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Rapid routine determination of some anti-epileptic drugs in serum by gas chromatography
CLINICA CHIMICAACTA
171
CGA 5062
RAPID
ROUTINE
DETERMINATION
OF SOME ANTI-EPILEPTIC
DRUGS
IN SERUM BY GAS CHROMATOGRAPHY
NIELS-ERIK LARSEN, JOERGEN NAESTOFT AND EIGILL HVIDBERG Department of Clinical Chemistry, Division ofClinical Pharmacology, Copenhagen-Glostrup (Denmark)
Glostrufi Hospital, DK-2 6 oo
(Received February 21, 1972)
SUMMARY
Procedures are described for the gas chromatographic determination of phenytoin (diphenylhydantoin, DPH), carbamazepine and phenobarbital in serum. The sensitivity, accuracy, and specificity are all comparable to those of other methods. The time involved for the procedures is about half an hour and the retention times I to 2 min. The capacity for routine analysis in the control of anti-epileptic treatment of patients is therefore considerably higher than that of other methods.
This paper presents a report on the determination of diphenylhydantoin (phenytoin, DPH), carbamazepine (Tegretol@) and phenobarbital in serum of patients by gas-liquid chromatography applied to routine analysis. Several procedures, most of them spectrophotometric, are available for the determination of these drugs. However, by gas chromatography it is possible to process large numbers of samples within a short time without loss of accuracy and specificity. MATERIALSAND METHODS
A@aratus Gas chromatograph: Pye Series 104, Model 64. Preheater temperature : 290”. Column temperature : 235”/290”. Detector temperature : 320’. Column: stainless steel, length 1.5 m, internal diameter 4 mm. Carrier substance: Celite J J CQ IOO/IZO mesh BS. Stationary phase : SE 52,3% (w/w). Amount of column material: 16 g. Column conditioned at 320~ for 48 h. Carrier gas flow: 40 ml N,/min. Hydrogen flow: 40 ml/min. CZin. Chim.
Acta, 40 (1972) 171-176
LARSEN
172 Air flow: 400 ml/min. Attenuation factor of the recorder:
104-2
et a&.
x 10~.
Dichloromethane, analytical grade, Merck. Ethyl acetate, analytical grade, Merck. Ethanol, 95%. 0.5 1V HCl, saturated with NaCl. IN H,SO, 6 N NaOH. Diazepam, ethanolic solution (400 mg/l). Enibomal, ethanolic solution {Soo mg/l). Procedures P~e~~~o~nand ~aybama~~~~~e.A quantity of 4.0 ml dichloromethane (DCIM) is added to 3.0 ml serum and carefully mixed for 3 min in a rotary mixer (20-30 rev. jmin). After centrifugation for 5 min at roooxg, the aqueous phase is removed. A 2.5-ml aliquot of the organic phase is transferred to another centrifuge tube and mixed for 3 min (20-30 rev. jmin) with 5 ml redistilled water and IOO ,d 6 N NaOH. After centrifugation 4.0 ml of the aqueous phase is mixed for 3 min (20-30 rev./min) with 1.0 ml IN H,SO, and 4. o ml DClM, andis centrifuged for 3 min. A zsoo-~1 aliquot of the DClM phase is then transferred to a tapered centrifuge tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue is dissolved in 25 ,d ethanol containing diazepam (internal standard 400 mg/l). A s-p1 quantity of this solution is injected into the gas chromatograph for the determination of DPH. With this method the normal therapeutic range for phenytoin is found to be 7-15 mg/l. A zooo-~1 aliquot of the organic phase from the original extract (see above) is TABLE
I
BASIS FOR THE
CONSTRUCTION
PHENOBARBITAL
IN
FIGURE
OF THE
Solution
Peak heights
drug mg/l
measured arbitrary
units
drug
intern. st.
