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Polarographic study of the hydrolysis of a triazolobenzodiazepine, Estazolam
533
Bioelectrochemistty and Bioenergetics, 19 (1988) 533-539 A section of J. Electroanal. Chem., and constituting Vol. 253 (1988) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Polarographic study of the hydrolysis of a triazolobenzodiazepine, Estazolam R.M. Jimenez, R.M. Alonso, E. Oleaga and F. Vicente Departamento de Quimica, Facultad de Ciencias, Unioersidad de1 Pais Vasco, Apdo 644, 48080 Bilbao (Spain) (Received 21 August 1987; in revised form 10 May 1988)
ABSTRACT The hydrolysis reaction in acidic medium of the triazolobenzodiazepine Estazolam has been studied by differential pulse polarography. Estazolam exhibits two well defined polarographic peaks, attributed to the compound pattern and the hydrolysis product, with a potential difference of 0.115 V, constant with pH. Kinetic studies of the hydrolytic process following the changes in current of both polarographic peaks indicated that the process is a first-order reaction with a reaction rate constant (mean value of k ohs = 0.155 min-‘) independent of pH. Arrhenius parameters were also calculated.
INTRODUCTION
Estazolam is a psychotropic drug, belonging to the triazolobenzodiazepine family, which undergoes a hydrolysis process in acidic media. The variation of the reduction peak of Estazolam and of its hydrolysis product with time has allowed the kinetic study of the hydrolysis of Estazolam by differential pulse polarography. EXPERIMENTAL
Apparatus A Princeton Applied Research Corporation (PAR) model 174 polarographic analyzer equipped with a PAR model 303 SMDE cell and a Houston Omnigraphic X-Y recorder was used. A thermostated cell of 15 cm3 capacity and a three-electrode system involving a dropping mercury electrode (capillary tube of 0.015 mm inside diameter as the indicator electrode and a platinum wire as the auxiliary electrode, were employed. 0302-4598/88/$03.50
0 1988 Elsevier Sequoia S.A.
534
Polarography was performed in the differential polarographic mode. A pulse of 25 mV was applied using a drop-time of 0.5 s. The electrode area was 0.096 cm*. Scans were recorded at a scan rate of 5 mV s-r with a full range of 1.5 V. pH measurements were carried out with a Radiometer pHM 64 digital pH-voltmeter using a combined glass/calomel electrode, Ingold gK 2301C. Reagents
Estazolam was kindly supplied by Europharma, Lab. Castejon (Madrid). A stock solution of the drug (3.39 X lop3 M) was prepared in Merck, p.a. methanol and stored in the dark under refrigeration. A stock Britton-Robinson (BR) buffer solution, which was 0.04 M in glacial acetic acid, phosphoric acid and boric acid, was prepared from analytical grade reagents. Buffer solutions were prepared by adding the necessary amount of HCl or KOH in order to obtain the appropriate pH value. The ionic strength was adjusted to p = 0.5 M by addition of potassium chloride. All the reagents were of analytical grade, and solutions were prepared using deionized, distilled water. The polarographic noise was acceptable at the current ranges employed. Procedure
An aliquot (10 cm3) of the appropriate blank solution was pipetted into the thermostated cell and oxygen was removed by bubbling nitrogen for 15 min. During this time, the solution attained the required temperature. The polarogram of the blank solution was recorded. Subsequently, 200 mm3 of the methanolic stock solution of Estazolam was inserted into the cell by means of a micropipette. Timing was begun when the Estazolam solution was added. Solutions were mixed thoroughly by bubbling nitrogen for about 30 s. The polarograms were then registrated at convenient time intervals. Time measurements used to construct peak current versus time plots, were the time at which the recordings started. The peak current of the hydrolysis product was measured from the base line obtained after the appearance of the second polarographic peak, because the potentials of the two polarographic peaks were very close. RESULTS
The hydrolysis reaction of Estazolam takes place at a pH value lower than 4. The kinetic study was carried out by means of differential pulse polarography up to a pH value of 2.5. At higher pH values, the hydrolysis product gave rise to a shoulder which was difficult to measure. Differential pulse polarograms were obtained for 6.80 X 10h5 M Estazolam solutions in B&ton-Robinson buffers at 20 + 0.5 o C over a period of time of 70 min. Figure 1 shows the variation of the polarograms as a function of time at pH = 0.77. The third polarographic peak, near the potential cut-off of the electrolyte, is a catalytic wave with little reproducibility; therefore this peak was not used for the kinetic study.
