Br. J. clin. Phannac. (1978), 6
LETTERSTO THE EDITORS
gave intravenous infusions of 15 mg amitriptyline to four normal volunteers and found half-lives ranging from 15.5 to 19.5 h which is consonant with our findings. A range of 9.0 to 25.3 h for amitriptyline half-life was observed by J0rgensen & Staehr (1976) in six patients at 'steady-state' following discontinuation of a sustained release preparation of amitriptyline. Both of these studies utilized a gas chromatographic assay with a lower limit of sensitivity of 5 ng ml-'. Our assay, having greater sensitivity, enabled estimations of the half-life to be made following a single, low oral dose of amitriptyline. By this technique the halflife would appear to be shorter than previously suggested by gas chromatographic assay.
20
lo
10
Q5
\
E\
O
6
12
18
24
183
30
Time (h)
Figure 1 Mean plasma concentrations of amitriptyline from nine volunteers. Fitted to two compartment open model with first order absorption and lag time, t' : C =Ae--a () + Be-P(-fl (A+l3)e-ka(t-t'). Computer estimates for mean data were A, 1. 18 ng ml-'; B, 13.86 ng ml-'; a, 6.55 h-1; ,B, 0.065 h-1; kat 1 .48 h-'; t', 1 .72 h.
points to fit a one compartment model adequately. The mean data for all nine subjects is plotted in Figure I and a two-compartment open model with first order absorption has been fitted by non-linear optimization (SIMPLEX) (Nelder & Mead, 1965). The half life as determined in our normal subjects by both methods of determination is much shorter than that reported in the two normal subjects studied by Braithwaite & Widdop (1971) using a gaschromatographic assay for amnitriptyline but longer than this group have reported using radioimmunoassay (Braithwaite et al., 1978). It is interesting to note that the peak plasma amitriptyline levels noted by these authors were in all but one case far higher than any found in our group of volunteers taking the same dose of amitriptyline. Jorgensen & Hansen (1976)
H.J. ROGERS, P.J. MORRISON & I.D. BRADBROOK' Departments of Clinical Pharmacology and Pharmacology and 'Forensic Medicine, Guy's Hospital Medical School, London SEI 9RT Received March 16, 1978 References
BRAITHWAITE, R.A. & WIDDOP, B. (1971). A specific gaschromatographic method for the measurement of 'steady-state' plasma levels of amitriptyline and nortriptyline in patients. Clin. Chim. Acta, 35, 461-472. BRAITHWAITE, R., MONTGOMERY, S. & ROBINSON, J.D.
(1978). A radioimmunoassay for amitriptyline and nortriptyline. Br. J. Pharmac., 63, 370-371P. FABER, D.B., MULDER, C. & MAN IN'T VELD, W.A. (1974). A thin-layer method for the determination of amitriptyline and nortriptyline in plasma. J. Chromatogr., 100, 55-61. HUCKER, H.B., STAUFFER, S.C., CLAYTON, F.G., NAKRA, B.R.S. & GAIND, R. (1975). Plasma levels of a new
pelietized form of amitriptyline for maintenance therapy. J. clin. Pharmac., 15, 168-172. J0RGENSEN, A. & STAEHR, P. (1976). On the biological half-life of amitriptyline. J. Pharm. Pharmac., 28, 62-64. J0RGENSEN, A. & HANSEN, V. (1976). Pharmacokinetics of amitriptyline infused intravenously in man. Eur. J. clin. Pharmac., 10, 337-341. NELDER, J.A. & MEAD, R. (1965). A simplex method for function minimization. Computer J., 7, 308-313.
DETERMINATION OF METFORMIN IN BIOLOGICAL SAMPLES A high incidence of fatal lactic acidosis associated with phenformin has led to the suggestion that its use be restricted and that metformin should be the biguanide of choice in the treatment of maturity-onset diabetes (British Medical Journal, 1977). In France, where metformin accounts for about three-quarters of biguanide prescriptions lactic acidosis is rare and renal failure has been present in most cases (Assan, Heuclin,
Ganeval, Bismuth, George & Girard, 1977). Sensitive and specific methods for the analysis of biguanides in biological fluids are essential to an evaluation of their safety and efficacy. Although a general method for their analysis, involving acylation and gas chromatography of the resulting diamine striazines, has been applied to studies of the pharmacokinetics of phenformin (Matin, Karam & Forsham,
Br. J. clin. Pharmac. (1978),6
LETTERS TO THE EDITORS
184
100-
100
E
100 _
°0)
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10
0
01) 0
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.I
I
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.L
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o 0.5 1
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Time (days)
Time (min) Figure 1 Recovery of unchanged drug in an isolated perfused rat liver preparation after addition of 0.3 mg metformin HCI. Mean data for four male, 200 g Wistar strain rats; bars indicate ranges. Recirculating system containing 150 ml perfusate. Biliary excretion after 1 h accounted for a further 0.22% of the dose. 0 liver, 0 perfusate.
