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Improved method for measurement of serum levels of phenobarbital, carbamazepine, primidone and diphenylhydantoin by gas-liquid chromatography
463
Clinica Chimica Acta, 13 (1976) 463-466 @ Elsevier/North-Holland Biomedical Press
CCA 8190
IMPROVED METHOD FOR MEASUREMENT OF SERUM LEVELS PHENOBARBITAL, CARBAMAZEPINE, PRIMIDONE AND DIPHENYLHYDANTOIN BY GAS-LIQUID CHROMATOGRAPHY
TOSHIHIRO
NISHINA
*, KOKO OKOSHI
and MOTOSHI
Department of Clinical Chemistry, Toranomon for Medical Research, Tokyo (Japan) (Received
OF
KITAMURA
Hospital and Okinaka Memorial Institute
July lst, 1976)
Summary A gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone, carbamazepine and diphenylhydantoin in human serum following therapeutic doses has been developed. After extraction with chloroform, the anticonvulsant drugs were methylated with phenyltrimethylammonium hydroxide in dimethylformamide at 85°C for gas-liquid chromatography. Liner temperature programming of a 1% OV-17 column was used to achieve separation and quantitation. The procedure described in the present paper is relatively simple, highly specific and sufficiently sensitive for use in routine clinical assays.
Introduction Serum levels of anticonvulsant drugs like phenobarbital (PB), primidone (Prim), carbamazepine (Carb) and diphenylhydantoin (DPH) have mainly been determined by gas-liquid chromatography (GLC). On-column alkylation procedures like methylation [l-8], ethylation [ 7-91 and hexylation [lo] for GLC have been proposed by several authors. These procedures have proven to be rapid and convenient for quantitative estimation. However, various manipulative factors may influence the quality of results, among them injection-port temperature, speed of sample injections, the length of the heated injection ports, concentration of alkylating reagent and injection volume have been reported to be major factors. [ 3,7,8,11]. Using dimethylformamide (DMF) as a solvent in place of dioxane and preincubating reaction mixture at 85°C for 10 min, we can obtain satisfactory and reproducible results with the on-column methylation. * To
whom
correspondence
should
be addressed.
Materials and methods Apparatus 11 gas chromatograph
(Model 4BM, Shimazu Ltd., Kyoto, Japan) eyuipped with a dual flame ionization detector was used for analysis. The coiled-glass columns 0.3 cm in diameter and 200 cm in length were packed with Chromosorb W (AW-DMCS, 100-120 mesh) coated with 1% OV-17. The analyses were performed under the following conditions: column temperature, initial 18O”C, programmed at the rate of G”C/min to 270°C. The gas flows were nitrogen as a carrier 60 ml/min, hydrogen 40 ml/min. Reagents Standard
stock solution (200 pg/ml). 20 mg each of PB, Prim, Carb and DPH were dissolved in 100 ml of absolute methanol. This stock solution was stored at 4°C for periods no longer than three months. Standard working solutions. 0.5-ml to 3.0-ml aliquots of the stock standard solution were each diluted to 10 ml with distilled water to provide solutions containing 10 to 60 /.ig of standard per ml. These working solutions were prepared fresh just prior to use. Cholestane internal standard solution (200 pg/ml), 20 mg of cholcstane obtained from the Sigma Chemical Co. (Missouri, U.S.A.) was dissolved in 100 ml of methanol. Phenyltrimethylammonium hydroxide (PTMAH) solution (0.1 M). PTMAH was obtained from the Eastman Kodak Co. (Rochester, U.S.A.) or Tokyo Kasei Co. (Tokyo, Japan) and the solution was diluted to 0.1 M with methanol. All solvents and reagents used were reagent grade. Procedure
Into a 15-ml glass-stoppered centrifuge tube, 0.5 ml of serum was pipetted and 0.5 ml of 0.2 N NaOH and 10 ml chloroform were added. The mixture was shaken for 3 min and centrifuged for 5 min at 3000 rpm. The organic solvent phase was transferred to a lo-ml centrifuge tube and evaporated. The chloroform residue (fraction 1) contained Prim and Carb. The stored alkaline soluble fraction, which was pippetted out when fraction 1 was taken, was acidified with 0.5 ml of concentrated HCl and then extracted with 10 ml of chloroform. If a wafer of insoluble material is present at the interface, it can be lifted out with a spatula. The chloroform layer was transferred to a 15-ml centrifuge tube, then successively extracted with 5.0 ml of 0.2 M tribasic sodium phosphate (NaJPO,) and reacidified with 0.2 ml of concentrated HCl and re-extracted with 10 ml of chloroform. The final chloroform fraction (fraction 2) contained PB and DPH, and was transferred to fraction 1 and evaporated to dryness. The inside of tube was washed down with 0.2 ml of methanol and 40 ~1 of DMF, 50 ~1 of 0.1 M PTMAH and 50 ~1 of cholestane solution (10 lg) were added. This reaction mixture was aliowed to stand for 10 min in an 85°C water bath to complete the reaction and evaporate the methanol. 2 ~1 of the DMF solution were injected into the gas chromatograph.