A
corrected
: Phenytoin
5.0 IO.0 15.0 20.0 B
STANDARD
2
34 57 89 116
53 47 50 51
31 60 89 IX.5
: Carbamazepine
5.0 10.0
25
15.0
2
20.0
101
C: Phenobarbitd 16 5.0 IO.0 31 15.0 20.0 2:
48 46 51 43
27 :4 108
16
50 49 47 51
C&z. Chim. Acta, 40 (1972)
32 42 60 171-176
CURVES
FOR
PHENYTOIN,
CARBAMAZEPINE,
AND
DETERMINATION
OF ANTI-EPILEPTIC
DRUGS IN SERUM APeok
I73
hs,ghls
1.w.
5
10
I5
20
Fig. I. Examples of chromatograms of serum samples containing phenytoin (B), and phenobarbital (C) with corresponding internal standards.
25
(A), carbamazepine
Fig. 2. Standard curves plotted on the basis of chromatograms of 6 serum samples containing varying amounts of phenytoin (A), carbamazepine (B), and phenobarbital (C), respectively (cf Table I), diazepam and enibomal being used as internal standards. The peak heights are corrected additively in relation to a randomly chosen peak height of the internal standard. (For details see ref. 3).
evaporated to dryness in a tapered centrifuge tube under a stream of nitrogen. The residue is dissolved in 25 ,ul ethanol containing diazepam (400 mg/l). A 5-~1 volume of this solution is injected for the determination of carbamazepine. With this method the normal therapeutic range for carbamazepine is found to be 5-11 mg/l. Phenobarbital. A s-ml serum sample is mixed with 5 ml of an acid-free saturated NaCl solution with 3.0 ml ethyl acetate for 5 min and centrifuged for 5 min at IOOOxg. A 15oo+l aliquot of the organic phase is transferred to a tapered centrifuge tube and the ethyl acetate evaporated to dryness under a stream of nitrogen at 50”. The residue is dissolved in 25 ~1 ethanol containing enibomal (internal standard 800 mg/l). A 5+1 volume of this solution is injected for the determination of phenobarbital. Calculations.The serum concentrations are read from standard curves with the aid of the corrected peak heights (Figs. I and 2, Table I). The standard curves are based on aqueous solutions run simultaneously with the samples. Serum samples containing known amounts of the drugs are run in parallel as controls. To begin with, standard curves were based on serum samples (Figs. I, z), but aqueous solutions turned out to give equal results. TABLE II REPRODUCIBILITY BARBITAL
AND
RECOVERY
EXPERIMENTS
WITH
PHENYTOIN,
CARBAMAZEPINE
AND
PHENO-
IN SERUM
Drug
No.
of
Added
serum sam@les n Phenytoin Carbamazepine Phenobarbital
I
I5 5 75
Phenytoin Carbamazepine Phenobarbital
I.5 I5 I5
mgll
Recovery (wll)
f
S.D.
r5.0
14.8
f
12.0
II.2
+
20.0
18.6
i
2.5 2.5 5.0
2.4 2.4 4.7
0.43 0.29 0.3’
5 0.05 f 0.05 * 0.12 Clin. Chim. Acta, 40 (1972)
171-176
LARSEN t2f al.
I74 RESULTS
Sensitivity
and re$roducibility
In the development of the procedures described attempts were made to adapt them for routine determinations. The lower limit for safe quantitation of each of the three drugs in serum is I mg/l. The reproducibility of the determinations is shown in Table II. Recovery determinations were performed on plasma on the upper as well as on the lower part of the usual concentration range. The average recovery is in each method close to 100% with a standard deviation within a few per cent. This should be considered acceptable for routine determinations. Selectivity
It is seen from Table III that the average retention time for carbamazepine on the stationary phase used here is quite close to that of phenytoin (ratio 0.87). Because of the short retention times the interval between the registration times of the two TABLE
III
RELATIONSHIPBETWEEN COLUMNTEMPERATURES
AND
RETENTION
TIMES
Drug
Column temperature “C
Retention time min
Phenytoin
290
1.3
Carbamazepine (Diazepam, int. stand.)
