Br. J. clin. Pharmac. (1979), 8,169-171
DETERMINATION OF VILOXAZINE IN PLASMA BY GLC T.R. NORMAN, G.D. BURROWS, B.M. DAVIES
J.M.E. WURM
D atment of Psychiatry, University of Melbourr a, Parkville, Victoria, 3052, Australia
1 A gas chromatographic procedure for the determination of viloxazine in therapeutic concentrations in human plasma, with use of a flame ionization detector, is described. 2 The drug is extracted at pH 12 into hexane, back extracted into HCI, re-extracted into hexane after alkalinization of the HCI and derivatized with acetic anhydride. 3 An imipramine-butriptyline mixture is used as an internal standard. The coefficients of variation for a concentration of 0.75 ig/ml are 9.4% and 13.1% for within day and day to day precision respectively. For a 1.75 pg/ml solution the within day precision was 5.7%. 4 The method has been applied to a patient receiving 300 mg/day viloxazine treatment.
IRWduction Viloxazine (Vivalan, I.C.I.) is a bicyclic compound with a pharmacological profile similar to that observed with the tricyclic antidepressants (Mallion, Greenwood, Bainbridge, Turner, Todd, Madinaveitia, Somerville & Whittle, 1972; Greenwood, 1975). Controlled clinical evaluations have confirmed that viloxazine is an effective antidepressant agent (Pinder, Brogden, Speight & Avery, 1977). Peet (1973) reported no relationship between the clinical effect of viloxazine and its concentration in plasma. Case (1973) has described a method, based on gas liquid chromatography (glc) with an electron capture detector, for the determination of viloxazine in blood and plasma. This method, while suitable for use in clinical studies, does not include the use of an internal standard to correct for procedural losses during the determination. The present paper descibes a method, based on glc with a flame ionization detector, for plasma viloxazine determinations. The method has been applied to the determination of samples from patients who took part in a clinical evaluation of the drug (Davies, Joshua, Burrows & Poynton, 1977). Methods Reagents
Hexane (Nanograde, Mallinckrodt), absolute ethanol
(pro analysi, Merck) triethanolamine (G.P.R., 0306-5251/79/080169 03 $01.00
Hopkin and Williams), chloroform (A.R., Univar) and acetic anhydride (A.R. Univar) were used without further purification. Butriptyline hydrochloride (Ayerst Laboratories Pty Ltd, Sydney), (Ciba-Geigy hydrochloride imipramine Pharmaceuticals, Sydney) and viloxazine hydrochloride (I.C.I., Melbourne) were used as received. Stock standards An imipramine hydrochloride-butriptyline hydrochloride internal standard solution was prepared by dissolving 22.6 mg imipramine and 22.6 mg butriptyline in 10 ml absolute ethanol. The solution was diluted with glass distilled water to contain 4 pg/ml of both compounds as their bases. A viloxazine hydrochloride solution was prepared by dissolving 23.1 mg of the drug in 20 ml of absolute ethanol. A dilution of 1 ml of this stock solution in 10 ml water gave a 100 pg/ml solution of viloxazine base. Plasma standards
Aliquots of the 100 jg/ml standard of viloxazine base solution were added to separate 4 ml portions of drug free plasma to produce concentrations in the range 0 to 2 pg/ml. These standards were prepared with each run. Two standards, one 0.75 pg/ml and the other 1.75 ig/ml, of viloxazine were stored frozen at -200C until required. Both standards were used to evaluate the precision of the assay. © Macmillan Journals Ltd 1979
T.R. NORMAN, G.D. BURROWS, B.M. DAVIES & J.M.E. WURM
170
Apparatus
Results
Analyses were performed using a Packard Model 419 Becker gas chromatograph equipped with a flame ionization detector. A 1.8 metre x 4 mm coiled glass column packed with 3% OV-17 on 80-100 mesh Gas Chrom Q (Applied Science Laboratories Inc.) was used to accomplish the separation. The chromatographic conditions used were: carrier gas, nitrogen with a flow rate of 20 ml/min; column oven 215°C; detector and injection port temperature 260°C; air flow to detector 250 ml/min; hydrogen flow to detector 40 ml/min. Retention times and peak areas were determined with a Hewlett-Packard Model 3380A reporting integrator.
