- Email: [email protected]
Ion-pair liquid chromatography of amitriptyline and metabolites in plasma
.iound of Chronzarogmpky, 157 (1978) 379-385 0 EIse+ier Scientic~Pi&lishhgCompany,Amsterdam-
CflROM.. 10,996
Printedin The Netherlands
_
IO&UR LIQUID CHROMATOGRAP& METABOLITES IN PLASMA
Department of Chical
P hramcrcologu, Kmolhka
OF. AMITRIP’kYLINE
Institute, Ehidi~e
AND
Hospital, S-141 86 Hm&ge
L%=W
@zcf5vedMarchSt.&1978)
SUMMARY
A method is describedfor the determinationof amitriptyline_and nortriptyline in the plasma of patientsduringtreatment.The method is based on liquid chromatography of the aminesas ion-pairswith perchlorate.The separationcolumn is packed with silica gel, and a mixtureof diisopropylether, dichloromethane,methanol and water containingthe counterion is used as eluent. High efficacyand stabilityof the . systemis achieved.The separationof metabolitcsis demonstrated.The-k- and transIO-hydroxyisomers of amitriptyline and nortriptyline are easily resolved with this chromatographic system.
INTRODUCTION
Amitriptyline and its N-demethylated metabolite (nortriptylinc) are two important ticyclic antidepressantdrugs in widespreadclinical use_ Studies .have shown that the therapeuticas well as the adverseeffectsof thesedrugs are relatedto the plasma concentrationsachievedduringtreatment14and sever@ liquid chromategraphic methods for the determinationof tricyclicantidepressantdrugs have been describeder2. Ion-pair partition chromatography is a highly suitable technique for the measurementof these drugs*~-‘s and the present study is a modification of this techniquethat offers advantageswith respectto stability and reproducibilitycompared with an earlier systemIs.By using this method, therapeuticplasma concentrationsof amitriptylineand nortriptylinehave been measured;the separationof the major metabolitesof the drue. is also described. EXPERIMENTAL Appamm The liquid chromatographincorporatedan LDC 711-26 solvent delivering . * Present address: PoisonsUnit, Guy’s Hospital, London SE 1, Great 33rhi.1~
system. The detectorunit was an Altex Model 153 analyticalUV detectoroperated at 254 run. The injectorwas a Rheodyne Model 70-10 sample injectionvalve (Altex i Scientific,Berkeley,Calif., U.S.A.). The volume of the injectionloop -was70 ,QI. Chromatographic system The separationcolumn (100 or 125 mm x 4.6 mm I.D.) was of stainlesssteel
and was packedwithPartisil5(H. ReeveAngel, Clifton, NJ., U.S.A.) by thebalanceddensityslurrytechnique16. Equal volumes of tetrabromoethane,carbon tetrachloride. and dioxan were used as suspendingmedium for the particles. The mobile phase consisted of perchloric acid, sodium perchlorate,methanol, dichloromethaneand diisopropyletherand was degassedin an ultrasonicbath for 45 min before use_ Reagents and chemicals
Diisopropyl ether was of synthesis grade (E. Merck, Darmstadt, G.F.R.; stabilized with 2,6-di-tert.-butyl4methyIphenol); all other chemicals and solvents were-of analyticalgrade. The internalstandard,2-(dibenz~b,5]azepin-5-yl)-N-methyl ethylamine(Fig_ l), was obtainedfrom Ciba Geigy (Basel, Switzerland).
Fig. 1. Stiucture of internalstandard.