290 290
I.1 1.8
Phenobarbital (Enibomal, int. stand.)
235 235
2.1 I.6
peaks is in the order of IO sec. This interval is very small and may cause faulty determinations of the last registered peak, if the concentrations of the two drugs differ very much. In the control of patients receiving both drugs it is therefore essential to enhance the selectivity. This has been achieved by the extraction procedures as phenytion will not be extracted by dichloromethane from alkalinised samples. The results from a series of determinations following addition to serum samples of all three drugs demonstrates the selectivity of the procedures (Table IV). Interference with other drugs is rare. Phenylbutazone will be determined as phenytoin; however, if the laboratory is informed of a simultaneous treatment with this drug, it can be removed quite easily (cf. ref. 3). Cyclobarbital (but not hexobarbital) will interfere with phenobarbital, but simultaneous treatment with these two barbiturates is quite uncommon. Many other drugs will not interfere with the analysis of phenytoin3. It should be mentioned that normal peroral doses of diazepam will not interfere with the determination, in spite of the use of this compound as internal standard. In ordinary treatment with diazepam plasma concentrations around IOO ,ug/l are common, and this isfar below the concentrations obtained by the addition of diazepam as internal standard, which corresponds to a concentration around II mg/l. Capacity
Results for all three drugs might be available to the clinical department within 30 min after the blood sample has been delivered to the laboratory. This implies, of Clin. Chim. Acta, 40 (1972) 171-176
DETERMINATION
TABLE
OF ANTI-EPILEPTIC
175
DRUGS IN SERUM
IV
SELECTIVITY
OF THE METKODS
Serum sanaple
Concentrut~on
9 10 I* 12 13 14 ‘5 16
WITH
MIXTURES
OF THE DRUGS _..
IN 16 SERUM
SAPnPLES
in mg/l
Phenytoin aed
8
DEMiONSTRATED
recovered
Carbamazepine
Phenobarbital
added
recovered
added
-
5.0
4.9
IS.0
-
10.0
IO.1
IS.0
-
15.3
15.0
-
19.7
15.0
-
15.0
-
5.0
5.0
-
IS.0
-
9.8 15.1
-
19.9
x5.0
-
IO.0 15.0
x5.0 5,O IO.0
-
20.0
5.1 9.9 ‘5.3
-
-
X5.0 20.0
-
4.7
15.0
10.0
15.0
X4.9 20.0
15.0
5.0
recovered
15.0 20.0
20.4
IO.0 15.0 20.0
15.0
-
1j.O
-
15.0 15.0
-
x5.0
-
10.0
15.0
-
15.0 15.0
x
20.0
1 -
15.0
5.0 15.0
-_
__._
4.8 9.7 15.2 19.3
course, that the procedure must be run daily as routine analysis. The maximum capacity is about x50 blood samples per working day per technician. To achieve a large analysing capacity like this it is necesary to operate with short retention times. These are tabulated together with the recommended column temperatures in Table III. DISCUSSION
Some of the analytical procedures described above have partly been reported separately elsewhere 3v4.However, a full description of the integrated procedures for use in the routine control of epileptic patients given two or three of these drugs simultaneously has not been published previously. Gas chromatography of phenytoin has the advantage of being more rapid, having a higher specificity and no less accuracy than the spectrophotometric methods of Dill e6 aL2 or of Svensmark and Kristensen’. Borg& et al.1 investigated the specificity of Dill’s method and found that several commonly used drugs interfered with the analysis, unless careful purification procedures were instituted. This reduced the capacity to about IO determinations per day per technician. As compared with this capacity the present gas chromatographic method represents an advantage in the routine analysis. One drawback of this procedure is the size of the blood sample needed for the determination (maximum 112 ml serum, i.e. 25-30 ml blood, for one duplicate determination of each of the three drugs). It has not been investigated whether this amount could be reduced. If so, however, a reduction of the capacity may be anticipated, because a longer retention time would be necessary as less available serum will reduce the amount of drug injected into the chromatograph. This, in turn, would require less attenuation of the signals from the detector. The amount of ethanol, however, would be unchanged. During such circumstances the ratio between the detector responses (ethanol/drug) would increase to a degree where the registration of the ethanol peak will disturb the drug peak, unless a considerable reduction of the column temperature is made. The result of this would be much longer retention times. C&s.
