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Determination of 4-methylpyrazol in serum with mass fragmentography
BIOCHEMICAL
12, 205-212 (1975)
MEDICINE
Determination with
of 4Aethylpyrazol Mass
in Serum
Fragmentography’
INGEMAR BJ~RKHEM, ROLF BLOMSTRAND, OLLE LANTTO, AND LENNART SVENSSON Department of Clinical Chemistry, Huddinge and Karolinska
Huddinge Instituter,
University Hospital, Solna, Sweden
Received June 24, 1974
The partial inhibition of ethanol oxidation in living rats and man by 4methylpyrazol is well established and might become of clinical interest (l-4). In connection with studies on the possible toxicity of 4-methylpyrazol (5) we have been interested in determinations of blood disappearance rate and half-life of 4-methylpyrazol in man under different physiological conditions. In a previous work by Rydberg, Buijten, and Neri (6) a gas chromatographic method was used for determination of half life of pyrazol and 4-methylpyrazol in rats. The technique used by these authors is however not sensitive enough for studies with human volunteers, to which only very small amounts of 4-methylpyrazol can be given at the present state of knowledge. In the present work a sensitive and accurate method for determination of 4-methylpyrazol in serum is described. 4-Methylpyrazol with three atoms of deuterium in the methyl group has been synthesized and added to a fixed amount of serum. After extraction with ether, the amount of unlabeled Cmethylpyrazol has been determined from the ratio between the molecular ions at m/e 82 and m/e 85 using a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). MATERIALS AND METHODS Unlabeled I-methylpyrazol was obtained from AB Labkemi (Gothenburg, Sweden) and was pure as shown by gas chromatography under the conditions described below. 4-[5-3HjMethylpyrazo1 was a generous gift from Dr. Berndt Sjoberg (Astra lakemedel AB Sodertalje). 4-[6-W,$I4ethypyrazol. 4-Trifluoromethylpyrazol, 25 mg, was dissolved in 20 ml of absolute ether and refluxed for 2 hr together with 100 1 This work was supported by the Bank of Sweden Tercentenary N.I.A.A.A., USA (A.A. 00323-01). 205 Copyr@ht @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
Fund and from
206
BJiiRKHEM
ET AL.
loo,z r al E ; .? 5 z aY
CH3 50.
-TJ A MW=82 I 20
za. c r al 5 !! ; _a tz
LO
I
I 60
80
100
m/e
loo-
CD3 50-
v I4 MW=85 II, 20
a0
60
:
_.I,, 80
, 100
mle
FIG. 1. Mass spectrum of unlabeled *H,]methylpyrazol (lower spectrum).
4-methylpyrazol
(upper spectrum) and 4{6-
mg of lithium aluminium deuteride (obtained from Merck, Darmstadt, Germany). The 4-trifluoromethylpyrazol was kindly given to us by Prof. S. Gronowitz, Lund, Sweden. The excess reagent in the reaction mixture was destroyed with ethyl acetate. After addition of a small volume of water the 4-[6-2H,]methylpyrazol was extracted from the mixture with ether five times. The combined ether extracts were then washed with a small volume with water and dried with anhydrous calcium chloride. The ether was carefully evaporated at room temperature. The material obtained, 18 mg, had the same gas-chromatographic properties as authentic 4-methylpyrazol and had a mass spectrum consistent with 4methylpyrazol with three atoms of deuterium in the molecule (Fig. 1). It is noteworthy that the peak in the mass spectrum of 4-[6-2H,]methylpyrazol corresponding to M-l was relatively small. There was however a prominent peak corresponding to M-2 in the mass spectrum of 4-[62H,]methylpyrazol, indicating that the major part of the M-l peak in the mass spectrum of unlabeled 4-methylpyrazol is due to loss of one atom of hydrogen from the methyl group. It was calculated (7) that the composition of the 4-[6-2H,]methylpyrazol was the following: unlabeled molecules, 1%; monodeuterated molecules, 0%; dideuterated molecules, 11%; trideuterated molecules, 88%. No traces of 4-trifluoromethylpyrazol or other contaminating compounds could be found in the material. Animal Experiments. Male rats of the Sprague-Dawley strain weighing about 100 g were used. The hydrochloride of 4-methylpyrazol (0.1, 0.5, and 1 mmole/kg body wt) was injected intraperitoneally dissolved in 1 ml
DETERMINATION
OF 4-METHYLPYRAZOL
207
m/e=85
I
.