AnaZyticaZprocedure
Plasma samples (1 ml) were extractedby methods previously described1SJ7. Briefly,50 ~1 of the internalstandardsolution (2.74 pM) and 1 ml of 2 M carbonate buffer(pH 10.5) wereadded to the samplesand extractionof the drugs into n-hexane (5 ml) was achievedby gentle shaking to avoid the formation of an emulsion: The compounds were back-extractedinto 1 ml of 0.25 N sulphuricacid and, after this solution had been made alkalinewith 0.2 ml of 2.5 M sodium hydroxide, they were finally extractedinto 0.1 ml of diisopropylether; this extractwas used for the liquid chromatography. RESULTS
AND
DISCUSSION
Chromatography
In previous ion-pair partitition chromatographicsystems for tricyclic antidepressant&I&~‘~, the stationaryaqueous phase was pre-coated on the support. In the presentsystem,a mobile phase that is under-saturatedwith the aqueous phase contain&g the counterion is used. This techniquegave highly stableand reproducible columns’*, without the need for thermostaticcontrol of the chromatographicsystem that is essentialwith precoated columns. Originally, a mixture of methanol and dichloromethanewas used as the organicsolventfor the mobile phase; however,the presenceof diisopropyletherwas found to be necessaryto retainthe compounds and for good column performance. _
381
ION-PAJEt Lc OF AMlTEupTyLINE
It is our hypothesis that the polar silica gel support partly adsorbs the more polar compouents of the mobile phase. Support for this theory was obtained by measurements of the interstitial volume (V&) of the column with tetmchloroetbylene as the marker during equilibration of a column. The results are illustrated in Fig. 2. The intercept value, 0.74, was obtained with dry dichloromethane as mobile phase and probably represents the total porosity (et) of the cAnun’*. The support availability for the mobile phase, represented by E,,,which is equal to WV,, where V0 is the volume of the empty column), decreasesto an equilibrium value of 0.68.or 0.62 after the passage of about 12 or 90 cohuuus volumes, respectively, indicating that adsorption of a stationary phase occurs. It can be cak~Iated from ct = E, _t E= (where .zsis given by VJVO is the volume of the stationary phase) that this corresponds to the presence of 0.12 or 0.21 ml of stationary phase, which is about one-fifth and onethird, respectively, of the volume for a precoated ion-pair partition system. The adsorbed phase probably consists of a mixture of the aqueous phase and methanol.
OS-
column volume mobile phase
-r
lcio
2iIo
Fig. 2. Curves of porosity VS.nur&er of column volumes of mobile phase. Column, 125 x 4.6 mm 1-D. Mobile phase, 0.01 M perchloric acid + 0.09 M sodium perchlorate-methanol-dichloromethane-diisopropyl ether; q , 0.9:10:12:30; a, 0.9:5.2:12:30. Flow-rate, 1.0 ml/r&~.
In straight-phaseion-pair chromatography the capacity factor (II’) is given by
Vs r=-Vm-E&,,-(X)
‘0)
where E& is the conditional ion-pair extraction constant and (X) .is the concentration of counter-ion in the stationary phase. The capacity factors decrease with increasiug concentration of perchlorate ions in accordance with eqn: 1 as illustrated in Fig. 3. However, the relationship is not rectilinear, partly because of the varying ionic strength and also because of the influence of other retention mechanisms.The possibility of regulating the capacity factors by adding methanol to the mobile phase is demonstrated in Fig. 4_ The main features are that the capacity factors decmase as the methanol concentration increases above 11.8% (for the mobile phase studied). Some selectivity changes,. especially between amitriptyliue and .the IO-hydroxy
382
CJ
I
0
Oil
1
42
0.3
OA
Qs
hl cro;
Fig. 3. Infiuence of counter-ion cor~centrationon capacity factor (k’). Column, as in Fig. 1. Mobile
phase, perchloric acid + sodium perchlorate+methanoLdicb-dichloromethan+diisopropyl ether (0.952: 12:30). Flow-rate, 1.1 ml/min. For abbreviations see Fig. 7. Bog
k’
05
0
.
-0s
.i
Fig. 4. fnauence of methanol concentration on capacity factor CK). Colrrmn, as in Fig. 1. Mobile phase, 0.01 M percbloric acid + 0_09A4 sodium .perchlora~~e-diisopropyl ether <0_9:8:30)-me&anoL Flow-rate, 1.1 ml/min_For abbreviations see Fig. 7.
metabolites of nortriptyline, can be seen. With decreasing~methanol concentration
below ll.S%, the mobile phase will approach saturation v&h respect to water and counter ion. The cohmm eflkiency with the system, calculated as the height of a theoretical plate, is between 0.02 and 0.04 mm.