Acetylation of viloxazine was complete in 30 s without the need for a catalyst such as pyridine. This was confirmed by using a series of eight tubes each containing 1 pg viloxazine, to which 100 pl acetic anhydride was added. Each tube was stoppered, vortexed for 30 s and placed in a water bath at 450C for periods of 0 to 120 min. No peaks due to free viloxazine were detected when the tubes were reconstituted in 40 1&l hexane and 5 pl aliquots injected into the chromatograph. Neither of the internal standards underwent acetylation, nor did they give rise to additional peaks in their individual chromatograms, when acetic anhydride was added. No peaks were observed in the chromatograms of ten drug free plasmas extracted. The relationship between the ratio of peak area of viloxazine acetate to peak area of imipramine and plasma viloxazine concentration was linear over the range 0-2 tg/ml (Spearman correlation, coefficient = 0.99; n = 10). The second internal standard was not used in quantitative determinations, but was used as a check of the reproducibility of each extraction. The ratio of the peak area of butriptyline to imipramine remained constant (± 10%) throughout an extraction and was constant (±20%) between runs at 0.95. For precision studies, the 0.75 tg/ml and the 1.75 pig/ml viloxazine standards were used. Eight aliquots of each standard were analysed on a single day. The within day precision at 0.75 jg/ml was 9.4% expressed as the coefficient of variation and at 1.75 jg/ml was 5.7%. Further aliquots of the 0.75 g/nml standard were thawed and analysed on five different days over a 30 day period, to determine day to day precision and sample stability. The coefficient of variation was 13.1%. The stability of the drug in plasma was established as at least one month when frozen at -20°C. The extracts were stable ovemight when stored at -4C in a freezer. No other drugs were screened for interference in the assay. In the case of the samples in this study, the patients were known to be receiving no other drugs. In clinical practice interference by other drugs, especially diazepam, may present some difficulties in viloxazine analysis. Acidic drugs, such as the barbiturates, would not be recovered under the alkaline extraction conditions of the assay. Other antidepressants would be extracted but it is not usual
Extraction procedure A 4 ml sample of plasma, 0.5 ml 5M sodium hydroxide and 0.3 ml internal standard were placed in a 20 ml glass stoppered test tube and carefully mixed by vortexing. The solution was extracted with 5 ml hexane by shaking on a horizontal mechanical shaker for 20 min. The tube was centrifuged at 1400 g for 5 min and the hexane phase separated. A further 5 ml hexane was added to the plasma and the extraction repeated. The two hexane extracts were combined and shaken with 1 ml lM hydrochloric acid for 15 min, centrifuged at 1400 g for 5 min and the organic phase discarded. The aqueous phase was transferred to a clean 5 ml glass stoppered test tube and made alkaline with 0.3 ml SM sodium hydroxide. The free bases were extracted into 2 ml hexane on a Matburn wheel and the solution centrifuged at 1400 g for 5 min. The hexane phase was separated, placed in a clean 5 ml glass test tube with 0.5 ml of a 0.04% triethanolamine solution in chloroform and evaporated to dryness in stream of air at 37°C. The remaining aqueous phase was re-extracted with 2 ml hexane, centrifuged and the hexane layer separated and added to the dried first extraction. This solution was vortexed for 30 s with 100 g4 acetic anhydride, evaporated to dryness at 37°C in a stream of air, stoppered and stored overnight in a freezer at - 4C. Samples were reconstituted in 40 pi hexane and 5 pl aliquots injected into the gas chromatograph. Peak area ratios of viloxazine to imiprane were determined and plasma concentrations calculated from the standard curve.