C/&R.. Acta, 40 (1972)
171-176
LARSEN
176
et al.
MaSee has developed a highly specific method based on a methylation of phenytoin. The total time for the entire procedure is about the same as described in the present publication, but the overall capacity will be less as the retention times of the chromatograms are about five times longer. The procedure for the determination of phenobarbital is somewhat different from the gas chromatographic method described by Munksgaard and Felby6. The most important difference, however, is in the longer retention time (2.1 instead of 6.1 min). The gas chromatographic methods described in this paper have proved to be reliable and convenient instruments in the control of epileptic patients in this hospital for about two years. REFERENCES I 0. BORG& L. LUND AND F. SJGQVIST, Likartidning, 66, Suppl. 3 (1969) 89. 2 W. A. DILL, A. KAZENKO, L. M. WOLF and A. J. GLASKO, J. Pharmacol. Exp. Therap., 118 (1956) 270. 3 N.-E. LARSEN, Med. Lab. Techn., 28 (1971) 377. 4 N.-E. LARSEN, J. WENDELBOE and L. BOHN, Stand. J. Clin. Lab. Invest., Suppl. IIO (1969) 35. 5 J. MACGEE Anal. Chem., 42 (1970) 421. 6 E. C. MUNKSGAARD AND S. FELBY, Dansk Tidsskr. Farm., 44 (1970) 343. 7 0. SVENSMARK AND ~.KRISTENSEN, J. Lab. Clin. Med., 63 (Ig63)5oI. C&n. Chim.
Acta, 40 (1972) 171-176
171
CGA 5062
RAPID
ROUTINE
DETERMINATION
OF SOME ANTI-EPILEPTIC
DRUGS
IN SERUM BY GAS CHROMATOGRAPHY
NIELS-ERIK LARSEN, JOERGEN NAESTOFT AND EIGILL HVIDBERG Department of Clinical Chemistry, Division ofClinical Pharmacology, Copenhagen-Glostrup (Denmark)
Glostrufi Hospital, DK-2 6 oo
(Received February 21, 1972)
SUMMARY
Procedures are described for the gas chromatographic determination of phenytoin (diphenylhydantoin, DPH), carbamazepine and phenobarbital in serum. The sensitivity, accuracy, and specificity are all comparable to those of other methods. The time involved for the procedures is about half an hour and the retention times I to 2 min. The capacity for routine analysis in the control of anti-epileptic treatment of patients is therefore considerably higher than that of other methods.
This paper presents a report on the determination of diphenylhydantoin (phenytoin, DPH), carbamazepine (Tegretol@) and phenobarbital in serum of patients by gas-liquid chromatography applied to routine analysis. Several procedures, most of them spectrophotometric, are available for the determination of these drugs. However, by gas chromatography it is possible to process large numbers of samples within a short time without loss of accuracy and specificity. MATERIALSAND METHODS
A@aratus Gas chromatograph: Pye Series 104, Model 64. Preheater temperature : 290”. Column temperature : 235”/290”. Detector temperature : 320’. Column: stainless steel, length 1.5 m, internal diameter 4 mm. Carrier substance: Celite J J CQ IOO/IZO mesh BS. Stationary phase : SE 52,3% (w/w). Amount of column material: 16 g. Column conditioned at 320~ for 48 h. Carrier gas flow: 40 ml N,/min. Hydrogen flow: 40 ml/min. CZin. Chim.