The system has been successfullyapplied to the measurement of plasma concentrations of amitriptyline and nortriptyline in patients receiving therapeutic doses of these drugs. Typical traces obtained from the analysis of l-ml plasma samples (a) containing no drug and (b) obtained from a patient receiving amitriptyline are shown in Fig. 5. Chlorimipramine and demethylchlorimpiramirk have also been. measured by using this chromatographic system and the extraction procedure described earlieP.
c 0
2
4
6
8
min
0
2
4
6
8 min
Fig. 5. Chromatogmms from plasma samples. Column, as in Fig. 1. MobiIephase, 0.1 MperchIoric acid-methanol-dichlorometham-diisopropyl ether (0.9:5.2:10:30). Flow-rate, 19 ml/ink (a) Blank human plasma, (b) plasma of a patient receiving amitriptyline. Peaks: a = demethyhto.tipt b = nortriptyline(462 n&4),c = internalstandard (135 n&Z), d = vnitriptyline (226 niW), e .G frmrrlO-hydroxynortriptyline. The reproducibility of the method determined from measurements.on 38 duplicate plasma samples and calculated as the coefficient of variation ‘Was 10.1% for amitriptyline and 5.2% for nortriptybne ; the concentration range was 90-900 r&f. A comparison between the proposed k&n&e and a mass fragmentographic methodLFwas carried out for the analyses of plasma nortriptybne in a group of 10
EON-PAIR Ix= OF m
.--
385
ACKNOWLEDGEMENTS We would like to thank Dr. A. Jorgensen, Lundbeck -4/S, Copenhagen, Denmark, for the supply of metabolites of amitriptyline and Merck Sharp & Dohme, Rahway, N-J., U.S.A., for the sample of trans-LO-hydroxynotiptyline. This research was su@potied by grants from the Swedish Medical Research Council (04X-03902, and 29X-0591.5) and by funds from the Karolinska Institute.
1 2 3 4 5
M. &berg, B. Cronholm, F. Sjijqv& and.D. Tuck, Bit. Med. J., 4 (1970) 18. M. &erg, B. Cronhobn, F. Sjiiqvist and D. Tuck, Bit. Med. J., 3 (1971) 331. R A. Braithwaite, R GouIding, G. Theano, J; Bailey and A. Coppen, Lancet, i (1972) 1297. P. Kragh-S+xesen, C. Eggert-Hansen and M. kberg, Lcurce&i (1973) 113. P.Kragh -Smense~, C. Eggert-Hansen, P. C. Baastrup and E. F. Hvidberg, Psycfwpharnzaw~ogia, 45 (1976) 305. 6 V. E. ZiegIer, B. T. Co, J. R. Taylor, P. J. Clayton and J. T. Brig& C&Z-Pharm. Theran, 19 (1976) 795. 7 V. E. Zieg!er, P. J. Clayton, J. R. Taylor, B. T. Co and J. T. Brim, Clrn.Pharm.2iierap.,20 (1976) 458. 8 S. Montgomery, R A. Bra&w&e and J. Cmmmer, Brit. Med. J., 3 (197.7) 166. 9 M. R. Detavernier, L. Dryon and D. L. Mass&z, J. Chromatog_, 128 (1976) 204. 10 I. D. Watson and M. J. Stewart, J. Cfuonza~ogr.,134 (1977) 182. 11 J. C. Kraak and P. Bijster, J. Chromatogr., 143 (1977) 499. 12 I_ H. M. van den Berg, H. J. J. M. de Ruwe and R S. De&k, J. Chromatofll, 138 (1977) 431. 13 B. Mellstriim and S. Eksborg, .K Chromatogr., 116 (1976) 475. 14 P.-O. Lagerstriim, I. Carkson and B.-A. Pessson, Acta Pfiarm. Suet., 13 (197a 157. 15 B. MelJstrijmand G. Tybring, J. Chromarogr., 143 (1977) 597. 16 R E. Majors, Anal. Chem., ti (1972) 1722. 17 S. Dawling and R. A. Braithwaite, J. Chronzutogr.,(1978). 18 J. H. M. van den Berg, J. Milley, N. Vonk and R. S. Deelder, J. Chromufogr., 132 (1977) 421. 19 0. Borg& L. P&&r, F. Sjaqvist and B. Holmstedt, Proc. 5th Inf. Congr. Pharmacol, San Francisco, 1972, Vol. 3, Karger, Basel, 1973,p. 56-68. 20 L. Bert&on and B. Alexaaderson, Eur. J. Clin. Phtznnawl., 4 (1972) 201. 21 P. C. Remy, W. A. van Saun, Jr., E. L. Engelbardt and B. H. Arison, J. Org. Chem., 38 (1973) 700.