Figure 2 Disposition of metformin after oral administration of 3 x 0.5 g Glucophage@ tablets to a normal subject (male, 28 years). Metformin concentration is expressed as HCI salt. A urine; A blood; U plasma; 0 saliva. Inset: - urine; --- faeces; total excretion.
1975; Alkalay, Khemani, Wagner & Bartlett, 1975) similar reports have not appeared in the case of metformin. Analysis of the latter presents more difficulties in extraction from biological fluids owing to its greater polarity. Garrett, Tsau & Hinderling (1972) found negligible extraction of metformin into polar organic solvents even at high alkalinities and attributed this to a low degree of molecular association of dialkylated compared to mono-substituted biguanides. In our hands, extraction of metformin from alkaline solutions using methylene chloride, as described by Matin et al. (1975), was not sufficiently reproducible when using buformin as internal standard. One solution to this problem is based upon protein precipitation with trichloroacetic acid followed by extraction of a 1:1 bromothymol blue-biguanide ion-pair from the supernatant into methylene chloride (Garrett et al., 1972). An alternative method, which we describe below, is simpler and combines protein precipitation and extraction into one step by the addition of a large volume ratio of acetonitrile. Analyses were performed on a Pye GCD gas chromatograph fitted with an electron-capture detector. The glass column (6 ft x 4 mm i.d.) used was packed with 3% OV-17 on 100/120 Gas Chrom Q (Field Instruments Co Ltd). Injection port, detector and oven temperatures were 2250C, 3250C and 2100C, respectively. Nitrogen carrier gas flow was 50ml/min. Metformin HCI and buformin HC1, the internal standard, were gifts from Winthrop Labs. The following procedure was used for the analysis of plasma, whole blood, liver perfusate, liver homogenate, bile, saliva (1:2 dilution), urine (up to 1 :1000 diludon) and faeces (up to 1:10,000 dilution). To 100-200 p1 of sample was added 200 ng internal
standard and 5.0 ml acetonitrile. After shaking and centrifuging the supernatant was removed and evaporated to dryness using a vortex-evaporator (Buchler). The residue was shaken with 100 gl amyl acetate and 10 jil monochlorodifluoroacetic anhydride (Fluorochem Ltd, redistilled over phosphorous pentoxide) and after a minute 500 1t1 4N NaOH were added to hydrolyze the excess anhydride and to retain it in aqueous solution as the acid. This mixture was centrifuged and 0.1-0.5 p1 of the organic layer were injected into the chromatograph. Single peaks were observed corresponding to the metformin and buformin s-triazines at 1.7 min and 3.2 min, respectively. Linear calibration graphs were obtained up to 400 ng using peak height ratios. The lower limit of sensitivity of the assay was about 2 ng with a 200 g1 sample. Using the same plasma spiked with 50 ng/ml or 2 gig/ml metformin eight replicate analyses at each concentration gave coefficients of variation of + 9% and ± 5%, respectively. Eight different plasma samples analyzed on different days gave coefficients of variation ranging from + 14% at 100 ng/ml to + 6% at 4 jg/ml. Similar results were obtained with samples of blood, saliva, liver perfusate and homogenate, bile, urine and faeces and the slopes of the calibration graphs were independent of the nature of the fluid. All unknowns were analyzed in duplicate and calibration points were included in each run. The application of the method is illustrated in Figures 1 and 2. Figure 1 shows recoveries of unchanged metformin in perfusate and liver after bolus injection of 0.3 mg into the reservoir of a recirculating isolated perfused rat liver preparation. Over 95% of the dose was recovered indicating little, if
Br. J. clin. Pharmac. (1978), 6
any, metabolism of the drug. About 90% was eliminated unchanged over 8 days in a normal volunteer given 1.5 g by mouth, with half appearing in the urine and half in the faeces (Figure 2). Plasma, whole blood and salivary drug concentrations are also indicated in Figure 2, as is the urinary excretion rate. M.S. LENNARD, C. CASEY, G.T. TUCKER & H.F. WOODS Department of Pharmacology and Therapeutics, University of Sheffield, Hallamshire Hospital, Sheffield S1O 2JF Received March 29, 1978
Requests for reprints to G.T.T.