Acta, 40 (1972) 171-176
LARSEN
172 Air flow: 400 ml/min. Attenuation factor of the recorder:
104-2
et a&.
x 10~.
Dichloromethane, analytical grade, Merck. Ethyl acetate, analytical grade, Merck. Ethanol, 95%. 0.5 1V HCl, saturated with NaCl. IN H,SO, 6 N NaOH. Diazepam, ethanolic solution (400 mg/l). Enibomal, ethanolic solution {Soo mg/l). Procedures P~e~~~o~nand ~aybama~~~~~e.A quantity of 4.0 ml dichloromethane (DCIM) is added to 3.0 ml serum and carefully mixed for 3 min in a rotary mixer (20-30 rev. jmin). After centrifugation for 5 min at roooxg, the aqueous phase is removed. A 2.5-ml aliquot of the organic phase is transferred to another centrifuge tube and mixed for 3 min (20-30 rev. jmin) with 5 ml redistilled water and IOO ,d 6 N NaOH. After centrifugation 4.0 ml of the aqueous phase is mixed for 3 min (20-30 rev./min) with 1.0 ml IN H,SO, and 4. o ml DClM, andis centrifuged for 3 min. A zsoo-~1 aliquot of the DClM phase is then transferred to a tapered centrifuge tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue is dissolved in 25 ,d ethanol containing diazepam (internal standard 400 mg/l). A s-p1 quantity of this solution is injected into the gas chromatograph for the determination of DPH. With this method the normal therapeutic range for phenytoin is found to be 7-15 mg/l. A zooo-~1 aliquot of the organic phase from the original extract (see above) is TABLE
I
BASIS FOR THE
CONSTRUCTION
PHENOBARBITAL
IN
FIGURE
OF THE
Solution
Peak heights
drug mg/l
measured arbitrary
units
drug
intern. st.
A
corrected
: Phenytoin
5.0 IO.0 15.0 20.0 B
STANDARD
2
34 57 89 116
53 47 50 51
31 60 89 IX.5
: Carbamazepine
5.0 10.0
25
15.0
2
20.0
101
C: Phenobarbitd 16 5.0 IO.0 31 15.0 20.0 2:
48 46 51 43
27 :4 108
16
50 49 47 51
C&z. Chim. Acta, 40 (1972)
32 42 60 171-176
CURVES
FOR
PHENYTOIN,
CARBAMAZEPINE,
AND
DETERMINATION
OF ANTI-EPILEPTIC
DRUGS IN SERUM APeok
I73
hs,ghls
1.w.
5
10
I5
20
Fig. I. Examples of chromatograms of serum samples containing phenytoin (B), and phenobarbital (C) with corresponding internal standards.
25
(A), carbamazepine
Fig. 2. Standard curves plotted on the basis of chromatograms of 6 serum samples containing varying amounts of phenytoin (A), carbamazepine (B), and phenobarbital (C), respectively (cf Table I), diazepam and enibomal being used as internal standards. The peak heights are corrected additively in relation to a randomly chosen peak height of the internal standard. (For details see ref. 3).
evaporated to dryness in a tapered centrifuge tube under a stream of nitrogen. The residue is dissolved in 25 ,ul ethanol containing diazepam (400 mg/l). A 5-~1 volume of this solution is injected for the determination of carbamazepine. With this method the normal therapeutic range for carbamazepine is found to be 5-11 mg/l. Phenobarbital. A s-ml serum sample is mixed with 5 ml of an acid-free saturated NaCl solution with 3.0 ml ethyl acetate for 5 min and centrifuged for 5 min at IOOOxg. A 15oo+l aliquot of the organic phase is transferred to a tapered centrifuge tube and the ethyl acetate evaporated to dryness under a stream of nitrogen at 50”. The residue is dissolved in 25 ~1 ethanol containing enibomal (internal standard 800 mg/l). A 5+1 volume of this solution is injected for the determination of phenobarbital. Calculations.The serum concentrations are read from standard curves with the aid of the corrected peak heights (Figs. I and 2, Table I). The standard curves are based on aqueous solutions run simultaneously with the samples. Serum samples containing known amounts of the drugs are run in parallel as controls. To begin with, standard curves were based on serum samples (Figs. I, z), but aqueous solutions turned out to give equal results. TABLE II REPRODUCIBILITY BARBITAL
AND
RECOVERY
EXPERIMENTS
WITH
PHENYTOIN,
CARBAMAZEPINE
AND
PHENO-
IN SERUM
Drug
No.