465
Calculations Each standard working solution to be analyzed was day’s unknown samples. The ratio of the peak height of cholestane was plotted against the drug concentration to tion curves used to calculate drug concentration in samples
processed with each each drug to that of generate the calibrafrom patients.
Results Methylation The methylation of anticonvulsant drugs with PTMAH in the present method appeared to be quantitative. A plot of peak height against the amount of anticonvulsant injected into the GLC produced a straight line. The chromatogram resolution from the methylation of equal amounts of anticonvulsant drugs and cholestane is shown in Figs. 1A and 1C. The five compounds were separated well and did not have tailing peaks. No interfering peaks were found with retention times similar to that of anticonvulsant drugs when 15 human sera, obtained from patients not receiving any of four anticonvulsant drugs were extracted, methylated and chromatographed (Fig. 1B). Occasional samples showed peaks coming out immediately after or before the PB peak. These unknown peaks were separated enough not to interfere with the calculation. A
5
0
IO
I5
min
1
jL!_&-i-II 5
0
II
10
15
min
C
Fig. 1. A. A chromatogram of anticonvulsant drugs added to water. B. A typical chromatogram of a negative blood specimen. C. A chromatogram of anticonvulsant drugs added to a negative blood specimen. Conditions: 1% OV-17 on Chromosorb W; nitrogen flow rate 60 mllmln; Initial column temperature 18O’C; temperature program rate 6’Clmin to 270°C.
466
Standard curues The peak height ratio of the ~ticonvul~nt drugs to chofestane, the internal standard, plotted against the amount of anticonvulsant drugs added to water is shown in Fig. 2. The plots were linear up to concentrations of 60 @g/ml for each drug, appearing to pass through the origin. Standard curve determinations were made on a daily basis since minor day to day variations were noted.
Precision The coefficients of variation tracted from different serum mean within-day value.
of five determinations at each concentration exsamples never exceeded more than 4% of the
Recoueries 10, 20 and 30 Erg of each drug were added to 0.5 ml of serum and measured. The mean recoveries from sera were 101 + 5% (range 96-105%). Serum samples taken from patients receiving the a~ticonvulsant drugs were
Cholestane
Standard
Curves
IIB
Concetiriition (uglml
Cholestane
I
Fig. 2. Calibration cuntes for anticonvulsant drugs. Each point represents the peak height ratio of anticonvulsant drugs to cholestane as an internal standard as a function of PB, Prim. Garb and DPH. Conditions: see Fig. 1. Fig. 3, Gas chromatograms of a methylated extract of plasma taken from a patient and DPH (B) and Prim and DPH (C). Condition; see Fig. 1.
receiving
Garb (A), PB
467
TABLE
I
Patient
Daily medication (mg/day) PB
U.O. T.A. H.I. S.I. A.M. T.M. M.K. K.Y. M.K. I.K. K.T. T.S. Y.M. H.S. F.M.