Table I Plsma viloxazine levels in a patient receiving 300 mg/day Day of therapy
Plasma evel (pg/ml) *
0 0
12 0.62
14 1.18
Each plasma value is the mean of two determinations
17 1.13
21
1.44
24 1.43
28 1.36
ASSAY OF VILOXAZINE BY GLC
practice to treat a patient with more than one such drug. Plasma from a patient receiving 100 mg viloxazine three times a day was collected between 11.00 h and 13.00 h, immediately before the second dose, during the treatment period. Viloxazine concentrations were determined in duplicate and the results are presented in Table 1.
Discussion No peaks, other than those arising from imipramine, butriptyline and viloxazine acetate, were detected in the chromatograms. The major metabolites of viloxazine in man have been identified as the hydroxy compounds (Case & Reeves, 1975). De-alkylation, which occurs in some animal species, does not occur in man (Case & Reeves, 1975). Hydroxyl derivatives being more polar than viloxazine itself, probably would not be extracted under the conditions of the proposed method. The absence of additional peaks in the chromatograms is in keeping with this contention. The levels of viloxazine observed are higher than
171
those usually found in patients who receive similar doses of the tricyclic antidepressants. Typically a dose of 150mg of secondary amine type antidepressant, such as nortriptyline, would result in a plasma concentration of around 170 ng/ml (Burrows, Davies & Scoggins, 1972). The maximum plasma concentrations observed after single oral doses of viloxazine are also about the levels observed in this study (Bayliss & Case, 1975). A steady state viloxazine concentration was established after 14 days. The sensitivity of the assay was evaluated using plasma solutions containing 0.05, 0.10, 0.20 and 0.25 jg/ml. No drug was detected below 0.20 pg/ml, which was considered to be the limit of detection of the assay. The sensitivity of the assay is adequate for routine plasma level determinations in patients receiving these doses of viloxazine. For pharmacokinetic studies of viloxazine the sensitivity of the assay may not be adequate. This work was supported by the National Health and Medical Research Council of Australia. We wish to thank Dr C. Proctor of I.C.I. Australia for samples of vioxazine hydrochloride, the staff of the Parkville Psychiatric Unit for patient care and Miss L. Byrt for technical assistance.
References BAYLISS, P.F.C. & CASE, D.E. (1975). Blood level studies with viloxazine hydrochloride in man. Br. J. clin. Pharmac., 2, 209-214. BURROWS, G.D., DAVIES, B. & SCOGGINS, B.A. (1972). Plasma concentration of nortriptyline and clinical response in depressive illness. Lancet, ii, 619-623. CASE, D.E. (1973). Gas liquid chromatographic estimation in blood of ICI 58834. J. Pharmn. Pharmac., 25, 800-802. CASE, D.E. & REEVES, P.R. (1975). The disposition and metabolism of ICI 58,834 in two human subjects. Xenobiotica, 5, 113-129. DAVIES, B.M., JOSHUA, S., BURROWS, G. & POYNTON, C.
(1977). A sequential trial of viloxazine (Vivalan) and imipramine in moderately depressed patients. Med. J. Aust. 1, 521-522. GREENWOOD, D.T. (1975). Animal pharmacology of
viloxazine (Vivalan). J. int. med. Res., 3 (Suppl. 3), 1828. MALLION, K.B., TODD, A.H., TURNER, R.W., BAINBRIDGE, J.G., GREENWOOD, D.T., MADINAVEITIA, J.,
SOMERVILLE, -A.R. & WHITrLE, B.A. (1972). 2-(2Ethoxyphenoxy-methyl) tetra-hydro-1,4-oxazine hydrochloride, a potential psychotropic agent. Nature, 238, 157-158. PEET, M. (1973). A clinical trial of ICI-58,834-a potential antidepressant. J. int. med. Res., 1, 624-626. PINDER, R.M., BROGDEN, R.N., SPEIGHT, T.M. & AVERY,
G.S. (1977). Viloxazine: A review of its pharmacological properties and therapeutic efficacy in depressive illness. Drugs, 13, 401-421.