of
Added
serum sam@les n Phenytoin Carbamazepine Phenobarbital
I
I5 5 75
Phenytoin Carbamazepine Phenobarbital
I.5 I5 I5
mgll
Recovery (wll)
f
S.D.
r5.0
14.8
f
12.0
II.2
+
20.0
18.6
i
2.5 2.5 5.0
2.4 2.4 4.7
0.43 0.29 0.3’
5 0.05 f 0.05 * 0.12 Clin. Chim. Acta, 40 (1972)
171-176
LARSEN t2f al.
I74 RESULTS
Sensitivity
and re$roducibility
In the development of the procedures described attempts were made to adapt them for routine determinations. The lower limit for safe quantitation of each of the three drugs in serum is I mg/l. The reproducibility of the determinations is shown in Table II. Recovery determinations were performed on plasma on the upper as well as on the lower part of the usual concentration range. The average recovery is in each method close to 100% with a standard deviation within a few per cent. This should be considered acceptable for routine determinations. Selectivity
It is seen from Table III that the average retention time for carbamazepine on the stationary phase used here is quite close to that of phenytoin (ratio 0.87). Because of the short retention times the interval between the registration times of the two TABLE
III
RELATIONSHIPBETWEEN COLUMNTEMPERATURES
AND
RETENTION
TIMES
Drug
Column temperature “C
Retention time min
Phenytoin
290
1.3
Carbamazepine (Diazepam, int. stand.)
290 290
I.1 1.8
Phenobarbital (Enibomal, int. stand.)
235 235
2.1 I.6
peaks is in the order of IO sec. This interval is very small and may cause faulty determinations of the last registered peak, if the concentrations of the two drugs differ very much. In the control of patients receiving both drugs it is therefore essential to enhance the selectivity. This has been achieved by the extraction procedures as phenytion will not be extracted by dichloromethane from alkalinised samples. The results from a series of determinations following addition to serum samples of all three drugs demonstrates the selectivity of the procedures (Table IV). Interference with other drugs is rare. Phenylbutazone will be determined as phenytoin; however, if the laboratory is informed of a simultaneous treatment with this drug, it can be removed quite easily (cf. ref. 3). Cyclobarbital (but not hexobarbital) will interfere with phenobarbital, but simultaneous treatment with these two barbiturates is quite uncommon. Many other drugs will not interfere with the analysis of phenytoin3. It should be mentioned that normal peroral doses of diazepam will not interfere with the determination, in spite of the use of this compound as internal standard. In ordinary treatment with diazepam plasma concentrations around IOO ,ug/l are common, and this isfar below the concentrations obtained by the addition of diazepam as internal standard, which corresponds to a concentration around II mg/l. Capacity
Results for all three drugs might be available to the clinical department within 30 min after the blood sample has been delivered to the laboratory. This implies, of Clin. Chim. Acta, 40 (1972) 171-176
DETERMINATION
TABLE
OF ANTI-EPILEPTIC
175
DRUGS IN SERUM
IV
SELECTIVITY
OF THE METKODS
Serum sanaple
Concentrut~on
9 10 I* 12 13 14 ‘5 16
WITH
MIXTURES
OF THE DRUGS _..