CflROM.. 10,996
Printedin The Netherlands
_
IO&UR LIQUID CHROMATOGRAP& METABOLITES IN PLASMA
Department of Chical
P hramcrcologu, Kmolhka
OF. AMITRIP’kYLINE
Institute, Ehidi~e
AND
Hospital, S-141 86 Hm&ge
L%=W
@zcf5vedMarchSt.&1978)
SUMMARY
A method is describedfor the determinationof amitriptyline_and nortriptyline in the plasma of patientsduringtreatment.The method is based on liquid chromatography of the aminesas ion-pairswith perchlorate.The separationcolumn is packed with silica gel, and a mixtureof diisopropylether, dichloromethane,methanol and water containingthe counterion is used as eluent. High efficacyand stabilityof the . systemis achieved.The separationof metabolitcsis demonstrated.The-k- and transIO-hydroxyisomers of amitriptyline and nortriptyline are easily resolved with this chromatographic system.
INTRODUCTION
Amitriptyline and its N-demethylated metabolite (nortriptylinc) are two important ticyclic antidepressantdrugs in widespreadclinical use_ Studies .have shown that the therapeuticas well as the adverseeffectsof thesedrugs are relatedto the plasma concentrationsachievedduringtreatment14and sever@ liquid chromategraphic methods for the determinationof tricyclicantidepressantdrugs have been describeder2. Ion-pair partition chromatography is a highly suitable technique for the measurementof these drugs*~-‘s and the present study is a modification of this techniquethat offers advantageswith respectto stability and reproducibilitycompared with an earlier systemIs.By using this method, therapeuticplasma concentrationsof amitriptylineand nortriptylinehave been measured;the separationof the major metabolitesof the drue. is also described. EXPERIMENTAL Appamm The liquid chromatographincorporatedan LDC 711-26 solvent delivering . * Present address: PoisonsUnit, Guy’s Hospital, London SE 1, Great 33rhi.1~
system. The detectorunit was an Altex Model 153 analyticalUV detectoroperated at 254 run. The injectorwas a Rheodyne Model 70-10 sample injectionvalve (Altex i Scientific,Berkeley,Calif., U.S.A.). The volume of the injectionloop -was70 ,QI. Chromatographic system The separationcolumn (100 or 125 mm x 4.6 mm I.D.) was of stainlesssteel
and was packedwithPartisil5(H. ReeveAngel, Clifton, NJ., U.S.A.) by thebalanceddensityslurrytechnique16. Equal volumes of tetrabromoethane,carbon tetrachloride. and dioxan were used as suspendingmedium for the particles. The mobile phase consisted of perchloric acid, sodium perchlorate,methanol, dichloromethaneand diisopropyletherand was degassedin an ultrasonicbath for 45 min before use_ Reagents and chemicals
Diisopropyl ether was of synthesis grade (E. Merck, Darmstadt, G.F.R.; stabilized with 2,6-di-tert.-butyl4methyIphenol); all other chemicals and solvents were-of analyticalgrade. The internalstandard,2-(dibenz~b,5]azepin-5-yl)-N-methyl ethylamine(Fig_ l), was obtainedfrom Ciba Geigy (Basel, Switzerland).
Fig. 1. Stiucture of internalstandard.