(Received October 4, 1978)
DETERMINATION OF VILOXAZINE IN PLASMA BY GLC T.R. NORMAN, G.D. BURROWS, B.M. DAVIES
J.M.E. WURM
D atment of Psychiatry, University of Melbourr a, Parkville, Victoria, 3052, Australia
1 A gas chromatographic procedure for the determination of viloxazine in therapeutic concentrations in human plasma, with use of a flame ionization detector, is described. 2 The drug is extracted at pH 12 into hexane, back extracted into HCI, re-extracted into hexane after alkalinization of the HCI and derivatized with acetic anhydride. 3 An imipramine-butriptyline mixture is used as an internal standard. The coefficients of variation for a concentration of 0.75 ig/ml are 9.4% and 13.1% for within day and day to day precision respectively. For a 1.75 pg/ml solution the within day precision was 5.7%. 4 The method has been applied to a patient receiving 300 mg/day viloxazine treatment.
IRWduction Viloxazine (Vivalan, I.C.I.) is a bicyclic compound with a pharmacological profile similar to that observed with the tricyclic antidepressants (Mallion, Greenwood, Bainbridge, Turner, Todd, Madinaveitia, Somerville & Whittle, 1972; Greenwood, 1975). Controlled clinical evaluations have confirmed that viloxazine is an effective antidepressant agent (Pinder, Brogden, Speight & Avery, 1977). Peet (1973) reported no relationship between the clinical effect of viloxazine and its concentration in plasma. Case (1973) has described a method, based on gas liquid chromatography (glc) with an electron capture detector, for the determination of viloxazine in blood and plasma. This method, while suitable for use in clinical studies, does not include the use of an internal standard to correct for procedural losses during the determination. The present paper descibes a method, based on glc with a flame ionization detector, for plasma viloxazine determinations. The method has been applied to the determination of samples from patients who took part in a clinical evaluation of the drug (Davies, Joshua, Burrows & Poynton, 1977). Methods Reagents
Hexane (Nanograde, Mallinckrodt), absolute ethanol
(pro analysi, Merck) triethanolamine (G.P.R., 0306-5251/79/080169 03 $01.00
Hopkin and Williams), chloroform (A.R., Univar) and acetic anhydride (A.R. Univar) were used without further purification. Butriptyline hydrochloride (Ayerst Laboratories Pty Ltd, Sydney), (Ciba-Geigy hydrochloride imipramine Pharmaceuticals, Sydney) and viloxazine hydrochloride (I.C.I., Melbourne) were used as received. Stock standards An imipramine hydrochloride-butriptyline hydrochloride internal standard solution was prepared by dissolving 22.6 mg imipramine and 22.6 mg butriptyline in 10 ml absolute ethanol. The solution was diluted with glass distilled water to contain 4 pg/ml of both compounds as their bases. A viloxazine hydrochloride solution was prepared by dissolving 23.1 mg of the drug in 20 ml of absolute ethanol. A dilution of 1 ml of this stock solution in 10 ml water gave a 100 pg/ml solution of viloxazine base. Plasma standards
Aliquots of the 100 jg/ml standard of viloxazine base solution were added to separate 4 ml portions of drug free plasma to produce concentrations in the range 0 to 2 pg/ml. These standards were prepared with each run. Two standards, one 0.75 pg/ml and the other 1.75 ig/ml, of viloxazine were stored frozen at -200C until required. Both standards were used to evaluate the precision of the assay. © Macmillan Journals Ltd 1979
T.R. NORMAN, G.D. BURROWS, B.M. DAVIES & J.M.E. WURM
170
Apparatus
Results
Analyses were performed using a Packard Model 419 Becker gas chromatograph equipped with a flame ionization detector. A 1.8 metre x 4 mm coiled glass column packed with 3% OV-17 on 80-100 mesh Gas Chrom Q (Applied Science Laboratories Inc.) was used to accomplish the separation. The chromatographic conditions used were: carrier gas, nitrogen with a flow rate of 20 ml/min; column oven 215°C; detector and injection port temperature 260°C; air flow to detector 250 ml/min; hydrogen flow to detector 40 ml/min. Retention times and peak areas were determined with a Hewlett-Packard Model 3380A reporting integrator.