535
-120
-1.00
-0.80
-0.60
-0.40
Fig. 1. Variation of differential pulse polarograms of Estazolam solutions (6.80X 10e5 M) at pH = 0.77 and at 20°C as a function of time. Ionic medium = 0.5 M in KCI. t = (1) 4.92; (2) 8.75; (3) 12.5; (4) 20.58 min.
The ratio of the currents of the first two peaks when equilibrium is reached changes slightly with pH. At more acidic media the hydrolysis product current is higher than the azomethine group one. The difference between the peak potentials of these two peaks is 0.115 V and is constant over the whole pH range studied.
0
Fig. 2. Peak current changes for a solution of Estazolam (6.80X 1O-5 M) at different pH values. (a) First polarographic peak. (b) Second polarographic peak. pH = (0) 0.553; (A) 0.927; (0) 1.587.
536
Plots of the current versus time were plotted at different pH values, as shown in Fig. 2 for Estazolam and its hydrolysis product, the corresponding benzophenone. Kinetic analysis The protonated form of Estazolam (HE+), which is the predominant species in acidic medium, undergoes a reversible hydrolysis giving rise to the ring-opened form (HE * ), the corresponding benzophenone. This hydrolysis reaction of Estazolam in acidic medium can be represented as: HE+ 2 HE* k-1
(I)
where k, and k_, are the reaction reactions (see Scheme 1).
rate
constants
of the forward
and
reverse
CL
HE+
Scheme
HE*
1. Hydrolysis
reaction
of Estazolam
The kinetic equation representing reverse reactions are first-order is:
where cO is the initial the hydrolysis product ln(c,-c,)=
in acidic medium.
rate when both the forward
and
concentration of the reactant and x is the concentration at time = t. This equation can be computed to
of
-(Z~,+k_,)t+ln(c~--c,)
the reaction
(3)
where c, is the reactant concentration when equilibrium is attained and c, is the concentration of the reactant at time = t. Assuming proportionality between the current peak with the concentration of the electroactive species [l], eqn. (3) becomes: ln(Z,-I,)=
-k,,t+In(Z,-I,)
(4)
where kobs = k, + k_,. Figure 3 shows plots of ln(Z, - Z,) versus time at 20 o C and at different pH values. A linear relation is observed up to 15 min, which indicates that the hydrolysis process can be described by a pseudo-first-order reaction. In Table 1 the reaction rate constants kobsr obtained from the slope of the plots of ln(Z, - Z,) versus time, are given at different pH values.
531 1.0
+1.0
00
0.0
-10
-1.0
-2.0
-2.0
-3.0
- 3.0 t/min
10
20
30
Fig. 3. Typical pseudo-first-order plots for hydrolysis OL nsrazotam at different pH values. Treatment from the current variation of the first polarographic peak. pH = (0) 0.553; (0) 0.927; (A) 1.587; (0) 2.478. Fig. 4. Typical pseudo-fist-order plots for hydrolysis of Estazolam at different pH values. Treatment from the current variation of the second polarograpbic peak. pH = (0) 0.553; (0) 0.927; (A) 1.587; (0) 2.478.
TABLE
1
Reaction rate constants obtained from the current variation of the first (kobs,) and second (kobs2) polarographic peaks or Estazolam solution (6.80 X 10e5 M) at 20 o C and at different pH values. Ionic medium: 0.5 M KC1 PH
0.553 0.927 1.587 2.478
kobslDin- ’ calculated according to eqn. (4)
k obsZ/tin’ calculated according to eqn. (5)
0.154 0.183 0.146 0.179
0.154 0.135 0.153 0.140
k obsl = 0.165
k obsz=0.145
538
-3D.-
t/min 4
10
A 16
Fig. 5. Typical pseudo-first-order (0) 20; (A) 10; (0) 5 ppm.
22
plots
for hydrolysis
The computed reaction rate equation product formation can be expressed as ln(Z,-I,)=
of Estazolam
obtained
at different
initial
as a function
concentrations.
of hydrolysis
-k,,,t+ln(Z,-I,)
Plots of ln(Z, - Z,) versus time are shown in Fig. 4. The kobs values obtained from these plots are collected in Table 1. Results obtained by both types of quantification show a good concordance. As can be observed from Table 1, the reaction rate constant is not dependent on the proton concentration over the time and pH range studied. The ordinates of both graphs do not have the same value for the whole pH range studied, although the initial Estazolam concentration was kept constant. This fact can be explained by the acid-base reaction taking place before the ring-opening reaction. The dissociation constant of the compound, pk, = 1.92, calculated from polarographic data [2], gives a different concentration of the protonated species at every pH value. A study of the influence of the initial concentration of Estazolam on the variation of the azomethine group current with time was carried out. In Fig. 5, plots of ln( Z, - Z,) versus time at different initial concentrations at 20 o C and pH = 0.77 are shown. The reaction rate constant is not dependent on the initial concentration, which confirms the earlier hypothesis.