1
I
I
I
.
,
01234567 Retention
time
(min)
Retention
time
(min)
FIG. 2. MID-recording of unlabeled 4-methylpyrazol (left) and 4j6-2H,lmethylpyrazo1 (right). For experimental details, see Materials and Methods.
01234567
i
3T-z-z
Retention time (min) Retention time (min) FIG. 3. MID-recording (left) and gas chromatographic recording (right) of an extract of serum 4-methylpyrazol together with 4{6-2H,]methylpyrazo1. For experimental details, see Materials and Methods.
BJijRKHEM
208
ET AL.
i
PM pt.4 L-Methylpyrozole
fl.
in serum
FIG. 4. Standard curve for determination of serum 4-methylpyrazol For experimental details, see Materials and Methods.
in the range I-20
of saline. Venous blood was collected from the tail veins at regular intervals (cf. Fig. 5). Human Experiments. A healthy male volunteer, age 39, weight 82 kg, was given 150 mg hydrochloride of 4-methylpyrazol (16 pmolekg body wt) intravenously in 200 ml of 5% glucose solution. The solution was continuously infused during 0.5 hr. Venous blood was collected after regular intervals as shown in Fig. 6. No subjective symptoms from the drug could be registered during or after the experiment. Extraction Procedure. In the experiments with rats and human, 0.1 and 2 ml, respectively, of serum was used for each analysis. In the experiment with rat serum, 1.9 ml of water was added prior to extraction. To the undiluted or diluted serum, 5 pg of 4-[6-2HJmethlpyrazole was added dissolved in 0.1 ml of water. The mixture was then extracted with 10 ml of ether in glass stoppered test tubes by automatically shaking for 0.5 hr. The ether phase was collected and dried over anhydrous sodium sulphate. The solvent was then evaporated at room temperature under a stream of nitrogen down to a volume of about 50 ~1. The evaporation was shown to be the critical step in the whole procedure. Thus it was shown with 4-[5-3H]methylpyra.zol that more than 80% of the material could be extracted from the water phase under the conditions employed whereas only about 10% of the material seas recovered after the evaporation due to the volatile nature of free 4-methylpyrazol. Substitution of the ether for chloroform-methanol (2 : 1, v/v) did not
DETERMINATION
OF
209
‘t-METHYLPYRAZOL
I
60
240
180
120
300
360
420
Time after the intraperitonial administration of 4-Mcthylpyrotolc (minutes)
FIG. 5. The concentration of 4-methylpyrazol (log scale) in rat serum during the first 7 hr after ip injection of 4-methylpyrazol (1 mmolekg).
increase the recovery in the extraction step and with this solvent considerable amounts with contaminating compounds were obtained in the extract that interfered with the gas chromatographic-mass spectrometric analysis. In preparation of the standard curve, serum from untreated humans were used to which unlabeled 4-methylpyrazol had been added to give final concentrations of either l-20 @ (human experiment) or 100-1000 @J4 (rat experiments). In each set of experiment samples corresponding to the appropriate standard curve were prepared and analyzed simultaneously with the samples obtained from the treated rats or human. Muss Fragmentography. About one-tenth of the above serum extract was analyzed by gas chromatography-mass spectrometry using an LKB 9000 instrument equipped with a 3.5% EGSS-X column (on Chromosorb W, 100-120 mesh, 2mm x 4 m). The carrier gas was helium and the flow rate 15 ml/mitt. The temperature of the column was 150°C and the temperature of the fiashheater and the ion source 290°C. The electron energy was set to 20 eV and the trap current to 60 ,uA. The electron mul-
I
2
1 Time
after
3 the
of 4-Methylpyrgzole
4
intravenous
5
6
7
administration (hours)
FIG. 6. The concentration of 4-methylpyrazol (log scale) in human serum during the first 6 hr after intravenous injection of 4-methylpyrazol(16 pmoleikg).