AnaZyticaZprocedure
Plasma samples (1 ml) were extractedby methods previously described1SJ7. Briefly,50 ~1 of the internalstandardsolution (2.74 pM) and 1 ml of 2 M carbonate buffer(pH 10.5) wereadded to the samplesand extractionof the drugs into n-hexane (5 ml) was achievedby gentle shaking to avoid the formation of an emulsion: The compounds were back-extractedinto 1 ml of 0.25 N sulphuricacid and, after this solution had been made alkalinewith 0.2 ml of 2.5 M sodium hydroxide, they were finally extractedinto 0.1 ml of diisopropylether; this extractwas used for the liquid chromatography. RESULTS
AND
DISCUSSION
Chromatography
In previous ion-pair partitition chromatographicsystems for tricyclic antidepressant&I&~‘~, the stationaryaqueous phase was pre-coated on the support. In the presentsystem,a mobile phase that is under-saturatedwith the aqueous phase contain&g the counterion is used. This techniquegave highly stableand reproducible columns’*, without the need for thermostaticcontrol of the chromatographicsystem that is essentialwith precoated columns. Originally, a mixture of methanol and dichloromethanewas used as the organicsolventfor the mobile phase; however,the presenceof diisopropyletherwas found to be necessaryto retainthe compounds and for good column performance. _
381
ION-PAJEt Lc OF AMlTEupTyLINE
It is our hypothesis that the polar silica gel support partly adsorbs the more polar compouents of the mobile phase. Support for this theory was obtained by measurements of the interstitial volume (V&) of the column with tetmchloroetbylene as the marker during equilibration of a column. The results are illustrated in Fig. 2. The intercept value, 0.74, was obtained with dry dichloromethane as mobile phase and probably represents the total porosity (et) of the cAnun’*. The support availability for the mobile phase, represented by E,,,which is equal to WV,, where V0 is the volume of the empty column), decreasesto an equilibrium value of 0.68.or 0.62 after the passage of about 12 or 90 cohuuus volumes, respectively, indicating that adsorption of a stationary phase occurs. It can be cak~Iated from ct = E, _t E= (where .zsis given by VJVO is the volume of the stationary phase) that this corresponds to the presence of 0.12 or 0.21 ml of stationary phase, which is about one-fifth and onethird, respectively, of the volume for a precoated ion-pair partition system. The adsorbed phase probably consists of a mixture of the aqueous phase and methanol.
OS-
column volume mobile phase
-r
lcio
2iIo
Fig. 2. Curves of porosity VS.nur&er of column volumes of mobile phase. Column, 125 x 4.6 mm 1-D. Mobile phase, 0.01 M perchloric acid + 0.09 M sodium perchlorate-methanol-dichloromethane-diisopropyl ether; q , 0.9:10:12:30; a, 0.9:5.2:12:30. Flow-rate, 1.0 ml/r&~.
In straight-phaseion-pair chromatography the capacity factor (II’) is given by
Vs r=-Vm-E&,,-(X)
‘0)
where E& is the conditional ion-pair extraction constant and (X) .is the concentration of counter-ion in the stationary phase. The capacity factors decrease with increasiug concentration of perchlorate ions in accordance with eqn: 1 as illustrated in Fig. 3. However, the relationship is not rectilinear, partly because of the varying ionic strength and also because of the influence of other retention mechanisms.The possibility of regulating the capacity factors by adding methanol to the mobile phase is demonstrated in Fig. 4_ The main features are that the capacity factors decmase as the methanol concentration increases above 11.8% (for the mobile phase studied). Some selectivity changes,. especially between amitriptyliue and .the IO-hydroxy
382
CJ
I
0
Oil
1
42
0.3
OA
Qs
hl cro;
Fig. 3. Infiuence of counter-ion cor~centrationon capacity factor (k’). Column, as in Fig. 1. Mobile
phase, perchloric acid + sodium perchlorate+methanoLdicb-dichloromethan+diisopropyl ether (0.952: 12:30). Flow-rate, 1.1 ml/min. For abbreviations see Fig. 7. Bog
k’
05
0
.
-0s
.i
Fig. 4. fnauence of methanol concentration on capacity factor CK). Colrrmn, as in Fig. 1. Mobile phase, 0.01 M percbloric acid + 0_09A4 sodium .perchlora~~e-diisopropyl ether <0_9:8:30)-me&anoL Flow-rate, 1.1 ml/min_For abbreviations see Fig. 7.
metabolites of nortriptyline, can be seen. With decreasing~methanol concentration
below ll.S%, the mobile phase will approach saturation v&h respect to water and counter ion. The cohmm eflkiency with the system, calculated as the height of a theoretical plate, is between 0.02 and 0.04 mm.
