In the course of studies on the plasma concentration of the antirheumatic drug azapropazone this approach was the choice. Initially, the method described by Schatz et aL3 was selected and compared with a gas chromatographic method of
The determination of azapropawne in blood plasma
Curr Med Res Opin Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
analysis and, in later studies, the simple colourimetricmethod describedby Farrier’ was also used.
Materials and methods Subjects All the subjects engaged in these experiments were young male and female volunteers aged between 19 and 26 years. They were in good health with no history of any gastric disorders. With the exception of one experiment when the dose of drug was taken before food all were instructed to take the dose of azapropazone after food, and they were asked to abstain from very fatty meals during the appropriate times during the experiment.
Drug Azapropazone in the form of 300 mg. capsules of azapropazone dihydrate was used. Within an experiment a single batch of capsules was used. Sampling Blood was taken from the antecubital vein and collected in lithium heparin anticoagulant. It was immediately centrifuged and the plasma removed to a siliconed glass vial. If the analysis could not be performed immediately the sample was stored at -20°C. Analytical methods (a) Gas chromatography. 2 ml. of plasma was added to 2 ml. of 0.1 M phosphoric acid and extracted by shaking vigorously with 20 ml. of dichloroethane. The solvent was removed to a clean tube and a second extraction with 20 ml. of solvent performed. The combined solvent extracts were then extracted with 10 ml. of 0.5 M sodium hydroxide in two 5 ml. portions. The combined alkali extracts were acidified with 5 N phosphoric acid and the azapropazone extracted into 10 ml. of dichloroethane. The solvent extract was evaporated to dryness in a fine pointed centrifuge tube or Dreyer’s tube in a heating block at 60°C using a stream of nitrogen to assist evaporation and exclude air. The residue was dissolved in 20 PI. of chloroform containing 1 mg. per ml. of C22hydrocarbon as an internal standard. Gas chromatography was done immediately and the concentration calculated from the relative peak areas of azapropazone and internal standard. A calibration curve was constructed by analysing plasma samples containing added azapropazone. The standards contained 20 pg., 50 pg. and 80 pg. azapropazone dihydrate per ml. and all were subjected to exactly the same analytical procedure as the test samples. Gas chromatography was done on 2.5% SE-30 on acid washed and silanised Chromosorb W. Columns were glass, 5 ft. (1.5 m.) in length and 0.25 inches (6 mm.) O.D. A Pye 104 instrument was used with flame ionisation detector. Gas flows were: carrier (nitrogen) 50 ml. per min; hydrogen 50 ml. per min; air 300 mi. per minute. 36
H.Leach
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The oven temperature was 225°C and the detector oven was maintained at 300°C. No injection point heater was used and samples were injected directly into the column packing. Under these conditions the retention time of azapropazone was 7.3 min. and the internal standard 3.4 min. (b) Spectrophotometric assay. The spectrophotometric procedure of Schatz et was used with minor modifications. 0.5 ml. of plasma was added to 4.5 ml. of methanol and, after shaking, the tube was closed with a screw cap and placed in a heating block at 100°C for 2 minutes with occasional shaking. After centrifuging until the supernatant was clear, the solvent was removed and scanned between 450 nm. and 225 nm. in a Unicam S.P.1800 spectrophotometer. An extract prepared from the zero time plasma sample was used as the blank. The concentration of azapropazone was determined from the absorbance reading at about 255 nm. A calibration curve was prepared from blank plasma samples containing added azapropazone from 10 to 100 pg. per ml. All results were the mean of parallel duplicates. Ethanol was also used as the protein precipitant and extractant with no significant change. Schatz et al. read the samples against methanol and subtracted the blank value from the test readings. It was found that more satisfactory precision was obtained if the blank plasma extract (zero time sample) was used as the reference solution. (c) Colourimetric assay method. This was the method of Farrier' with slight modification. To 6 ml. of methanol, 2 ml. of plasma was added and after vigorous mechanical shaking for 3 min. the mixture was centrifuged and the supernatant decanted into a clean centrifuge tube. The protein precipitate was extracted with a further 5 ml. of methanol and centrifuged. The combined extracts were evaporated to dryness in a heating block at 70°C with the aid of a stream of nitrogen and finally dried over phosphorus pentoxide in a dessicator for 1 hour. The surfactant BBS-3 (Beckman Instruments Company) was diluted 1 in 3 with methanol: 2 ml. of this solution was added to the residue and solution assisted by heating to 70°C; I ml. of sodium hypochlorite solution (6 % available chlorine) was added and after gentle mixing and centrifuging the supernatant was removed for photometry. The solutions were read at 450 nm. against a blank prepared from the zero time sample. A calibration curve of azapropazone dihydrate showed a straight line relationship between absorbance and concentration to above 200 pg. per ml. (d) Thin-layer chromatography for metabolites. Plasma samples taken after the administration of azapropazone for several days were pooled and 5 volumes of methanol added. After heating to boiling and frequent shaking the mixture was Centrifuged and the clear supernatant removed. The precipitate was extracted with a further 5 volumes of methanol and the combined extracts evaporated to a small volume in a rotary vacuum evaporator. The solution was transferred to a small tube and evaporation completed in a stream of nitrogen. 37
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The determination of azapropazone in blood plasma
The residue was dissolved in 50 pg, of methanol and centrifuged. The whole of the extract was applied to a Merck Kieselgel F254plate and reference spots of azar ropazone and 6-hydroxyazapropazone were applied either side of the sample streak. The plate was developed in a solvent mixture of ethyl acetate: ethanol: methylchloride: formic acid (40:5:45:10) until the solvent had run about 10 cm. The dried plates were then examined with short wave ultra-violet light and then sprayed with a location reagent of ferric chloride-potassium ferricyanide.