210
BJiiRKHEM
ET AL.
tiplier sensitivity was set to 130. The first channel of the MID-unit was focused on the ion at m/e 82 and the amplification was 900x. The second channel was focused on the ion at m/e 85 and the amplification was 300x. The filter setting was 0.5 Hz for both channels and the measure time was 20 msec. The MID-recordings were made on uv-paper, and the ratio between the peak heights of the two recordings were determined (cf. Fig. 4). RESULTS Figure 2 shows a typical MID-recording of the ions at m/e 82 and m/e 85 of an extract of unlabeled 4-methylpyrazol and 4-[6-2HJmethylpyrazol. Figure 3 shows a typical MID-recording and the corresponding gas chromatogram of an extract of a mixture of unlabeled and 6-2H,labeled 4-methylpyrazol. The ratio between the peaks at m/e 82 and m/e 85 obtained in the MID-recordings from extracts of different standard mixtures of unlabeled 4-methylpyrazol with 5 pg of 4-[6-2H,]methylpyrazol was found to be approximately linear with the amount of unlabeled 4-methylpyrazol added in the range 1 to 20 fl (Fig. 4). In connection with experiments with rats, in which higher amounts of 4-methylpyrazol were given and in which 20-fold diluted serum was used, the standard curves were prepared by adding 4-methylpyrazol to give concentrations ranging from 10 to 1000 fl in undiluted serum. Also in this case the ratio between m/e 82 and m/e 85 was found to be linear with the amounts of unlabeled 4-methylpyrazol added. No corrections were necessary for the very small deviation from linearity due to the small contribution at m/e 85 from unlabeled 4-methylpyrazol and the small contribution at m/e 82 from 4-[6-2H,]methylpyrazol. In general it was not possible to determine unlabeled 4-methylpyrazol in concentrations lower than about 1 $U in undiluted serum and about 10 fl in diluted serum due to occasional presence of contaminating compounds with ions either at m/e 82 or m/e 85 in their mass spectra with gas chromatographic properties similar to 4-methylpyrazol (cf. Fig. 3). The concentration of these compounds were however so low that they did not significantly influence on the determinations in which the amount of unlabeled 4-methylpyrazol exceeded 1 M. The relative standard deviation of the method as calculated from duplicate analysis of eight different plasma samples containing 4-methylpyrazol in the range 4-21 fl was only 1.4%. The relative standard -deviation of the method as calculated from four different plasma samples containing 4-methylpyrazol in the range l-3 @ was however 15.2%. In Fig. 5, a typical elimination curve for 4-methylpyrazol in the rat is shown. The elimination might possibly be of first order during the first seven hours after the injection. Extrapolation from the straight line
DETERMINATION
OF
‘t-METHYLPYRAZOL
211
shown in Fig. 5 gave a half life of about 650 minutes as calculated from 18 measurements during the first eight hours after the injection. When lower doses were given (0.5 and 0.1 mmole/kg) no first order kinetics could be demonstrated and the elimination was more rapid than with the higher dose. Thus after administration of 0.1 mmole/kg the plasma concentration of 4-methylpyrazol decreased from about 100 fl to about 50 fl during the tirst 90 minutes after the injection. When 0.01 mmole/kg was given, measurable amounts of 4-methylpyrazol could be detected only for about 0.5 hours after the injection. In Fig. 6, the results of the human experiment are shown. In spite of the small amount of 4-methylpyrazol given, 16 pmole/kg, the presence of 4-methylpyrazol could be followed for the first six hours after the administration, The elimination was rather slow for the first three hours and then increased. The curve obtained was not conclusive with respect to determination of order of elimination. DISCUSSION In the present work a method for determination of 4-methylpyrazol is described with a sensitivity sufficient for future studies on serum levels of 4-methylpyrazol in humans. It is surprising that such relatively high sensitivity could be obtained in view of the low mass number of the molecular ion of 4-methylpyrazol. The specificity of mass fragmentographic techniques in general decrease with decreasing mass number of the fragment chosen. This is due to that the probability of occurence of two compounds with the same or similar gas-chromatographic properties and with the same peak in their mass spectrum increase with decreasing mass number of the fragment. As could be expected the sensitivity of the method was limited not by the sensitivity of the instrument but by the presence of compounds with similar gas chromatographic properties as 4-methylpyrazol and containing peaks at m/e 82 or 85 in their mass spectrum. An increased sensitivity might have been obtained in the present study if it had been possible to prepare a derivative of 4methylpyrazol with a suitable peak of higher molecular weight in the mass spectrum. A drawback of the technique used in the present study was the extensive loss of 4-methylpyrazol during the concentration of the ether extract of the material. It was clearly shown, however, that the deuterated standard was lost during the concentration to exactly the same degree as the unlabeled 4-methylpyrazol. Thus varying degree of evaporation of the solvent did not have any effect on the ratio between m/e 82 and m/e 85 determined in the mass spectrometric analysis. Due to the specific volatile nature of 4-methylpyrazol it was not possible to use other pyrazol derivatives such as 4-trifluoromethylpyrazol as internal standard.