539
The Arrhenius parameters for the hydrolysis of Estazolam were obtained from the slope and the intercept of the plot of the logarithm of the apparent first-order rate constant, kobs, versus the reciprocal of the absolute temperature, T, given by In k = In A - E,,,/RT
(6)
In A is related to the entropy of activation, AS,,, = R [In A - ln( k’T/h)
ASaCt, by
- I]
where h and k’ are the Planck and Boltzmann constants, respectively; related to the enthalpy of activation of the hydrolysis AH,,,, by AH,,, = E,,, - RT
(7) and E,,, is
(8)
The Arrhenius parameters for the hydrolysis of Estazolam at pH = 0.77 are: E,,, = 64.0951 kJ mol-‘; In A = 24.51; AS,,, = - 49.24 J K-’ mol-‘; AH,,, = 61.63 kJ mol-‘. Kinetic studies on the hydrolysis of Estazolam indicated that the reaction followed a pseudo-first-order process with a reaction rate constant independent of PH. The hydrolysis behaviour of Estalozam is similar to that of other benzodiazepine derivatives studied by electrochemical methods [3,4], but differs from the Triazolam hydrolysis process, whose kinetic constants are dependent on pH [5]. The reduction peaks obtained for this compound allows its polarographic determination. REFERENCES 1 A.J. Bard and L.R. Faulkner, Electrochemical Methods, Fundamentals and Applications, Wiley, New York, 1980, p. 194. 2 R.M. Jimenez, R.M. Alonso, E. Oleaga, F. Vicente and L. Hemtidez, Fresenius Z. Anal. Chem., 329 (1987) 468. 3 J.C. Vire and G.J. Patriarche, J. Electroanal. Chem., 214 (1986) 275. 4 W.F. Smyth and J.A. Groves, Anal. Chim. Acta, 134 (1982) 227. 5 R. Jimenez, R. Alonso, F. Vicente and L. Hemtidez, Bull. Sot. Chim. Belg., 96 (1987) 265.
Bioelectrochemistty and Bioenergetics, 19 (1988) 533-539 A section of J. Electroanal. Chem., and constituting Vol. 253 (1988) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Polarographic study of the hydrolysis of a triazolobenzodiazepine, Estazolam R.M. Jimenez, R.M. Alonso, E. Oleaga and F. Vicente Departamento de Quimica, Facultad de Ciencias, Unioersidad de1 Pais Vasco, Apdo 644, 48080 Bilbao (Spain) (Received 21 August 1987; in revised form 10 May 1988)
ABSTRACT The hydrolysis reaction in acidic medium of the triazolobenzodiazepine Estazolam has been studied by differential pulse polarography. Estazolam exhibits two well defined polarographic peaks, attributed to the compound pattern and the hydrolysis product, with a potential difference of 0.115 V, constant with pH. Kinetic studies of the hydrolytic process following the changes in current of both polarographic peaks indicated that the process is a first-order reaction with a reaction rate constant (mean value of k ohs = 0.155 min-‘) independent of pH. Arrhenius parameters were also calculated.
INTRODUCTION
Estazolam is a psychotropic drug, belonging to the triazolobenzodiazepine family, which undergoes a hydrolysis process in acidic media. The variation of the reduction peak of Estazolam and of its hydrolysis product with time has allowed the kinetic study of the hydrolysis of Estazolam by differential pulse polarography. EXPERIMENTAL
Apparatus A Princeton Applied Research Corporation (PAR) model 174 polarographic analyzer equipped with a PAR model 303 SMDE cell and a Houston Omnigraphic X-Y recorder was used. A thermostated cell of 15 cm3 capacity and a three-electrode system involving a dropping mercury electrode (capillary tube of 0.015 mm inside diameter as the indicator electrode and a platinum wire as the auxiliary electrode, were employed. 0302-4598/88/$03.50
0 1988 Elsevier Sequoia S.A.