LETTERS TO THE EDITORS
185
References ALKALAY, D., KHEMANI, L., WAGNER, W.E. &
BARTLETr, M.F. (1975). Pharmacokinetics of phenformin in man. J. clin. Pharnac., 15, 446-448. ASSAN, R., HEUCLIN, Ch., GANEVAL, D., BISMUTH, Ch.,
GEORGE, J. & GIRARD, J.R. (1977). Metformin-induced lactic acidosis in the presence of acute renal failure. Diabetologia, 13, 211-217. BRITISH MEDICAL JOURNAL(I977). Biguanides and lactic acidosis in diabetes. Br. med. J., 2, 1436. GARRETT, E.R., TSAU, J. & HINDERLING, P.H. (1972). Application of ion-pair methods to drug extraction from biological fluids. IL: Quantitative determination of biguanides in biological fluids and comparison of protein binding estimates. J. pharm. Sci., 61, 1411-1418. MATIN, S.K., KARAM, J.H. & FORSHAM, P.H. (1975). Simple electron capture gas chromatographic method for the determination of oral hypoglycemic biguanides in biological fluids. Analyt. Chem., 47, 545-548.
THE URINARY EXCRETION OF PHENELZINE The substituted hydrazine drug, phenelzine, prescribed under the name Nardil, has been used for some years in the treatment of neurotic depression (Medical Research Council, 1965). While there is some evidence that clinical efficacy and side effects of the drug may be related to acetylator status, it is only since analytical techniques have become available (Caddy, Tilstone & Johnstone, 1976; Caddy & Stead, 1977) which enable levels of this drug in body fluids to be determined, that its excretion can be examined in more detail. The present paper describes the application of an iodate oxidation procedure to the analysis of phenelzine levels in the urine of five patients.
Chemicals, apparatus and analyticalprocedure The assay of phenelzine in urine by its oxidation to,Bphenylethanol with acidic potassium iodate has been adequately discussed in an earlier publication (Caddy & Stead, 1977). Drug regimen and acetylator status Following the oral administration of phenelzine (30 mg three times daily) at regular intervals, urine samples for assay of the drug were collected from five hospitalized patients on days 1 and 13 of a continuous course of treatment. The time of sampling and the volume of urine excreted are given in Table 1. Acetylator status was determined by the method of Evans (1969). The half-life of the drug was calculated from the gradient (G) obtained from a plot of loge (excretion rate) against time, using the equation: 0.693 G
The times of sampling (1 and 13 days) were chosen because it was necessary to compare analytical results obtained before with those obtained during treatment. It was also assumed that 13 days was sufficient time for a steady state pharmacokinetic equilibrium to have been established. The results of analyses of the urine samples obtained from the five patients are summarized in Table 1. The pH of all such urine samples lay in the range 6 to 7. Note that the large urine outputs are symptomatic of the high fluid intake resulting from the dry mouth experienced with phenelzine chemotherapy. The drug excretion profiles in these patients show a marked change in pharmacokinetic behaviour during the course of treatment. The time of maximum excretion rate, the amount excreted over the 8 h period between dosing and the acetylator phenotypes of each patient are recorded in Table 2. The pharmacokinetic data are consistent with the expected changes resulting from a successful treatment of depression with phenelzine. Since it can be postulated that, during the 13 days of drug administration, monoamine oxidase activity has been reduced by irreversible inhibition of the enzyme, it is to be expected that at the beginning of the course of treatment the half-lives would be less. Examination of the data shows this to be true; the averaged half-lives of the five patients studied at the beginning of treatment was calculated to be 0.87 + 0.47 h, which is significantly different (P < 0.05 Mann-Whitney U Test) from the half-lives at the end of the course of treatment. There is little variation of the times of maximum excretion rate amongst depressed patients for days 1 and 13, these values being 1.70 + 0.37 h and 2.06 + 0.55 h respectively.