IN 16 SERUM
SAPnPLES
in mg/l
Phenytoin aed
8
DEMiONSTRATED
recovered
Carbamazepine
Phenobarbital
added
recovered
added
-
5.0
4.9
IS.0
-
10.0
IO.1
IS.0
-
15.3
15.0
-
19.7
15.0
-
15.0
-
5.0
5.0
-
IS.0
-
9.8 15.1
-
19.9
x5.0
-
IO.0 15.0
x5.0 5,O IO.0
-
20.0
5.1 9.9 ‘5.3
-
-
X5.0 20.0
-
4.7
15.0
10.0
15.0
X4.9 20.0
15.0
5.0
recovered
15.0 20.0
20.4
IO.0 15.0 20.0
15.0
-
1j.O
-
15.0 15.0
-
x5.0
-
10.0
15.0
-
15.0 15.0
x
20.0
1 -
15.0
5.0 15.0
-_
__._
4.8 9.7 15.2 19.3
course, that the procedure must be run daily as routine analysis. The maximum capacity is about x50 blood samples per working day per technician. To achieve a large analysing capacity like this it is necesary to operate with short retention times. These are tabulated together with the recommended column temperatures in Table III. DISCUSSION
Some of the analytical procedures described above have partly been reported separately elsewhere 3v4.However, a full description of the integrated procedures for use in the routine control of epileptic patients given two or three of these drugs simultaneously has not been published previously. Gas chromatography of phenytoin has the advantage of being more rapid, having a higher specificity and no less accuracy than the spectrophotometric methods of Dill e6 aL2 or of Svensmark and Kristensen’. Borg& et al.1 investigated the specificity of Dill’s method and found that several commonly used drugs interfered with the analysis, unless careful purification procedures were instituted. This reduced the capacity to about IO determinations per day per technician. As compared with this capacity the present gas chromatographic method represents an advantage in the routine analysis. One drawback of this procedure is the size of the blood sample needed for the determination (maximum 112 ml serum, i.e. 25-30 ml blood, for one duplicate determination of each of the three drugs). It has not been investigated whether this amount could be reduced. If so, however, a reduction of the capacity may be anticipated, because a longer retention time would be necessary as less available serum will reduce the amount of drug injected into the chromatograph. This, in turn, would require less attenuation of the signals from the detector. The amount of ethanol, however, would be unchanged. During such circumstances the ratio between the detector responses (ethanol/drug) would increase to a degree where the registration of the ethanol peak will disturb the drug peak, unless a considerable reduction of the column temperature is made. The result of this would be much longer retention times. C&s.
C/&R.. Acta, 40 (1972)
171-176
LARSEN
176
et al.
MaSee has developed a highly specific method based on a methylation of phenytoin. The total time for the entire procedure is about the same as described in the present publication, but the overall capacity will be less as the retention times of the chromatograms are about five times longer. The procedure for the determination of phenobarbital is somewhat different from the gas chromatographic method described by Munksgaard and Felby6. The most important difference, however, is in the longer retention time (2.1 instead of 6.1 min). The gas chromatographic methods described in this paper have proved to be reliable and convenient instruments in the control of epileptic patients in this hospital for about two years. REFERENCES I 0. BORG& L. LUND AND F. SJGQVIST, Likartidning, 66, Suppl. 3 (1969) 89. 2 W. A. DILL, A. KAZENKO, L. M. WOLF and A. J. GLASKO, J. Pharmacol. Exp. Therap., 118 (1956) 270. 3 N.-E. LARSEN, Med. Lab. Techn., 28 (1971) 377. 4 N.-E. LARSEN, J. WENDELBOE and L. BOHN, Stand. J. Clin. Lab. Invest., Suppl. IIO (1969) 35. 5 J. MACGEE Anal. Chem., 42 (1970) 421. 6 E. C. MUNKSGAARD AND S. FELBY, Dansk Tidsskr. Farm., 44 (1970) 343. 7 0. SVENSMARK AND ~.KRISTENSEN, J. Lab. Clin. Med., 63 (Ig63)5oI. C&n. Chim.
Acta, 40 (1972) 171-176