40 66 50 30 50 66 33 90 40 42
Pri
Garb
Blood level (/%/ml) DPH
PB
Pri 17.6 8.8 12.0 2.0
133 100 80 100 133 100 99 100 125
44.8 16.8 24.4 6.8 14.2 14.4 26.4 34.8 10.8 4.8 30.6 10.0 12.4
750 375 500 150
120 250
500 300 200 400 400
Garb
4.8 5.8
8.8 2.8 3.2 6.0 8.6
DPH
3.6 2.8 14.4 4.8 4.0 2.6 3.2 1.6 2.6
also analyzed. The concentrations of the drugs are shown in Table I. Typical chromatograms of a patient receiving Prim, Carb and DPH are shown in Figs. 3A, B and C. Since Prim is converted in part to PB as described by other authors, significant PB levels were detected although it was not given to patients receiving only Prim. Their observations were seen in patients U.O., T.A. and H.I. as shown in Table I. Discussion The on-column methylation has proven to be a rapid and convenient technique for quantitative estimation of various anticonvulsant drugs. However, it has been found to give some variability in results, in that on some occasions extra peaks are formed and relative peak heights are not constant. It was suggested that DMF as a dipolar aprotic solvent would be preferable to other solvents like dioxan and methanol because using DMF would cause a larger production of free methyl groups from PTMAH than previous methods and should require shorter reaction times and milder conditions for the methylation [12]. Using DMF as a reaction solvent in place of dioxane and preincubation of the reaction mixture at 85°C for 10 min, we can obtain satisfactory and reproducible results. This procedure has two advantages over previous methods using GLC. First, by preincubating at 85°C for 10 min, the peak height of cholestane was constant because the other solvents were evaporated and the volume of DMF was constant. Secondly, the coefficient of variation, on a day-to-day basis, of the present method could be kept to the low range of between 4.5% and 6.2% because complete methylation could be obtained so easily. The present method allows the analysis of about 15 samples per day. Thus, for rapidity, accuracy and specificity, the method proposed here is suitable for the monitoring of epileptic patients taking PB, Prim, Carb and DPH. This method may be easily widened to other anticonvulsant drugs.
468
Acknowledgements We are grateful to Dr. K. Miyamoto of National Musashi Research Institute for Mental and Nervous Diseases (Tokyo) for his encouragement and advice. We wish also to thank Dr. Misako Inui for helpful discussions. References 1
Stevenson,
2
MacGee.
G.W.
3
Brochmann-Hanssen,
4
Street,
H.V.
5
Kupferberg,
6
Miyamoto, drugs Co.
H.J. K.,
Inc.,
7
Friel,
MacGee,
P. and
9
Kumps,
Clin.
Clin. M.,
(1969)
Acta Chim.
Ikeda.
et al.,
305-314
T.O.
J. Pharm.
Sci.
58,370-371
357-364
Acta
Y.
eds.),
34.
29,
283-288
and Yamagami,
pp.
106-110.
Clin.
Chem.
H. (1973) Excerpta
in Methods
Medica.
Troupin.
A.S.
Clin.
J.J.
11
Vree,
Breimer,
(1975)
Chem.
and Mardens,
Givannielle.
Y.
and Pecc.
17,
Kimura,
and
of anti-epileptic American
Elsevier
(1975)
J. (1976)
D.D.,
Van
21,
751-754
587-591 Clin. Clin.
Ginneken,
Chin
Acta
Chim. C.A.M.
62,371-376
Acta
67,
and
7-13
Van
Rossum,
365-372 12
of analysis
Amsterdam
York
10
T.B..
38,1948-1949 14.
Oke,
Chin
(1970) Seine,
J. (1971) A.
Chem.
Biochem.
E. and
J.W.A.
New
Anal.
Anal.
(1971)
(M&x,
8
(1966)
J. (1966)
M. and Nishina.
T. (1967)
Chem.
Pham.
Bull.