LETTERSTO THE EDITORS
gave intravenous infusions of 15 mg amitriptyline to four normal volunteers and found half-lives ranging from 15.5 to 19.5 h which is consonant with our findings. A range of 9.0 to 25.3 h for amitriptyline half-life was observed by J0rgensen & Staehr (1976) in six patients at 'steady-state' following discontinuation of a sustained release preparation of amitriptyline. Both of these studies utilized a gas chromatographic assay with a lower limit of sensitivity of 5 ng ml-'. Our assay, having greater sensitivity, enabled estimations of the half-life to be made following a single, low oral dose of amitriptyline. By this technique the halflife would appear to be shorter than previously suggested by gas chromatographic assay.
20
lo
10
Q5
\
E\
O
6
12
18
24
183
30
Time (h)
Figure 1 Mean plasma concentrations of amitriptyline from nine volunteers. Fitted to two compartment open model with first order absorption and lag time, t' : C =Ae--a () + Be-P(-fl (A+l3)e-ka(t-t'). Computer estimates for mean data were A, 1. 18 ng ml-'; B, 13.86 ng ml-'; a, 6.55 h-1; ,B, 0.065 h-1; kat 1 .48 h-'; t', 1 .72 h.
points to fit a one compartment model adequately. The mean data for all nine subjects is plotted in Figure I and a two-compartment open model with first order absorption has been fitted by non-linear optimization (SIMPLEX) (Nelder & Mead, 1965). The half life as determined in our normal subjects by both methods of determination is much shorter than that reported in the two normal subjects studied by Braithwaite & Widdop (1971) using a gaschromatographic assay for amnitriptyline but longer than this group have reported using radioimmunoassay (Braithwaite et al., 1978). It is interesting to note that the peak plasma amitriptyline levels noted by these authors were in all but one case far higher than any found in our group of volunteers taking the same dose of amitriptyline. Jorgensen & Hansen (1976)
H.J. ROGERS, P.J. MORRISON & I.D. BRADBROOK' Departments of Clinical Pharmacology and Pharmacology and 'Forensic Medicine, Guy's Hospital Medical School, London SEI 9RT Received March 16, 1978 References
BRAITHWAITE, R.A. & WIDDOP, B. (1971). A specific gaschromatographic method for the measurement of 'steady-state' plasma levels of amitriptyline and nortriptyline in patients. Clin. Chim. Acta, 35, 461-472. BRAITHWAITE, R., MONTGOMERY, S. & ROBINSON, J.D.
(1978). A radioimmunoassay for amitriptyline and nortriptyline. Br. J. Pharmac., 63, 370-371P. FABER, D.B., MULDER, C. & MAN IN'T VELD, W.A. (1974). A thin-layer method for the determination of amitriptyline and nortriptyline in plasma. J. Chromatogr., 100, 55-61. HUCKER, H.B., STAUFFER, S.C., CLAYTON, F.G., NAKRA, B.R.S. & GAIND, R. (1975). Plasma levels of a new
pelietized form of amitriptyline for maintenance therapy. J. clin. Pharmac., 15, 168-172. J0RGENSEN, A. & STAEHR, P. (1976). On the biological half-life of amitriptyline. J. Pharm. Pharmac., 28, 62-64. J0RGENSEN, A. & HANSEN, V. (1976). Pharmacokinetics of amitriptyline infused intravenously in man. Eur. J. clin. Pharmac., 10, 337-341. NELDER, J.A. & MEAD, R. (1965). A simplex method for function minimization. Computer J., 7, 308-313.
DETERMINATION OF METFORMIN IN BIOLOGICAL SAMPLES A high incidence of fatal lactic acidosis associated with phenformin has led to the suggestion that its use be restricted and that metformin should be the biguanide of choice in the treatment of maturity-onset diabetes (British Medical Journal, 1977). In France, where metformin accounts for about three-quarters of biguanide prescriptions lactic acidosis is rare and renal failure has been present in most cases (Assan, Heuclin,
Ganeval, Bismuth, George & Girard, 1977). Sensitive and specific methods for the analysis of biguanides in biological fluids are essential to an evaluation of their safety and efficacy. Although a general method for their analysis, involving acylation and gas chromatography of the resulting diamine striazines, has been applied to studies of the pharmacokinetics of phenformin (Matin, Karam & Forsham,
Br. J. clin. Pharmac. (1978),6
LETTERS TO THE EDITORS
184
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°0)
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0)
0.01 .