212
BJtiRKHEM
ET AL.
The half-life of 4-methylpyrazol in the rat was determined to be about 650 minutes under the conditions employed in the present work. Under essentially the same conditions Rydberg et aE. (6) determined the halflife of 4-methylpyrazol in the rat to be about 540 minutes using a gas chromatographic assay. The present work shows that the capacity to eliminate 4-methylpyrazol is different in rat and man. Studies on the metabolic basis of this difference are in progress. SUMMARY
A mass fragmentographic method for determination of 4-methylpyrazol in serum is described. 4-Methylpyrazol with three atoms of deuterium in the methyl group is added to a fixed amount of serum as internal standard. After extraction of the serum with ether, the amount of unlabeled 4-methylpyrazol is determined from the ratio between the recordings at m/e 82 and m/e 85 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). With this method 4-methylpyrazol could be determined in concentrations down to about 0.1 &ml corresponding to about 1 a. The relative standard deviation of the method in the range 4-21 m was less than 2%. The method could be used for determination of disappearance rate of 4-methylpyrazol in experimental animals as well as humans. REFERENCES 1. Blomstrand, R., and Theorell, H., Life Sciences 9, 631 (1970). 2. Blomstrand, R., in “Metabolic changes induced by alcohol,” p. 38 (G. A. Martine and C. H. Bode, Eds.). Springer Verlag, Berlin, 1971. 3. Blomstrand, R., and Forssell, L., Life Sciences 10, 523 (1971). 4. Blomstrand, R., and Kager, L., Life Sciences 13, 113 (1973). 5. Kager, L., and Ericsson, J. L. E., Acta Path. Micrub. Stand., 82, 534 (1974). 6. Rydberg, U., Buijten, J., and Neri, A., J. Pharm. Pharmuc. 24, 651 (1972). 7. Biemann, K., in “Mass Spectrometry,” p. 223. McGraw-Hill Book Company Inc., New York, 1962.
12, 205-212 (1975)
MEDICINE
Determination with
of 4Aethylpyrazol Mass
in Serum
Fragmentography’
INGEMAR BJ~RKHEM, ROLF BLOMSTRAND, OLLE LANTTO, AND LENNART SVENSSON Department of Clinical Chemistry, Huddinge and Karolinska
Huddinge Instituter,
University Hospital, Solna, Sweden
Received June 24, 1974
The partial inhibition of ethanol oxidation in living rats and man by 4methylpyrazol is well established and might become of clinical interest (l-4). In connection with studies on the possible toxicity of 4-methylpyrazol (5) we have been interested in determinations of blood disappearance rate and half-life of 4-methylpyrazol in man under different physiological conditions. In a previous work by Rydberg, Buijten, and Neri (6) a gas chromatographic method was used for determination of half life of pyrazol and 4-methylpyrazol in rats. The technique used by these authors is however not sensitive enough for studies with human volunteers, to which only very small amounts of 4-methylpyrazol can be given at the present state of knowledge. In the present work a sensitive and accurate method for determination of 4-methylpyrazol in serum is described. 4-Methylpyrazol with three atoms of deuterium in the methyl group has been synthesized and added to a fixed amount of serum. After extraction with ether, the amount of unlabeled Cmethylpyrazol has been determined from the ratio between the molecular ions at m/e 82 and m/e 85 using a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). MATERIALS AND METHODS Unlabeled I-methylpyrazol was obtained from AB Labkemi (Gothenburg, Sweden) and was pure as shown by gas chromatography under the conditions described below. 4-[5-3HjMethylpyrazo1 was a generous gift from Dr. Berndt Sjoberg (Astra lakemedel AB Sodertalje). 4-[6-W,$I4ethypyrazol. 4-Trifluoromethylpyrazol, 25 mg, was dissolved in 20 ml of absolute ether and refluxed for 2 hr together with 100 1 This work was supported by the Bank of Sweden Tercentenary N.I.A.A.A., USA (A.A. 00323-01). 205 Copyr@ht @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
Fund and from
206
BJiiRKHEM
ET AL.
loo,z r al E ; .? 5 z aY
CH3 50.