15.1315-1321
J.M.
(1971)
Clin.
Chim.
Acta
34,
Clinica Chimica Acta, 13 (1976) 463-466 @ Elsevier/North-Holland Biomedical Press
CCA 8190
IMPROVED METHOD FOR MEASUREMENT OF SERUM LEVELS PHENOBARBITAL, CARBAMAZEPINE, PRIMIDONE AND DIPHENYLHYDANTOIN BY GAS-LIQUID CHROMATOGRAPHY
TOSHIHIRO
NISHINA
*, KOKO OKOSHI
and MOTOSHI
Department of Clinical Chemistry, Toranomon for Medical Research, Tokyo (Japan) (Received
OF
KITAMURA
Hospital and Okinaka Memorial Institute
July lst, 1976)
Summary A gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone, carbamazepine and diphenylhydantoin in human serum following therapeutic doses has been developed. After extraction with chloroform, the anticonvulsant drugs were methylated with phenyltrimethylammonium hydroxide in dimethylformamide at 85°C for gas-liquid chromatography. Liner temperature programming of a 1% OV-17 column was used to achieve separation and quantitation. The procedure described in the present paper is relatively simple, highly specific and sufficiently sensitive for use in routine clinical assays.
Introduction Serum levels of anticonvulsant drugs like phenobarbital (PB), primidone (Prim), carbamazepine (Carb) and diphenylhydantoin (DPH) have mainly been determined by gas-liquid chromatography (GLC). On-column alkylation procedures like methylation [l-8], ethylation [ 7-91 and hexylation [lo] for GLC have been proposed by several authors. These procedures have proven to be rapid and convenient for quantitative estimation. However, various manipulative factors may influence the quality of results, among them injection-port temperature, speed of sample injections, the length of the heated injection ports, concentration of alkylating reagent and injection volume have been reported to be major factors. [ 3,7,8,11]. Using dimethylformamide (DMF) as a solvent in place of dioxane and preincubating reaction mixture at 85°C for 10 min, we can obtain satisfactory and reproducible results with the on-column methylation. * To
whom
correspondence
should
be addressed.
Materials and methods Apparatus 11 gas chromatograph
(Model 4BM, Shimazu Ltd., Kyoto, Japan) eyuipped with a dual flame ionization detector was used for analysis. The coiled-glass columns 0.3 cm in diameter and 200 cm in length were packed with Chromosorb W (AW-DMCS, 100-120 mesh) coated with 1% OV-17. The analyses were performed under the following conditions: column temperature, initial 18O”C, programmed at the rate of G”C/min to 270°C. The gas flows were nitrogen as a carrier 60 ml/min, hydrogen 40 ml/min. Reagents Standard
stock solution (200 pg/ml). 20 mg each of PB, Prim, Carb and DPH were dissolved in 100 ml of absolute methanol. This stock solution was stored at 4°C for periods no longer than three months. Standard working solutions. 0.5-ml to 3.0-ml aliquots of the stock standard solution were each diluted to 10 ml with distilled water to provide solutions containing 10 to 60 /.ig of standard per ml. These working solutions were prepared fresh just prior to use. Cholestane internal standard solution (200 pg/ml), 20 mg of cholcstane obtained from the Sigma Chemical Co. (Missouri, U.S.A.) was dissolved in 100 ml of methanol. Phenyltrimethylammonium hydroxide (PTMAH) solution (0.1 M). PTMAH was obtained from the Eastman Kodak Co. (Rochester, U.S.A.) or Tokyo Kasei Co. (Tokyo, Japan) and the solution was diluted to 0.1 M with methanol. All solvents and reagents used were reagent grade. Procedure
Into a 15-ml glass-stoppered centrifuge tube, 0.5 ml of serum was pipetted and 0.5 ml of 0.2 N NaOH and 10 ml chloroform were added. The mixture was shaken for 3 min and centrifuged for 5 min at 3000 rpm. The organic solvent phase was transferred to a lo-ml centrifuge tube and evaporated. The chloroform residue (fraction 1) contained Prim and Carb. The stored alkaline soluble fraction, which was pippetted out when fraction 1 was taken, was acidified with 0.5 ml of concentrated HCl and then extracted with 10 ml of chloroform. If a wafer of insoluble material is present at the interface, it can be lifted out with a spatula. The chloroform layer was transferred to a 15-ml centrifuge tube, then successively extracted with 5.0 ml of 0.2 M tribasic sodium phosphate (NaJPO,) and reacidified with 0.2 ml of concentrated HCl and re-extracted with 10 ml of chloroform. The final chloroform fraction (fraction 2) contained PB and DPH, and was transferred to fraction 1 and evaporated to dryness. The inside of tube was washed down with 0.2 ml of methanol and 40 ~1 of DMF, 50 ~1 of 0.1 M PTMAH and 50 ~1 of cholestane solution (10 lg) were added. This reaction mixture was aliowed to stand for 10 min in an 85°C water bath to complete the reaction and evaporate the methanol. 2 ~1 of the DMF solution were injected into the gas chromatograph.