.I
I
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.L
60
o 0.5 1
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Time (days)
Time (min) Figure 1 Recovery of unchanged drug in an isolated perfused rat liver preparation after addition of 0.3 mg metformin HCI. Mean data for four male, 200 g Wistar strain rats; bars indicate ranges. Recirculating system containing 150 ml perfusate. Biliary excretion after 1 h accounted for a further 0.22% of the dose. 0 liver, 0 perfusate.
Figure 2 Disposition of metformin after oral administration of 3 x 0.5 g Glucophage@ tablets to a normal subject (male, 28 years). Metformin concentration is expressed as HCI salt. A urine; A blood; U plasma; 0 saliva. Inset: - urine; --- faeces; total excretion.
1975; Alkalay, Khemani, Wagner & Bartlett, 1975) similar reports have not appeared in the case of metformin. Analysis of the latter presents more difficulties in extraction from biological fluids owing to its greater polarity. Garrett, Tsau & Hinderling (1972) found negligible extraction of metformin into polar organic solvents even at high alkalinities and attributed this to a low degree of molecular association of dialkylated compared to mono-substituted biguanides. In our hands, extraction of metformin from alkaline solutions using methylene chloride, as described by Matin et al. (1975), was not sufficiently reproducible when using buformin as internal standard. One solution to this problem is based upon protein precipitation with trichloroacetic acid followed by extraction of a 1:1 bromothymol blue-biguanide ion-pair from the supernatant into methylene chloride (Garrett et al., 1972). An alternative method, which we describe below, is simpler and combines protein precipitation and extraction into one step by the addition of a large volume ratio of acetonitrile. Analyses were performed on a Pye GCD gas chromatograph fitted with an electron-capture detector. The glass column (6 ft x 4 mm i.d.) used was packed with 3% OV-17 on 100/120 Gas Chrom Q (Field Instruments Co Ltd). Injection port, detector and oven temperatures were 2250C, 3250C and 2100C, respectively. Nitrogen carrier gas flow was 50ml/min. Metformin HCI and buformin HC1, the internal standard, were gifts from Winthrop Labs. The following procedure was used for the analysis of plasma, whole blood, liver perfusate, liver homogenate, bile, saliva (1:2 dilution), urine (up to 1 :1000 diludon) and faeces (up to 1:10,000 dilution). To 100-200 p1 of sample was added 200 ng internal
standard and 5.0 ml acetonitrile. After shaking and centrifuging the supernatant was removed and evaporated to dryness using a vortex-evaporator (Buchler). The residue was shaken with 100 gl amyl acetate and 10 jil monochlorodifluoroacetic anhydride (Fluorochem Ltd, redistilled over phosphorous pentoxide) and after a minute 500 1t1 4N NaOH were added to hydrolyze the excess anhydride and to retain it in aqueous solution as the acid. This mixture was centrifuged and 0.1-0.5 p1 of the organic layer were injected into the chromatograph. Single peaks were observed corresponding to the metformin and buformin s-triazines at 1.7 min and 3.2 min, respectively. Linear calibration graphs were obtained up to 400 ng using peak height ratios. The lower limit of sensitivity of the assay was about 2 ng with a 200 g1 sample. Using the same plasma spiked with 50 ng/ml or 2 gig/ml metformin eight replicate analyses at each concentration gave coefficients of variation of + 9% and ± 5%, respectively. Eight different plasma samples analyzed on different days gave coefficients of variation ranging from + 14% at 100 ng/ml to + 6% at 4 jg/ml. Similar results were obtained with samples of blood, saliva, liver perfusate and homogenate, bile, urine and faeces and the slopes of the calibration graphs were independent of the nature of the fluid. All unknowns were analyzed in duplicate and calibration points were included in each run. The application of the method is illustrated in Figures 1 and 2. Figure 1 shows recoveries of unchanged metformin in perfusate and liver after bolus injection of 0.3 mg into the reservoir of a recirculating isolated perfused rat liver preparation. Over 95% of the dose was recovered indicating little, if
Br. J. clin. Pharmac. (1978), 6
any, metabolism of the drug. About 90% was eliminated unchanged over 8 days in a normal volunteer given 1.5 g by mouth, with half appearing in the urine and half in the faeces (Figure 2). Plasma, whole blood and salivary drug concentrations are also indicated in Figure 2, as is the urinary excretion rate. M.S. LENNARD, C. CASEY, G.T. TUCKER & H.F. WOODS Department of Pharmacology and Therapeutics, University of Sheffield, Hallamshire Hospital, Sheffield S1O 2JF Received March 29, 1978
Requests for reprints to G.T.T.