-TJ A MW=82 I 20
za. c r al 5 !! ; _a tz
LO
I
I 60
80
100
m/e
loo-
CD3 50-
v I4 MW=85 II, 20
a0
60
:
_.I,, 80
, 100
mle
FIG. 1. Mass spectrum of unlabeled *H,]methylpyrazol (lower spectrum).
4-methylpyrazol
(upper spectrum) and 4{6-
mg of lithium aluminium deuteride (obtained from Merck, Darmstadt, Germany). The 4-trifluoromethylpyrazol was kindly given to us by Prof. S. Gronowitz, Lund, Sweden. The excess reagent in the reaction mixture was destroyed with ethyl acetate. After addition of a small volume of water the 4-[6-2H,]methylpyrazol was extracted from the mixture with ether five times. The combined ether extracts were then washed with a small volume with water and dried with anhydrous calcium chloride. The ether was carefully evaporated at room temperature. The material obtained, 18 mg, had the same gas-chromatographic properties as authentic 4-methylpyrazol and had a mass spectrum consistent with 4methylpyrazol with three atoms of deuterium in the molecule (Fig. 1). It is noteworthy that the peak in the mass spectrum of 4-[6-2H,]methylpyrazol corresponding to M-l was relatively small. There was however a prominent peak corresponding to M-2 in the mass spectrum of 4-[62H,]methylpyrazol, indicating that the major part of the M-l peak in the mass spectrum of unlabeled 4-methylpyrazol is due to loss of one atom of hydrogen from the methyl group. It was calculated (7) that the composition of the 4-[6-2H,]methylpyrazol was the following: unlabeled molecules, 1%; monodeuterated molecules, 0%; dideuterated molecules, 11%; trideuterated molecules, 88%. No traces of 4-trifluoromethylpyrazol or other contaminating compounds could be found in the material. Animal Experiments. Male rats of the Sprague-Dawley strain weighing about 100 g were used. The hydrochloride of 4-methylpyrazol (0.1, 0.5, and 1 mmole/kg body wt) was injected intraperitoneally dissolved in 1 ml
DETERMINATION
OF 4-METHYLPYRAZOL
207
m/e=85
I
.
1
I
I
I
.
,
01234567 Retention
time
(min)
Retention
time
(min)
FIG. 2. MID-recording of unlabeled 4-methylpyrazol (left) and 4j6-2H,lmethylpyrazo1 (right). For experimental details, see Materials and Methods.
01234567
i
3T-z-z
Retention time (min) Retention time (min) FIG. 3. MID-recording (left) and gas chromatographic recording (right) of an extract of serum 4-methylpyrazol together with 4{6-2H,]methylpyrazo1. For experimental details, see Materials and Methods.
BJijRKHEM
208
ET AL.
i
PM pt.4 L-Methylpyrozole
fl.
in serum
FIG. 4. Standard curve for determination of serum 4-methylpyrazol For experimental details, see Materials and Methods.
in the range I-20
of saline. Venous blood was collected from the tail veins at regular intervals (cf. Fig. 5). Human Experiments. A healthy male volunteer, age 39, weight 82 kg, was given 150 mg hydrochloride of 4-methylpyrazol (16 pmolekg body wt) intravenously in 200 ml of 5% glucose solution. The solution was continuously infused during 0.5 hr. Venous blood was collected after regular intervals as shown in Fig. 6. No subjective symptoms from the drug could be registered during or after the experiment. Extraction Procedure. In the experiments with rats and human, 0.1 and 2 ml, respectively, of serum was used for each analysis. In the experiment with rat serum, 1.9 ml of water was added prior to extraction. To the undiluted or diluted serum, 5 pg of 4-[6-2HJmethlpyrazole was added dissolved in 0.1 ml of water. The mixture was then extracted with 10 ml of ether in glass stoppered test tubes by automatically shaking for 0.5 hr. The ether phase was collected and dried over anhydrous sodium sulphate. The solvent was then evaporated at room temperature under a stream of nitrogen down to a volume of about 50 ~1. The evaporation was shown to be the critical step in the whole procedure. Thus it was shown with 4-[5-3H]methylpyra.zol that more than 80% of the material could be extracted from the water phase under the conditions employed whereas only about 10% of the material seas recovered after the evaporation due to the volatile nature of free 4-methylpyrazol. Substitution of the ether for chloroform-methanol (2 : 1, v/v) did not
DETERMINATION
OF
209
‘t-METHYLPYRAZOL
I
60
240
180
120
300
360
420
Time after the intraperitonial administration of 4-Mcthylpyrotolc (minutes)
FIG. 5. The concentration of 4-methylpyrazol (log scale) in rat serum during the first 7 hr after ip injection of 4-methylpyrazol (1 mmolekg).