Acetylation of viloxazine was complete in 30 s without the need for a catalyst such as pyridine. This was confirmed by using a series of eight tubes each containing 1 pg viloxazine, to which 100 pl acetic anhydride was added. Each tube was stoppered, vortexed for 30 s and placed in a water bath at 450C for periods of 0 to 120 min. No peaks due to free viloxazine were detected when the tubes were reconstituted in 40 1&l hexane and 5 pl aliquots injected into the chromatograph. Neither of the internal standards underwent acetylation, nor did they give rise to additional peaks in their individual chromatograms, when acetic anhydride was added. No peaks were observed in the chromatograms of ten drug free plasmas extracted. The relationship between the ratio of peak area of viloxazine acetate to peak area of imipramine and plasma viloxazine concentration was linear over the range 0-2 tg/ml (Spearman correlation, coefficient = 0.99; n = 10). The second internal standard was not used in quantitative determinations, but was used as a check of the reproducibility of each extraction. The ratio of the peak area of butriptyline to imipramine remained constant (± 10%) throughout an extraction and was constant (±20%) between runs at 0.95. For precision studies, the 0.75 tg/ml and the 1.75 pig/ml viloxazine standards were used. Eight aliquots of each standard were analysed on a single day. The within day precision at 0.75 jg/ml was 9.4% expressed as the coefficient of variation and at 1.75 jg/ml was 5.7%. Further aliquots of the 0.75 g/nml standard were thawed and analysed on five different days over a 30 day period, to determine day to day precision and sample stability. The coefficient of variation was 13.1%. The stability of the drug in plasma was established as at least one month when frozen at -20°C. The extracts were stable ovemight when stored at -4C in a freezer. No other drugs were screened for interference in the assay. In the case of the samples in this study, the patients were known to be receiving no other drugs. In clinical practice interference by other drugs, especially diazepam, may present some difficulties in viloxazine analysis. Acidic drugs, such as the barbiturates, would not be recovered under the alkaline extraction conditions of the assay. Other antidepressants would be extracted but it is not usual
Extraction procedure A 4 ml sample of plasma, 0.5 ml 5M sodium hydroxide and 0.3 ml internal standard were placed in a 20 ml glass stoppered test tube and carefully mixed by vortexing. The solution was extracted with 5 ml hexane by shaking on a horizontal mechanical shaker for 20 min. The tube was centrifuged at 1400 g for 5 min and the hexane phase separated. A further 5 ml hexane was added to the plasma and the extraction repeated. The two hexane extracts were combined and shaken with 1 ml lM hydrochloric acid for 15 min, centrifuged at 1400 g for 5 min and the organic phase discarded. The aqueous phase was transferred to a clean 5 ml glass stoppered test tube and made alkaline with 0.3 ml SM sodium hydroxide. The free bases were extracted into 2 ml hexane on a Matburn wheel and the solution centrifuged at 1400 g for 5 min. The hexane phase was separated, placed in a clean 5 ml glass test tube with 0.5 ml of a 0.04% triethanolamine solution in chloroform and evaporated to dryness in stream of air at 37°C. The remaining aqueous phase was re-extracted with 2 ml hexane, centrifuged and the hexane layer separated and added to the dried first extraction. This solution was vortexed for 30 s with 100 g4 acetic anhydride, evaporated to dryness at 37°C in a stream of air, stoppered and stored overnight in a freezer at - 4C. Samples were reconstituted in 40 pi hexane and 5 pl aliquots injected into the gas chromatograph. Peak area ratios of viloxazine to imiprane were determined and plasma concentrations calculated from the standard curve.