465
Calculations Each standard working solution to be analyzed was day’s unknown samples. The ratio of the peak height of cholestane was plotted against the drug concentration to tion curves used to calculate drug concentration in samples
processed with each each drug to that of generate the calibrafrom patients.
Results Methylation The methylation of anticonvulsant drugs with PTMAH in the present method appeared to be quantitative. A plot of peak height against the amount of anticonvulsant injected into the GLC produced a straight line. The chromatogram resolution from the methylation of equal amounts of anticonvulsant drugs and cholestane is shown in Figs. 1A and 1C. The five compounds were separated well and did not have tailing peaks. No interfering peaks were found with retention times similar to that of anticonvulsant drugs when 15 human sera, obtained from patients not receiving any of four anticonvulsant drugs were extracted, methylated and chromatographed (Fig. 1B). Occasional samples showed peaks coming out immediately after or before the PB peak. These unknown peaks were separated enough not to interfere with the calculation. A
5
0
IO
I5
min
1
jL!_&-i-II 5
0
II
10
15
min
C
Fig. 1. A. A chromatogram of anticonvulsant drugs added to water. B. A typical chromatogram of a negative blood specimen. C. A chromatogram of anticonvulsant drugs added to a negative blood specimen. Conditions: 1% OV-17 on Chromosorb W; nitrogen flow rate 60 mllmln; Initial column temperature 18O’C; temperature program rate 6’Clmin to 270°C.
466
Standard curues The peak height ratio of the ~ticonvul~nt drugs to chofestane, the internal standard, plotted against the amount of anticonvulsant drugs added to water is shown in Fig. 2. The plots were linear up to concentrations of 60 @g/ml for each drug, appearing to pass through the origin. Standard curve determinations were made on a daily basis since minor day to day variations were noted.
Precision The coefficients of variation tracted from different serum mean within-day value.
of five determinations at each concentration exsamples never exceeded more than 4% of the
Recoueries 10, 20 and 30 Erg of each drug were added to 0.5 ml of serum and measured. The mean recoveries from sera were 101 + 5% (range 96-105%). Serum samples taken from patients receiving the a~ticonvulsant drugs were
Cholestane
Standard
Curves
IIB
Concetiriition (uglml
Cholestane
I
Fig. 2. Calibration cuntes for anticonvulsant drugs. Each point represents the peak height ratio of anticonvulsant drugs to cholestane as an internal standard as a function of PB, Prim. Garb and DPH. Conditions: see Fig. 1. Fig. 3, Gas chromatograms of a methylated extract of plasma taken from a patient and DPH (B) and Prim and DPH (C). Condition; see Fig. 1.
receiving
Garb (A), PB
467
TABLE
I
Patient
Daily medication (mg/day) PB
U.O. T.A. H.I. S.I. A.M. T.M. M.K. K.Y. M.K. I.K. K.T. T.S. Y.M. H.S. F.M.