increase the recovery in the extraction step and with this solvent considerable amounts with contaminating compounds were obtained in the extract that interfered with the gas chromatographic-mass spectrometric analysis. In preparation of the standard curve, serum from untreated humans were used to which unlabeled 4-methylpyrazol had been added to give final concentrations of either l-20 @ (human experiment) or 100-1000 @J4 (rat experiments). In each set of experiment samples corresponding to the appropriate standard curve were prepared and analyzed simultaneously with the samples obtained from the treated rats or human. Muss Fragmentography. About one-tenth of the above serum extract was analyzed by gas chromatography-mass spectrometry using an LKB 9000 instrument equipped with a 3.5% EGSS-X column (on Chromosorb W, 100-120 mesh, 2mm x 4 m). The carrier gas was helium and the flow rate 15 ml/mitt. The temperature of the column was 150°C and the temperature of the fiashheater and the ion source 290°C. The electron energy was set to 20 eV and the trap current to 60 ,uA. The electron mul-
I
2
1 Time
after
3 the
of 4-Methylpyrgzole
4
intravenous
5
6
7
administration (hours)
FIG. 6. The concentration of 4-methylpyrazol (log scale) in human serum during the first 6 hr after intravenous injection of 4-methylpyrazol(16 pmoleikg).
210
BJiiRKHEM
ET AL.
tiplier sensitivity was set to 130. The first channel of the MID-unit was focused on the ion at m/e 82 and the amplification was 900x. The second channel was focused on the ion at m/e 85 and the amplification was 300x. The filter setting was 0.5 Hz for both channels and the measure time was 20 msec. The MID-recordings were made on uv-paper, and the ratio between the peak heights of the two recordings were determined (cf. Fig. 4). RESULTS Figure 2 shows a typical MID-recording of the ions at m/e 82 and m/e 85 of an extract of unlabeled 4-methylpyrazol and 4-[6-2HJmethylpyrazol. Figure 3 shows a typical MID-recording and the corresponding gas chromatogram of an extract of a mixture of unlabeled and 6-2H,labeled 4-methylpyrazol. The ratio between the peaks at m/e 82 and m/e 85 obtained in the MID-recordings from extracts of different standard mixtures of unlabeled 4-methylpyrazol with 5 pg of 4-[6-2H,]methylpyrazol was found to be approximately linear with the amount of unlabeled 4-methylpyrazol added in the range 1 to 20 fl (Fig. 4). In connection with experiments with rats, in which higher amounts of 4-methylpyrazol were given and in which 20-fold diluted serum was used, the standard curves were prepared by adding 4-methylpyrazol to give concentrations ranging from 10 to 1000 fl in undiluted serum. Also in this case the ratio between m/e 82 and m/e 85 was found to be linear with the amounts of unlabeled 4-methylpyrazol added. No corrections were necessary for the very small deviation from linearity due to the small contribution at m/e 85 from unlabeled 4-methylpyrazol and the small contribution at m/e 82 from 4-[6-2H,]methylpyrazol. In general it was not possible to determine unlabeled 4-methylpyrazol in concentrations lower than about 1 $U in undiluted serum and about 10 fl in diluted serum due to occasional presence of contaminating compounds with ions either at m/e 82 or m/e 85 in their mass spectra with gas chromatographic properties similar to 4-methylpyrazol (cf. Fig. 3). The concentration of these compounds were however so low that they did not significantly influence on the determinations in which the amount of unlabeled 4-methylpyrazol exceeded 1 M. The relative standard deviation of the method as calculated from duplicate analysis of eight different plasma samples containing 4-methylpyrazol in the range 4-21 fl was only 1.4%. The relative standard -deviation of the method as calculated from four different plasma samples containing 4-methylpyrazol in the range l-3 @ was however 15.2%. In Fig. 5, a typical elimination curve for 4-methylpyrazol in the rat is shown. The elimination might possibly be of first order during the first seven hours after the injection. Extrapolation from the straight line
DETERMINATION
OF
‘t-METHYLPYRAZOL
211