LETTERS TO THE EDITORS
185
References ALKALAY, D., KHEMANI, L., WAGNER, W.E. &
BARTLETr, M.F. (1975). Pharmacokinetics of phenformin in man. J. clin. Pharnac., 15, 446-448. ASSAN, R., HEUCLIN, Ch., GANEVAL, D., BISMUTH, Ch.,
GEORGE, J. & GIRARD, J.R. (1977). Metformin-induced lactic acidosis in the presence of acute renal failure. Diabetologia, 13, 211-217. BRITISH MEDICAL JOURNAL(I977). Biguanides and lactic acidosis in diabetes. Br. med. J., 2, 1436. GARRETT, E.R., TSAU, J. & HINDERLING, P.H. (1972). Application of ion-pair methods to drug extraction from biological fluids. IL: Quantitative determination of biguanides in biological fluids and comparison of protein binding estimates. J. pharm. Sci., 61, 1411-1418. MATIN, S.K., KARAM, J.H. & FORSHAM, P.H. (1975). Simple electron capture gas chromatographic method for the determination of oral hypoglycemic biguanides in biological fluids. Analyt. Chem., 47, 545-548.
THE URINARY EXCRETION OF PHENELZINE The substituted hydrazine drug, phenelzine, prescribed under the name Nardil, has been used for some years in the treatment of neurotic depression (Medical Research Council, 1965). While there is some evidence that clinical efficacy and side effects of the drug may be related to acetylator status, it is only since analytical techniques have become available (Caddy, Tilstone & Johnstone, 1976; Caddy & Stead, 1977) which enable levels of this drug in body fluids to be determined, that its excretion can be examined in more detail. The present paper describes the application of an iodate oxidation procedure to the analysis of phenelzine levels in the urine of five patients.
Chemicals, apparatus and analyticalprocedure The assay of phenelzine in urine by its oxidation to,Bphenylethanol with acidic potassium iodate has been adequately discussed in an earlier publication (Caddy & Stead, 1977). Drug regimen and acetylator status Following the oral administration of phenelzine (30 mg three times daily) at regular intervals, urine samples for assay of the drug were collected from five hospitalized patients on days 1 and 13 of a continuous course of treatment. The time of sampling and the volume of urine excreted are given in Table 1. Acetylator status was determined by the method of Evans (1969). The half-life of the drug was calculated from the gradient (G) obtained from a plot of loge (excretion rate) against time, using the equation: 0.693 G
The times of sampling (1 and 13 days) were chosen because it was necessary to compare analytical results obtained before with those obtained during treatment. It was also assumed that 13 days was sufficient time for a steady state pharmacokinetic equilibrium to have been established. The results of analyses of the urine samples obtained from the five patients are summarized in Table 1. The pH of all such urine samples lay in the range 6 to 7. Note that the large urine outputs are symptomatic of the high fluid intake resulting from the dry mouth experienced with phenelzine chemotherapy. The drug excretion profiles in these patients show a marked change in pharmacokinetic behaviour during the course of treatment. The time of maximum excretion rate, the amount excreted over the 8 h period between dosing and the acetylator phenotypes of each patient are recorded in Table 2. The pharmacokinetic data are consistent with the expected changes resulting from a successful treatment of depression with phenelzine. Since it can be postulated that, during the 13 days of drug administration, monoamine oxidase activity has been reduced by irreversible inhibition of the enzyme, it is to be expected that at the beginning of the course of treatment the half-lives would be less. Examination of the data shows this to be true; the averaged half-lives of the five patients studied at the beginning of treatment was calculated to be 0.87 + 0.47 h, which is significantly different (P < 0.05 Mann-Whitney U Test) from the half-lives at the end of the course of treatment. There is little variation of the times of maximum excretion rate amongst depressed patients for days 1 and 13, these values being 1.70 + 0.37 h and 2.06 + 0.55 h respectively.