534
Polarography was performed in the differential polarographic mode. A pulse of 25 mV was applied using a drop-time of 0.5 s. The electrode area was 0.096 cm*. Scans were recorded at a scan rate of 5 mV s-r with a full range of 1.5 V. pH measurements were carried out with a Radiometer pHM 64 digital pH-voltmeter using a combined glass/calomel electrode, Ingold gK 2301C. Reagents
Estazolam was kindly supplied by Europharma, Lab. Castejon (Madrid). A stock solution of the drug (3.39 X lop3 M) was prepared in Merck, p.a. methanol and stored in the dark under refrigeration. A stock Britton-Robinson (BR) buffer solution, which was 0.04 M in glacial acetic acid, phosphoric acid and boric acid, was prepared from analytical grade reagents. Buffer solutions were prepared by adding the necessary amount of HCl or KOH in order to obtain the appropriate pH value. The ionic strength was adjusted to p = 0.5 M by addition of potassium chloride. All the reagents were of analytical grade, and solutions were prepared using deionized, distilled water. The polarographic noise was acceptable at the current ranges employed. Procedure
An aliquot (10 cm3) of the appropriate blank solution was pipetted into the thermostated cell and oxygen was removed by bubbling nitrogen for 15 min. During this time, the solution attained the required temperature. The polarogram of the blank solution was recorded. Subsequently, 200 mm3 of the methanolic stock solution of Estazolam was inserted into the cell by means of a micropipette. Timing was begun when the Estazolam solution was added. Solutions were mixed thoroughly by bubbling nitrogen for about 30 s. The polarograms were then registrated at convenient time intervals. Time measurements used to construct peak current versus time plots, were the time at which the recordings started. The peak current of the hydrolysis product was measured from the base line obtained after the appearance of the second polarographic peak, because the potentials of the two polarographic peaks were very close. RESULTS
The hydrolysis reaction of Estazolam takes place at a pH value lower than 4. The kinetic study was carried out by means of differential pulse polarography up to a pH value of 2.5. At higher pH values, the hydrolysis product gave rise to a shoulder which was difficult to measure. Differential pulse polarograms were obtained for 6.80 X 10h5 M Estazolam solutions in B&ton-Robinson buffers at 20 + 0.5 o C over a period of time of 70 min. Figure 1 shows the variation of the polarograms as a function of time at pH = 0.77. The third polarographic peak, near the potential cut-off of the electrolyte, is a catalytic wave with little reproducibility; therefore this peak was not used for the kinetic study.
535
-120
-1.00
-0.80
-0.60
-0.40
Fig. 1. Variation of differential pulse polarograms of Estazolam solutions (6.80X 10e5 M) at pH = 0.77 and at 20°C as a function of time. Ionic medium = 0.5 M in KCI. t = (1) 4.92; (2) 8.75; (3) 12.5; (4) 20.58 min.
The ratio of the currents of the first two peaks when equilibrium is reached changes slightly with pH. At more acidic media the hydrolysis product current is higher than the azomethine group one. The difference between the peak potentials of these two peaks is 0.115 V and is constant over the whole pH range studied.
0
Fig. 2. Peak current changes for a solution of Estazolam (6.80X 1O-5 M) at different pH values. (a) First polarographic peak. (b) Second polarographic peak. pH = (0) 0.553; (A) 0.927; (0) 1.587.
536
Plots of the current versus time were plotted at different pH values, as shown in Fig. 2 for Estazolam and its hydrolysis product, the corresponding benzophenone. Kinetic analysis The protonated form of Estazolam (HE+), which is the predominant species in acidic medium, undergoes a reversible hydrolysis giving rise to the ring-opened form (HE * ), the corresponding benzophenone. This hydrolysis reaction of Estazolam in acidic medium can be represented as: HE+ 2 HE* k-1
(I)
where k, and k_, are the reaction reactions (see Scheme 1).
rate
constants
of the forward
and
reverse
CL
HE+
Scheme
HE*
1. Hydrolysis
reaction
of Estazolam
The kinetic equation representing reverse reactions are first-order is:
where cO is the initial the hydrolysis product ln(c,-c,)=
in acidic medium.
rate when both the forward
and
concentration of the reactant and x is the concentration at time = t. This equation can be computed to
of
-(Z~,+k_,)t+ln(c~--c,)
the reaction
(3)
where c, is the reactant concentration when equilibrium is attained and c, is the concentration of the reactant at time = t. Assuming proportionality between the current peak with the concentration of the electroactive species [l], eqn. (3) becomes: ln(Z,-I,)=
-k,,t+In(Z,-I,)
(4)
where kobs = k, + k_,. Figure 3 shows plots of ln(Z, - Z,) versus time at 20 o C and at different pH values. A linear relation is observed up to 15 min, which indicates that the hydrolysis process can be described by a pseudo-first-order reaction. In Table 1 the reaction rate constants kobsr obtained from the slope of the plots of ln(Z, - Z,) versus time, are given at different pH values.