40 66 50 30 50 66 33 90 40 42
Pri
Garb
Blood level (/%/ml) DPH
PB
Pri 17.6 8.8 12.0 2.0
133 100 80 100 133 100 99 100 125
44.8 16.8 24.4 6.8 14.2 14.4 26.4 34.8 10.8 4.8 30.6 10.0 12.4
750 375 500 150
120 250
500 300 200 400 400
Garb
4.8 5.8
8.8 2.8 3.2 6.0 8.6
DPH
3.6 2.8 14.4 4.8 4.0 2.6 3.2 1.6 2.6
also analyzed. The concentrations of the drugs are shown in Table I. Typical chromatograms of a patient receiving Prim, Carb and DPH are shown in Figs. 3A, B and C. Since Prim is converted in part to PB as described by other authors, significant PB levels were detected although it was not given to patients receiving only Prim. Their observations were seen in patients U.O., T.A. and H.I. as shown in Table I. Discussion The on-column methylation has proven to be a rapid and convenient technique for quantitative estimation of various anticonvulsant drugs. However, it has been found to give some variability in results, in that on some occasions extra peaks are formed and relative peak heights are not constant. It was suggested that DMF as a dipolar aprotic solvent would be preferable to other solvents like dioxan and methanol because using DMF would cause a larger production of free methyl groups from PTMAH than previous methods and should require shorter reaction times and milder conditions for the methylation [12]. Using DMF as a reaction solvent in place of dioxane and preincubation of the reaction mixture at 85°C for 10 min, we can obtain satisfactory and reproducible results. This procedure has two advantages over previous methods using GLC. First, by preincubating at 85°C for 10 min, the peak height of cholestane was constant because the other solvents were evaporated and the volume of DMF was constant. Secondly, the coefficient of variation, on a day-to-day basis, of the present method could be kept to the low range of between 4.5% and 6.2% because complete methylation could be obtained so easily. The present method allows the analysis of about 15 samples per day. Thus, for rapidity, accuracy and specificity, the method proposed here is suitable for the monitoring of epileptic patients taking PB, Prim, Carb and DPH. This method may be easily widened to other anticonvulsant drugs.
468
Acknowledgements We are grateful to Dr. K. Miyamoto of National Musashi Research Institute for Mental and Nervous Diseases (Tokyo) for his encouragement and advice. We wish also to thank Dr. Misako Inui for helpful discussions. References 1
Stevenson,
2
MacGee.
G.W.
3
Brochmann-Hanssen,
4
Street,
H.V.
5
Kupferberg,
6
Miyamoto, drugs Co.
H.J. K.,
Inc.,
7
Friel,
MacGee,
P. and
9
Kumps,
Clin.
Clin. M.,
(1969)
Acta Chim.
Ikeda.
et al.,
305-314
T.O.
J. Pharm.
Sci.
58,370-371
357-364
Acta
Y.
eds.),
34.
29,
283-288
and Yamagami,
pp.
106-110.
Clin.
Chem.
H. (1973) Excerpta
in Methods
Medica.
Troupin.
A.S.
Clin.
J.J.
11
Vree,
Breimer,
(1975)
Chem.
and Mardens,
Givannielle.
Y.
and Pecc.
17,
Kimura,
and
of anti-epileptic American
Elsevier
(1975)
J. (1976)
D.D.,
Van
21,
751-754
587-591 Clin. Clin.
Ginneken,
Chin
Acta
Chim. C.A.M.
62,371-376
Acta
67,
and
7-13
Van
Rossum,
365-372 12
of analysis
Amsterdam
York
10
T.B..
38,1948-1949 14.
Oke,
Chin
(1970) Seine,
J. (1971) A.
Chem.
Biochem.
E. and
J.W.A.
New
Anal.
Anal.
(1971)
(M&x,
8
(1966)
J. (1966)
M. and Nishina.
T. (1967)
Chem.
Pham.
Bull.
15.1315-1321
J.M.
(1971)
Clin.
Chim.
Acta
34,