The system has been successfullyapplied to the measurement of plasma concentrations of amitriptyline and nortriptyline in patients receiving therapeutic doses of these drugs. Typical traces obtained from the analysis of l-ml plasma samples (a) containing no drug and (b) obtained from a patient receiving amitriptyline are shown in Fig. 5. Chlorimipramine and demethylchlorimpiramirk have also been. measured by using this chromatographic system and the extraction procedure described earlieP.
c 0
2
4
6
8
min
0
2
4
6
8 min
Fig. 5. Chromatogmms from plasma samples. Column, as in Fig. 1. MobiIephase, 0.1 MperchIoric acid-methanol-dichlorometham-diisopropyl ether (0.9:5.2:10:30). Flow-rate, 19 ml/ink (a) Blank human plasma, (b) plasma of a patient receiving amitriptyline. Peaks: a = demethyhto.tipt b = nortriptyline(462 n&4),c = internalstandard (135 n&Z), d = vnitriptyline (226 niW), e .G frmrrlO-hydroxynortriptyline. The reproducibility of the method determined from measurements.on 38 duplicate plasma samples and calculated as the coefficient of variation ‘Was 10.1% for amitriptyline and 5.2% for nortriptybne ; the concentration range was 90-900 r&f. A comparison between the proposed k&n&e and a mass fragmentographic methodLFwas carried out for the analyses of plasma nortriptybne in a group of 10
EON-PAIR Ix= OF m
.--
385
ACKNOWLEDGEMENTS We would like to thank Dr. A. Jorgensen, Lundbeck -4/S, Copenhagen, Denmark, for the supply of metabolites of amitriptyline and Merck Sharp & Dohme, Rahway, N-J., U.S.A., for the sample of trans-LO-hydroxynotiptyline. This research was su@potied by grants from the Swedish Medical Research Council (04X-03902, and 29X-0591.5) and by funds from the Karolinska Institute.
1 2 3 4 5
M. &berg, B. Cronholm, F. Sjijqv& and.D. Tuck, Bit. Med. J., 4 (1970) 18. M. &erg, B. Cronhobn, F. Sjiiqvist and D. Tuck, Bit. Med. J., 3 (1971) 331. R A. Braithwaite, R GouIding, G. Theano, J; Bailey and A. Coppen, Lancet, i (1972) 1297. P. Kragh-S+xesen, C. Eggert-Hansen and M. kberg, Lcurce&i (1973) 113. P.Kragh -Smense~, C. Eggert-Hansen, P. C. Baastrup and E. F. Hvidberg, Psycfwpharnzaw~ogia, 45 (1976) 305. 6 V. E. ZiegIer, B. T. Co, J. R. Taylor, P. J. Clayton and J. T. Brig& C&Z-Pharm. Theran, 19 (1976) 795. 7 V. E. Zieg!er, P. J. Clayton, J. R. Taylor, B. T. Co and J. T. Brim, Clrn.Pharm.2iierap.,20 (1976) 458. 8 S. Montgomery, R A. Bra&w&e and J. Cmmmer, Brit. Med. J., 3 (197.7) 166. 9 M. R. Detavernier, L. Dryon and D. L. Mass&z, J. Chromatog_, 128 (1976) 204. 10 I. D. Watson and M. J. Stewart, J. Cfuonza~ogr.,134 (1977) 182. 11 J. C. Kraak and P. Bijster, J. Chromatogr., 143 (1977) 499. 12 I_ H. M. van den Berg, H. J. J. M. de Ruwe and R S. De&k, J. Chromatofll, 138 (1977) 431. 13 B. Mellstriim and S. Eksborg, .K Chromatogr., 116 (1976) 475. 14 P.-O. Lagerstriim, I. Carkson and B.-A. Pessson, Acta Pfiarm. Suet., 13 (197a 157. 15 B. MelJstrijmand G. Tybring, J. Chromarogr., 143 (1977) 597. 16 R E. Majors, Anal. Chem., ti (1972) 1722. 17 S. Dawling and R. A. Braithwaite, J. Chronzutogr.,(1978). 18 J. H. M. van den Berg, J. Milley, N. Vonk and R. S. Deelder, J. Chromufogr., 132 (1977) 421. 19 0. Borg& L. P&&r, F. Sjaqvist and B. Holmstedt, Proc. 5th Inf. Congr. Pharmacol, San Francisco, 1972, Vol. 3, Karger, Basel, 1973,p. 56-68. 20 L. Bert&on and B. Alexaaderson, Eur. J. Clin. Phtznnawl., 4 (1972) 201. 21 P. C. Remy, W. A. van Saun, Jr., E. L. Engelbardt and B. H. Arison, J. Org. Chem., 38 (1973) 700.