364
LETTERS TO THE EDITORS
A SIMPLE METHOD FOR THE DETERMINATION OF BECLAMIDE IN PLASMA BY GAS CHROMATOGRAPHY Beclamide (N-benzyl-3-chlorpropionamide, NydraneR) is a drug possessing anticonvulsant activity (Harned, Cunningham, Clark, Hine, Kane, Smith, Vessey, Yuda & Zabransky, 1953) which is available for the management of epilepsy, particularly grand mal seizures (Wilson, Walton & Newell, 1959) and for behavioural disorders (Sharpe, Dutton & Mirrey, 1958). The determination of beclamide in body fluids is hampered by the low molar-absorptivity (e = 190) of the compound and like many amides, it presents difficulty when gas chromatography is used for analysis. A gas chromatographic method for the estimation of plasma concentrations of some anti-epileptic drugs has previously been described by Gardner-Thorpe, Parsonage, Smethurst & Toothill (1972) but the method has generally proved to lack sensitivity and reproducibility in the determination of beclamide. In an attempt to quantify the amount of beclamide in solvent extracts of plasma using gas chromatography, most of the common stationary phases (e.g. SE 30, OV-1, 0-17, Apiezon-L and OV-225) were found to give two peaks on the chromatogram. Although this enabled semiquantitative data to be obtained, it was apparent that further development of the method was required to ensure greater analytical precision. Following the use of a variety of stationary phases at different loadings, it was found that a polyamide stationary phase (Poly-A 103) gave single symmetrical peaks and therefore extraction of beclamide from plasma was followed by gas chromatography on this stationary phase. Plasma (2 ml) and 1.0 M phosphate buffer (I ml) at pH 7.4 were introduced into a 50 ml glass stoppered centrifuge tube, followed by 0.4 ml of the internal standard (10 mg/100 ml barbitone). To each tube, chloroform (45 ml) was added and the stoppered tubes were mixed on a rotary mixer for 0.5 hours. After centrifugation, the aqueous layer was removed as completely as possible and traces of water were removed with anhydrous sodium sulphate (about 0.5 g). The solvent was decanted into a beaker and evaporated to dryness in a vacumn desiccator or on a low heat. The residue was dissolved in a little chloroform and washed into a small tapered tube. After evaporation to dryness, carbon disulphide (0.2 ml) was added and 1-2 ,ul was injected into a Pye senres 104 gas chromatograph fitted with a flame ionization detector. Columns were 1.5 m borosilicate glass with an outside
S
B
Injection Figure 1 Gas chromatogram for the quantitative estimation of beclamide (B) against internal standard
(S).
diameter of 6 mm. They were packed with 3% Poly-A 103 on 100/120 mesh Gas Chrom Q. Argon was used as the carrier gas with a flow rate of 40 ml/min, hydrogen and air for the flame ionization detector being set to give maximum sensitivity with minimum noise. The oven temperature was 210 C and no detector or injection point heating was used. The sample was injected into the column packing. Under these conditions, the retention time of beclamide was 2.5 min and barbitone, the internal standard, was 5.0 min (Figure 1). Quantification was by
LETTERS TO THE EDITORS
measurement of peak areas and calculation of the ratio between the test and internal standard. Standard solutions were prepared by adding known amounts of beclamide to human plasma samples. After extraction and development of the chromatograms, a linear relationship was observed between the ratio of the recorded area under the curve of beclamide to internal standard, and concentration of beclamide in plasma over the range studied. In ten experiments where beclamide (10 pg/ml) was added to normal plasma, recoveries of over 98% were obtained. Using the above procedure, plasma levels of beclamide were determined in two adult volunteers who had received a single oral dose of beclamide (1.0 g) after being fasted overnight. Beclamide was not detected in the plasma before dosing but appeared in both subjects at 0.5 h reaching a maximum plasma concentration of 12.4 and 10.2 ,g/ml at 2.0 h and declining to 4.5 and 4.2 ;g/ml at 8.0 hours. No beclamide was detected 24 h after dosing. The method is simple to perform and large numbers of samples can be handled without difficulty. It also has a sufficient degree of sensitivity (0.2,g/ml) to make it suitable for pharmacokinetic studies. No interfering peaks were found in blood samples from volunteers who had ingested beclamide and no significant interference was found when random blood samples from hospital patients not given beclamide were analysed by this method. Plasma extracts contain some normal blood constituents such as cholesterol which might be expected to interfere with the method. However, cholesterol has a long retention time on the Poly-A 103 column and will not normally interfere. However, it is advisable to allow the column to recover if