531 1.0
+1.0
00
0.0
-10
-1.0
-2.0
-2.0
-3.0
- 3.0 t/min
10
20
30
Fig. 3. Typical pseudo-first-order plots for hydrolysis OL nsrazotam at different pH values. Treatment from the current variation of the first polarographic peak. pH = (0) 0.553; (0) 0.927; (A) 1.587; (0) 2.478. Fig. 4. Typical pseudo-fist-order plots for hydrolysis of Estazolam at different pH values. Treatment from the current variation of the second polarograpbic peak. pH = (0) 0.553; (0) 0.927; (A) 1.587; (0) 2.478.
TABLE
1
Reaction rate constants obtained from the current variation of the first (kobs,) and second (kobs2) polarographic peaks or Estazolam solution (6.80 X 10e5 M) at 20 o C and at different pH values. Ionic medium: 0.5 M KC1 PH
0.553 0.927 1.587 2.478
kobslDin- ’ calculated according to eqn. (4)
k obsZ/tin’ calculated according to eqn. (5)
0.154 0.183 0.146 0.179
0.154 0.135 0.153 0.140
k obsl = 0.165
k obsz=0.145
538
-3D.-
t/min 4
10
A 16
Fig. 5. Typical pseudo-first-order (0) 20; (A) 10; (0) 5 ppm.
22
plots
for hydrolysis
The computed reaction rate equation product formation can be expressed as ln(Z,-I,)=
of Estazolam
obtained
at different
initial
as a function
concentrations.
of hydrolysis
-k,,,t+ln(Z,-I,)
Plots of ln(Z, - Z,) versus time are shown in Fig. 4. The kobs values obtained from these plots are collected in Table 1. Results obtained by both types of quantification show a good concordance. As can be observed from Table 1, the reaction rate constant is not dependent on the proton concentration over the time and pH range studied. The ordinates of both graphs do not have the same value for the whole pH range studied, although the initial Estazolam concentration was kept constant. This fact can be explained by the acid-base reaction taking place before the ring-opening reaction. The dissociation constant of the compound, pk, = 1.92, calculated from polarographic data [2], gives a different concentration of the protonated species at every pH value. A study of the influence of the initial concentration of Estazolam on the variation of the azomethine group current with time was carried out. In Fig. 5, plots of ln( Z, - Z,) versus time at different initial concentrations at 20 o C and pH = 0.77 are shown. The reaction rate constant is not dependent on the initial concentration, which confirms the earlier hypothesis.
539
The Arrhenius parameters for the hydrolysis of Estazolam were obtained from the slope and the intercept of the plot of the logarithm of the apparent first-order rate constant, kobs, versus the reciprocal of the absolute temperature, T, given by In k = In A - E,,,/RT
(6)
In A is related to the entropy of activation, AS,,, = R [In A - ln( k’T/h)
ASaCt, by
- I]
where h and k’ are the Planck and Boltzmann constants, respectively; related to the enthalpy of activation of the hydrolysis AH,,,, by AH,,, = E,,, - RT
(7) and E,,, is
(8)
The Arrhenius parameters for the hydrolysis of Estazolam at pH = 0.77 are: E,,, = 64.0951 kJ mol-‘; In A = 24.51; AS,,, = - 49.24 J K-’ mol-‘; AH,,, = 61.63 kJ mol-‘. Kinetic studies on the hydrolysis of Estazolam indicated that the reaction followed a pseudo-first-order process with a reaction rate constant independent of PH. The hydrolysis behaviour of Estalozam is similar to that of other benzodiazepine derivatives studied by electrochemical methods [3,4], but differs from the Triazolam hydrolysis process, whose kinetic constants are dependent on pH [5]. The reduction peaks obtained for this compound allows its polarographic determination. REFERENCES 1 A.J. Bard and L.R. Faulkner, Electrochemical Methods, Fundamentals and Applications, Wiley, New York, 1980, p. 194. 2 R.M. Jimenez, R.M. Alonso, E. Oleaga, F. Vicente and L. Hemtidez, Fresenius Z. Anal. Chem., 329 (1987) 468. 3 J.C. Vire and G.J. Patriarche, J. Electroanal. Chem., 214 (1986) 275. 4 W.F. Smyth and J.A. Groves, Anal. Chim. Acta, 134 (1982) 227. 5 R. Jimenez, R. Alonso, F. Vicente and L. Hemtidez, Bull. Sot. Chim. Belg., 96 (1987) 265.