Table I Plsma viloxazine levels in a patient receiving 300 mg/day Day of therapy
Plasma evel (pg/ml) *
0 0
12 0.62
14 1.18
Each plasma value is the mean of two determinations
17 1.13
21
1.44
24 1.43
28 1.36
ASSAY OF VILOXAZINE BY GLC
practice to treat a patient with more than one such drug. Plasma from a patient receiving 100 mg viloxazine three times a day was collected between 11.00 h and 13.00 h, immediately before the second dose, during the treatment period. Viloxazine concentrations were determined in duplicate and the results are presented in Table 1.
Discussion No peaks, other than those arising from imipramine, butriptyline and viloxazine acetate, were detected in the chromatograms. The major metabolites of viloxazine in man have been identified as the hydroxy compounds (Case & Reeves, 1975). De-alkylation, which occurs in some animal species, does not occur in man (Case & Reeves, 1975). Hydroxyl derivatives being more polar than viloxazine itself, probably would not be extracted under the conditions of the proposed method. The absence of additional peaks in the chromatograms is in keeping with this contention. The levels of viloxazine observed are higher than
171
those usually found in patients who receive similar doses of the tricyclic antidepressants. Typically a dose of 150mg of secondary amine type antidepressant, such as nortriptyline, would result in a plasma concentration of around 170 ng/ml (Burrows, Davies & Scoggins, 1972). The maximum plasma concentrations observed after single oral doses of viloxazine are also about the levels observed in this study (Bayliss & Case, 1975). A steady state viloxazine concentration was established after 14 days. The sensitivity of the assay was evaluated using plasma solutions containing 0.05, 0.10, 0.20 and 0.25 jg/ml. No drug was detected below 0.20 pg/ml, which was considered to be the limit of detection of the assay. The sensitivity of the assay is adequate for routine plasma level determinations in patients receiving these doses of viloxazine. For pharmacokinetic studies of viloxazine the sensitivity of the assay may not be adequate. This work was supported by the National Health and Medical Research Council of Australia. We wish to thank Dr C. Proctor of I.C.I. Australia for samples of vioxazine hydrochloride, the staff of the Parkville Psychiatric Unit for patient care and Miss L. Byrt for technical assistance.
References BAYLISS, P.F.C. & CASE, D.E. (1975). Blood level studies with viloxazine hydrochloride in man. Br. J. clin. Pharmac., 2, 209-214. BURROWS, G.D., DAVIES, B. & SCOGGINS, B.A. (1972). Plasma concentration of nortriptyline and clinical response in depressive illness. Lancet, ii, 619-623. CASE, D.E. (1973). Gas liquid chromatographic estimation in blood of ICI 58834. J. Pharmn. Pharmac., 25, 800-802. CASE, D.E. & REEVES, P.R. (1975). The disposition and metabolism of ICI 58,834 in two human subjects. Xenobiotica, 5, 113-129. DAVIES, B.M., JOSHUA, S., BURROWS, G. & POYNTON, C.
(1977). A sequential trial of viloxazine (Vivalan) and imipramine in moderately depressed patients. Med. J. Aust. 1, 521-522. GREENWOOD, D.T. (1975). Animal pharmacology of
viloxazine (Vivalan). J. int. med. Res., 3 (Suppl. 3), 1828. MALLION, K.B., TODD, A.H., TURNER, R.W., BAINBRIDGE, J.G., GREENWOOD, D.T., MADINAVEITIA, J.,
SOMERVILLE, -A.R. & WHITrLE, B.A. (1972). 2-(2Ethoxyphenoxy-methyl) tetra-hydro-1,4-oxazine hydrochloride, a potential psychotropic agent. Nature, 238, 157-158. PEET, M. (1973). A clinical trial of ICI-58,834-a potential antidepressant. J. int. med. Res., 1, 624-626. PINDER, R.M., BROGDEN, R.N., SPEIGHT, T.M. & AVERY,
G.S. (1977). Viloxazine: A review of its pharmacological properties and therapeutic efficacy in depressive illness. Drugs, 13, 401-421.
(Received October 4, 1978)