365
baseline drift becomes noticeable and the gas chromatograph should remain on with a slow flow of carrier gas overnight to aid in cleaning interfering material from the column. In addithe oven temperature can be raised to tiona3 225 C during the periods between analyses. We wish to thank Rona Laboratories Ltd for supplying beclamide (NydraneR). O.P. JONES, H. LEACH, D.K. LUSCOMBE & P.J. NICHOLLS Area Laboratory, Department of Clinical Chemistry, Caernarvon & Anglesey General Hospital, Bangor, Gwynedd; and the Welsh School of Pharmacy, UWIST, Cathays Park, Cardiff. CF1 3NU Received February 27, 1975
References M.J., PARSONAGE, C., GARDNER-THORPE, SMETHURST, P.F. & TOOTHILL, C. (1972). A
comprehensive gas chromatographic scheme for the estimation of antiepileptic drugs. Clin. chim. Acta, 36, 223-230. HARNED, B.K., CUNNINGHAM, R.W., CLARK, M.C., HINE, C.H., KANE, M.M., SMITH, F.H., VESSEY, R.E., YUDA, N.N. & ZABRANSKY, F.W. (1953). The
pharmacology of N-benzyl-,-chloropropionamide (Hibicon), a new anticonvulsant. J. Pharmac., 107, 403423. SHARPE, D.S., DUTTON, G. & MIRREY, J.R. (1958). Effects of Nydrane on epilepsy in mental defectives. Br. med. J., 1, 1044-1046. WILSON, J., WALTON, J.N. & NEWELL, D.J. (1959). Beclamide in intractable epilepsy: a controlled trial. Br. med. J., 1, 1275-1278.
LETTERS TO THE EDITORS
A SIMPLE METHOD FOR THE DETERMINATION OF BECLAMIDE IN PLASMA BY GAS CHROMATOGRAPHY Beclamide (N-benzyl-3-chlorpropionamide, NydraneR) is a drug possessing anticonvulsant activity (Harned, Cunningham, Clark, Hine, Kane, Smith, Vessey, Yuda & Zabransky, 1953) which is available for the management of epilepsy, particularly grand mal seizures (Wilson, Walton & Newell, 1959) and for behavioural disorders (Sharpe, Dutton & Mirrey, 1958). The determination of beclamide in body fluids is hampered by the low molar-absorptivity (e = 190) of the compound and like many amides, it presents difficulty when gas chromatography is used for analysis. A gas chromatographic method for the estimation of plasma concentrations of some anti-epileptic drugs has previously been described by Gardner-Thorpe, Parsonage, Smethurst & Toothill (1972) but the method has generally proved to lack sensitivity and reproducibility in the determination of beclamide. In an attempt to quantify the amount of beclamide in solvent extracts of plasma using gas chromatography, most of the common stationary phases (e.g. SE 30, OV-1, 0-17, Apiezon-L and OV-225) were found to give two peaks on the chromatogram. Although this enabled semiquantitative data to be obtained, it was apparent that further development of the method was required to ensure greater analytical precision. Following the use of a variety of stationary phases at different loadings, it was found that a polyamide stationary phase (Poly-A 103) gave single symmetrical peaks and therefore extraction of beclamide from plasma was followed by gas chromatography on this stationary phase. Plasma (2 ml) and 1.0 M phosphate buffer (I ml) at pH 7.4 were introduced into a 50 ml glass stoppered centrifuge tube, followed by 0.4 ml of the internal standard (10 mg/100 ml barbitone). To each tube, chloroform (45 ml) was added and the stoppered tubes were mixed on a rotary mixer for 0.5 hours. After centrifugation, the aqueous layer was removed as completely as possible and traces of water were removed with anhydrous sodium sulphate (about 0.5 g). The solvent was decanted into a beaker and evaporated to dryness in a vacumn desiccator or on a low heat. The residue was dissolved in a little chloroform and washed into a small tapered tube. After evaporation to dryness, carbon disulphide (0.2 ml) was added and 1-2 ,ul was injected into a Pye senres 104 gas chromatograph fitted with a flame ionization detector. Columns were 1.5 m borosilicate glass with an outside
S
B
Injection Figure 1 Gas chromatogram for the quantitative estimation of beclamide (B) against internal standard
(S).