shown in Fig. 5 gave a half life of about 650 minutes as calculated from 18 measurements during the first eight hours after the injection. When lower doses were given (0.5 and 0.1 mmole/kg) no first order kinetics could be demonstrated and the elimination was more rapid than with the higher dose. Thus after administration of 0.1 mmole/kg the plasma concentration of 4-methylpyrazol decreased from about 100 fl to about 50 fl during the tirst 90 minutes after the injection. When 0.01 mmole/kg was given, measurable amounts of 4-methylpyrazol could be detected only for about 0.5 hours after the injection. In Fig. 6, the results of the human experiment are shown. In spite of the small amount of 4-methylpyrazol given, 16 pmole/kg, the presence of 4-methylpyrazol could be followed for the first six hours after the administration, The elimination was rather slow for the first three hours and then increased. The curve obtained was not conclusive with respect to determination of order of elimination. DISCUSSION In the present work a method for determination of 4-methylpyrazol is described with a sensitivity sufficient for future studies on serum levels of 4-methylpyrazol in humans. It is surprising that such relatively high sensitivity could be obtained in view of the low mass number of the molecular ion of 4-methylpyrazol. The specificity of mass fragmentographic techniques in general decrease with decreasing mass number of the fragment chosen. This is due to that the probability of occurence of two compounds with the same or similar gas-chromatographic properties and with the same peak in their mass spectrum increase with decreasing mass number of the fragment. As could be expected the sensitivity of the method was limited not by the sensitivity of the instrument but by the presence of compounds with similar gas chromatographic properties as 4-methylpyrazol and containing peaks at m/e 82 or 85 in their mass spectrum. An increased sensitivity might have been obtained in the present study if it had been possible to prepare a derivative of 4methylpyrazol with a suitable peak of higher molecular weight in the mass spectrum. A drawback of the technique used in the present study was the extensive loss of 4-methylpyrazol during the concentration of the ether extract of the material. It was clearly shown, however, that the deuterated standard was lost during the concentration to exactly the same degree as the unlabeled 4-methylpyrazol. Thus varying degree of evaporation of the solvent did not have any effect on the ratio between m/e 82 and m/e 85 determined in the mass spectrometric analysis. Due to the specific volatile nature of 4-methylpyrazol it was not possible to use other pyrazol derivatives such as 4-trifluoromethylpyrazol as internal standard.
212
BJtiRKHEM
ET AL.
The half-life of 4-methylpyrazol in the rat was determined to be about 650 minutes under the conditions employed in the present work. Under essentially the same conditions Rydberg et aE. (6) determined the halflife of 4-methylpyrazol in the rat to be about 540 minutes using a gas chromatographic assay. The present work shows that the capacity to eliminate 4-methylpyrazol is different in rat and man. Studies on the metabolic basis of this difference are in progress. SUMMARY
A mass fragmentographic method for determination of 4-methylpyrazol in serum is described. 4-Methylpyrazol with three atoms of deuterium in the methyl group is added to a fixed amount of serum as internal standard. After extraction of the serum with ether, the amount of unlabeled 4-methylpyrazol is determined from the ratio between the recordings at m/e 82 and m/e 85 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). With this method 4-methylpyrazol could be determined in concentrations down to about 0.1 &ml corresponding to about 1 a. The relative standard deviation of the method in the range 4-21 m was less than 2%. The method could be used for determination of disappearance rate of 4-methylpyrazol in experimental animals as well as humans. REFERENCES 1. Blomstrand, R., and Theorell, H., Life Sciences 9, 631 (1970). 2. Blomstrand, R., in “Metabolic changes induced by alcohol,” p. 38 (G. A. Martine and C. H. Bode, Eds.). Springer Verlag, Berlin, 1971. 3. Blomstrand, R., and Forssell, L., Life Sciences 10, 523 (1971). 4. Blomstrand, R., and Kager, L., Life Sciences 13, 113 (1973). 5. Kager, L., and Ericsson, J. L. E., Acta Path. Micrub. Stand., 82, 534 (1974). 6. Rydberg, U., Buijten, J., and Neri, A., J. Pharm. Pharmuc. 24, 651 (1972). 7. Biemann, K., in “Mass Spectrometry,” p. 223. McGraw-Hill Book Company Inc., New York, 1962.