Results and discussions In the course of some early studies, 6 volunteers ingested 600 mg. azapropazone and blood samples were collected at zero time for the blank and after 2.5 hrs., 5 hrs., 8 hrs. and 48 hrs. Plasma levels of azapropazone were determined in duplicate by the ultra-violet spectrophotometric method described. The mean plasma levels found are shown in Figure 1. Figure 1. Mean plasma concentration of azapropazone from 6 subjects after the administration of 600 mg. of the drug, (mean dose 8.4 mg./kg.): ultra-violet spectrophotometry 50
1
Hour.\ after close
Samples of pooled plasma from this experiment were also examined by the gas chromatographic method and no significant difference was found between the two methods. There was no evidence in the gas chromatogram of any substance other than azapropazone and it appeared unlikely that any metabolites were contributing to the results obtained by spectrophotometry. However, it was observed that although azapropazone in plasma appeared stable when the samples were kept at
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H. Leach
-2O"C, the extracts showed a rapid loss of azapropazone when kept at room temperature while awaiting chromatography and it was necessary to prepare the extracts immediately prior to chromatography. This conversion of azapropazone to compounds with a shorter retention time was investigated further during later experiments. A similar study was conducted with the ingestion of 600 mg. azapropazone before and after food. Blood samples were taken after 2 hrs., 5 hrs. and 24 hrs. In view of the instability of extracts prepared for gas chromatography, these were prepared in batches of four just beforechromatographywas done. The results by gas chromatography were compared with those obtained by ultra-violet spectrophotometry, (Table I). Table I. Comparison of results obtained by gas chrornotography and by ultra-violet spectrophotometry:600 mg. azapropazone
Time
Gas chromatography
Ultra-violet spectrophotometry
Pg./ml.
Mean
Wml.
Mean
Before Food 2 hrs. 5 hrs. 24 hrs.
23-70 41-74 15-23
46 51 20
21-73 40-17 18-26
41 59 21
After Food 2 hrs. 5 hrs. 24 hrs.
22-71 52-73 10-22
50 60 15
26-73 51-78 9-24
53 61 16
The close agreement between the two methods suggests that the spectrophotometric method determines azapropazone alone and that metabolites cause no significant interference. The remaining plasma samples were pooled and examined for the presence of the major metabolite - 6-hydroxyazapropazone - by thin-layer chromatography. No band corresponding to this substance was detected and the only substance giving the same reaction as azapropazone had a Rf value identical with authentic material. On standing in air, the extracts and the material on the thin-layer plate developed a yellow colour. The material was eluted and re-chromatographed. The second plate showed some unchanged azapropazone and a spot with a Rf corresponding to the compound described by Mixich2 as the product of alkaline oxidation of azapropazone, 3-dimethylamino-7-methyl-1,2,4-benzotriazine. Repeated dose study Six subjects ingested 300 mg. azapropazone 3-times daily. Blood samples were taken at 24 hr. intervals and 12 hrs., 36 hrs. and 60 hrs. after the last dose. In addition to ultra-violet spectrophotometry and gas chromatography, the samples were also examined using the colourimetric procedure described by Farrier.' The results of this experiment are shown in Table 11. It will be seen that a plateau is reached after 4 to 5 days and the half-life of azapropazone is approximately 12 hrs. 39
The determinationof azapropazone in blood plasma
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Table II. Comparison of results obtained by ultra-violetspectrophotometry,gas chromatography, and colourimetric assay: 1200 mg. azapropazone daily. Mean figures (pg./ml.) for 6 subjects Day
Ultra-violet spectrophotometry
Gas chromatography
Colourimetric method
1 2 3 4
36.8 79.0 84.6 89.3 94.6 96.5 48.0 15.7 5.2
34.0 74.5 75.0 84.5 81.0 89.0 41.O 14.5 5.0
34.0 75.6 82.8 86.2 89.5 91.5 44.2 16.0 below 10.0
5
6 12 hrs. after last dose 36 hrs. after last dose 60 hrs. after last dose
There was close agreement between the results obtained by the three methods of analysis and although the spectrophotometric method gives rather higher results than the other two assays the differences are not significant. The plasma samples from this experiment were examined for the presence of metabolites by gas and thin-layer chromatography. No detectable amounts of any metabolite were found.