diameter of 6 mm. They were packed with 3% Poly-A 103 on 100/120 mesh Gas Chrom Q. Argon was used as the carrier gas with a flow rate of 40 ml/min, hydrogen and air for the flame ionization detector being set to give maximum sensitivity with minimum noise. The oven temperature was 210 C and no detector or injection point heating was used. The sample was injected into the column packing. Under these conditions, the retention time of beclamide was 2.5 min and barbitone, the internal standard, was 5.0 min (Figure 1). Quantification was by
LETTERS TO THE EDITORS
measurement of peak areas and calculation of the ratio between the test and internal standard. Standard solutions were prepared by adding known amounts of beclamide to human plasma samples. After extraction and development of the chromatograms, a linear relationship was observed between the ratio of the recorded area under the curve of beclamide to internal standard, and concentration of beclamide in plasma over the range studied. In ten experiments where beclamide (10 pg/ml) was added to normal plasma, recoveries of over 98% were obtained. Using the above procedure, plasma levels of beclamide were determined in two adult volunteers who had received a single oral dose of beclamide (1.0 g) after being fasted overnight. Beclamide was not detected in the plasma before dosing but appeared in both subjects at 0.5 h reaching a maximum plasma concentration of 12.4 and 10.2 ,g/ml at 2.0 h and declining to 4.5 and 4.2 ;g/ml at 8.0 hours. No beclamide was detected 24 h after dosing. The method is simple to perform and large numbers of samples can be handled without difficulty. It also has a sufficient degree of sensitivity (0.2,g/ml) to make it suitable for pharmacokinetic studies. No interfering peaks were found in blood samples from volunteers who had ingested beclamide and no significant interference was found when random blood samples from hospital patients not given beclamide were analysed by this method. Plasma extracts contain some normal blood constituents such as cholesterol which might be expected to interfere with the method. However, cholesterol has a long retention time on the Poly-A 103 column and will not normally interfere. However, it is advisable to allow the column to recover if
365
baseline drift becomes noticeable and the gas chromatograph should remain on with a slow flow of carrier gas overnight to aid in cleaning interfering material from the column. In addithe oven temperature can be raised to tiona3 225 C during the periods between analyses. We wish to thank Rona Laboratories Ltd for supplying beclamide (NydraneR). O.P. JONES, H. LEACH, D.K. LUSCOMBE & P.J. NICHOLLS Area Laboratory, Department of Clinical Chemistry, Caernarvon & Anglesey General Hospital, Bangor, Gwynedd; and the Welsh School of Pharmacy, UWIST, Cathays Park, Cardiff. CF1 3NU Received February 27, 1975
References M.J., PARSONAGE, C., GARDNER-THORPE, SMETHURST, P.F. & TOOTHILL, C. (1972). A
comprehensive gas chromatographic scheme for the estimation of antiepileptic drugs. Clin. chim. Acta, 36, 223-230. HARNED, B.K., CUNNINGHAM, R.W., CLARK, M.C., HINE, C.H., KANE, M.M., SMITH, F.H., VESSEY, R.E., YUDA, N.N. & ZABRANSKY, F.W. (1953). The
pharmacology of N-benzyl-,-chloropropionamide (Hibicon), a new anticonvulsant. J. Pharmac., 107, 403423. SHARPE, D.S., DUTTON, G. & MIRREY, J.R. (1958). Effects of Nydrane on epilepsy in mental defectives. Br. med. J., 1, 1044-1046. WILSON, J., WALTON, J.N. & NEWELL, D.J. (1959). Beclamide in intractable epilepsy: a controlled trial. Br. med. J., 1, 1275-1278.