Recoveries andprecision of methods The recovery of azapropazone added to plasma at a concentration of 50 pg. per ml. was 92 % with gas chromatography, 107 % with ultra-violet spectrophotometry, and 96% with the colourimetric method. Results of the analysis of plasma samples containing 50 pg. and 100 pg. per ml. and calculated from a series of standards taken through the entire analytical procedure are given in Table 111. Table Ill. Recoveries and precision of the three different assay methods Method
Gas chromatography Ultra-violet spectrophotometry Colourimetricmethod
Mean values f standard deviation
50 pg. per ml.
100 pg. per ml.
46.6&2.3 53.9 +3.05 48.0k4.05
98.2k3.9 107+5.15 97.2k6.1
Comparison of assay methods As can be seen from Figure 2 showing a gas chromatogram of the 6-hydroxy metabolite of azapropazone, this is well removed from azapropazone and its detection should present no problem if it is present in solvent extracts of plasma. Although the specificity of the gas chromatographic assay method is better than can be obtained with either of the two photometric methods, it is technically far more demanding and prone to error. If extracts are not processed soon after preparation or stored in the dark in the absence of air while awaiting chromatography the azapropazone will be low or even absent. Figure 3 shows two gas chromato-
H. Leach
Figure 2. Gas chromatogram of azapropazone reference material and internal standard and of 6-hydroxy metabolite of azapropazone
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grams of the same plasma extract containing 40 pg per ml. azapropazone. Figure 3a is the extract immediately after preparation and the chromatogram is unencumbered by any significant peaks apart from azapropazone and the internal standard. In the second chromatogram, (Figure 3b) the sample has been allowed to stand at room temperature for I hour. The azapropazone peak has almost disappeared and has been replaced by 3 more rapidly eluted products. One of the peaks corresponds to the oxidation product described by MLxich2 and studies are now in progress on a new method based on the conversion of azapropazone to the oxidation product prior to gas chromatography. Preliminary work suggests that this procedure will give some increase in sensitivity with no loss of specificity or precision. Results obtained with the ultra-violet spectrophotometric method are higher than those obtained with the colourimetric assay or by gas chromatography. This cannot be associated with the presence of contributing metabolites since, even after several days administration of the drug, attempts to detect metabolites have been unsuccessful. While metabolites may not appear in relatively non-polar solvent 41
The determination of azapropazone in blood plasma
Figure 3. Gas chromatograms of azapropazone extracted from plasma showiiig appearance of extract immediately after preparation (a) and after standing at room temperature (b) for 1 hour n
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extracts, it would be expected that they would be easily extractable by methanol if present in any significant amount and would be detected by chromatography. Farrier’ demonstrated that the oxidation product of azapropazone had an absorbance maximum at 450 nm. while the 6-hydroxy metabolite is oxidised to a substance with a maximum at 370 nm. and which faded rapidly. Consequently, the colourimetric method is not affected by the presence of the major metabolite of azapropazone. For the determination of plasma levels of the drug, there is little to choose between the two spectrophotometric methods although the ultra-violet assay is more sensitive at low concentrations. Both methods will give results of acceptable accuracy and have the virtue of analytical simplicity. The more demanding nature of the gas chromatographic method makes it more suitable for use as a reference method when the number of samples is small enough to permit rapid processing of the extracts and where the presence of metabolites must be considered. The results of the analyses following oral dosage with azapropazone indicate that peak plasma levels are reached 2 to 6 hours after ingestion and that significant 42
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H. Leach
amounts remain in the blood after 24 hours. On repeated doses of 300 mg. 3-times daily, a plateau is reached after approximately 4 days with plasma levels between 61 and 144 pg. per ml. and a mean of approximately 90 pg. in the 6 subjects involved. The half-life of azapropazone is about 12 hours. The results obtained when plasma levels of the drug taken after food are compared with those after ingestion on an empty stomach are interesting in that little difference is discernible between the two sets of results. The individual variation within each experiment was considerably greater than the slight difference between the two sets of results.
Acknowledgement
I am indebted to Dr. Stewart Templeton, Mr. Francis Walker and Mr. Christopher Jones of A. H. Robins Company Limited for providing material for these studies and for helpful discussions.
References
1. Farrier, D. S.,(1974).Spectroscopic methods for the determination of azapropazone in serum developed and evaluated using radio-isotopic techniques. Avzneini.-fovsch., 24,747. 2. Mixich, G.,(1968). Zum chemischen Verhalten des Antiphlogisticums “Azapropazon” (Mi 85)=3-Dimethylamino-7-methyl-l,2-(n-propylmalonyl)-1,2-dihydro-l,2,4-benzotriazin. Helv. chinz. Acra, 51,532. 3. Schatz, F.,Adrian, R. W., Mixich, G., Molnarova, M., Reller, J., and Jahn U.,(1970). Pharmakokinetische Untersuchungen mit dem Antiphlogistikum Azapropazon (Prolixan 300) am Menschen. Therupiewoche, 20,39.
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