Influence of medium and temperature on the hydrolysis kinetics of propacetamol hydrochloride: Determination using derivative spectrophotometry

Article abstract of DOI:10.1248/cpb.53.277

Propacetamol hydrochloride (PRO) is a water-soluble prodrug of paracetamol (PA) which can be parenterally administered as analgesic for the treatment of postoperative pain, acute trauma, and gastric and/or intestinal disorders where oral administration is not possible. In these circumstances, PRO can be administered in physiologic or glucose solutions since it is rapidly and quantitatively hydrolyzed into PA by plasma estearases. We have studied the degradation kinetics of PRO in 5% glucose and 0.9% saline solutions at 4 °C and 25 °C (storage and room temperatures, respectively). The analytic technique used to determine PRO and PA quantitatively was first-derivative spectrophotometry. The degradation process of PRO can be best fitted to a second-order kinetics with independence of the medium used (saline or glucose solution). The hydrolysis kinetics of PRO conversion into PA depends on the temperature but not on the assay medium (saline or glucose solution). The degradation rate constants obtained for PRO were approximately 4.5 times higher at 25 °C than at 4 °C. The values of t_90% for PRO were 3.17 h and 3.61 h at 25 °C, and 13.42 h and 12.36 h at 4 °C when the tests were performed in 5% glucose and 0.9% saline solutions, respectively.

Full text of DOI:10.1248/cpb.53.277

March 2005
Chem. Pharm. Bull. 53(3) 277—280 (2005)
277
Influence of Medium and Temperature on the Hydrolysis Kinetics of
Propacetamol Hydrochloride: Determination Using Derivative
Spectrophotometry
a
Emilia BARCIA,*,a Alicia MARTIN,a Ma Luz AZUARA,b and Sofia NEGRO
a Department of Pharmaceutics, University Complutense of Madrid, School of Pharmacy; 28040 Madrid, Spain: and
b Palliative Care Unit, Spanish Cancer Association, “La Paz” Hospital; Madrid, Spain.
Received July 27, 2004; accepted November 2, 2004
Propacetamol hydrochloride (PRO) is a water-soluble prodrug of paracetamol (PA) which can be parenter-
ally administered as analgesic for the treatment of postoperative pain, acute trauma, and gastric and/or intesti-
nal disorders where oral administration is not possible. In these circumstances, PRO can be administered in
physiologic or glucose solutions since it is rapidly and quantitatively hydrolyzed into PA by plasma estearases.
We have studied the degradation kinetics of PRO in 5% glucose and 0.9% saline solutions at 4 °C and 25 °C
(storage and room temperatures, respectively). The analytic technique used to determine PRO and PA quantita-
tively was first-derivative spectrophotometry. The degradation process of PRO can be best fitted to a second-
order kinetics with independence of the medium used (saline or glucose solution). The hydrolysis kinetics of PRO
conversion into PA depends on the temperature but not on the assay medium (saline or glucose solution). The
degradation rate constants obtained for PRO were approximately 4.5 times higher at 25 °C than at 4 °C. The
values of t90% for PRO were 3.17 h and 3.61 h at 25 °C, and 13.42 h and 12.36 h at 4 °C when the tests were per-
formed in 5% glucose and 0.9% saline solutions, respectively.
Key words propacetamol; paracetamol; first-derivative spectrophotometry; degradation kinetics
Paracetamol (acetaminophen; PA) is a poorly water-solu- ologic or glucose solutions since it is rapidly and quantita-
ble analgesic that is widely used in hospital and ambulatory tively hydrolyzed into PA by plasma estearases within 7 min
practice for the relief of mild to moderate pain associated after intravenous injection.8) With the exception of absorp-
with headache, backache, arthritis pain, and postoperative tion, PRO exhibits similar disposition kinetics when given by
pain.1—3) It has been used in the treatment of pain in combi- either the intravenous route as PA or by mouth as conven-
nation with aspirin, caffeine, opiates and/or other agents. tional tablets.14)
Paracetamol also has antipyretic effects that do not differ sig-
nificantly from those of aspirin, although it has only weak lytically determined by first-derivative spectrophotometry.15)
antiinflammatory effects.4,5)
This technique has been utilized to investigate the kinetics of
Screening of the literature revealed that PRO can be ana-
Paracetamol is a step 1 agent in the analgesic ladder of drug degradation processes since it is an analytic tool that
WHO.6) During the advanced stages of cancer, nearly 75% of offers a convenient solution to a number of problems, espe-
the patients have pain that is moderate or severe, and accord- cially its potential for increasing the detection sensitivity of
ing to the WHO analgesic ladder, the first step is used for the minor spectral features.15—17) The first-derivative spectropho-
treatment of mild to moderate pain starting with either tometric technique has the advantages of the speed, low cost,
NSAIDs or PA.
and environmental protection. The derivative methods cir-
In recommended therapeutic dosage regimens, PA is usu- cumvent the problem of overlapping spectral bands, allowing
ally well tolerated. The most common side effect is light- for the simultaneous determination of PRO and PA without
headedness. Skin rash and other allergic reactions occur prior separation, and providing accurate, precise, rapid,
occasionally. Overdoses of PA may cause nausea, vomiting, reproducible results. Therefore this was the analytic tech-
sweating, and exhaustion. The most serious adverse effect of nique chosen for the present study.
acute overdosage is a dose-dependent, potentially fatal
Since PRO is frequently used as an intravenously adminis-
tered analgesic we considered it interesting to determine the
hepatic necrosis.7)
Due to its poor water solubility that prevents parenteral degradation kinetics of PRO in 5% glucose and 0.9% saline
administration in a soluble form, the clinical indications for solutions at 4 °C and 25 °C (storage and room temperatures,
PA are limited, especially in the postoperative setting. Con- respectively).
trary to the latter, propacetamol hydrochloride [N-N-diethyl,
4-acetylamino) phenyl ester monohydrochloride] (PRO) is a
Experimental
Materials Pro-efferalgan vials were purchased from Upsa Medica Lab.,
Spain (batch 2E61952. The vials contain 2.00 g of PRO hydrochloride.
PA powder was purchased from Guinama S.A., Spain (batch 018). Physi-
water-soluble prodrug of paracetamol, which can be par-
enterally administered as analgesic8,9) for the treatment
of postoperative pain, acute trauma, and gastric and/or
intestinal disorders where oral administration is not possible.
PRO has also shown efficacy in the symptomatic control of
fever in the fields of oncology, infectious disease, and
hematology.10—13)
ologic solutions of 0.9% sodium chloride or 5% glucose were purchased
from Grifols, Spain. Absolute ethanol for analysis was purchased from
Merck (Germany).
Methods. Instrumentation A double-beam Beckman DU-7 UV–visi-
ble spectrophotometer connected to a computer was used. Previous tests
were carried out to determine operating conditions. For example, different
In these circumstances, PRO can be administered in physi- scanning speeds were tried out ranging from 60 to 120 nm minꢀ1. The best
∗ To whom correspondence should be addressed. e-mail: ebarcia@farm.ucm.es
© 2005 Pharmaceutical Society of Japan

278
Vol. 53, No. 3
results were obtained for a scanning speed of 60 nm minꢀ1. The spectral Results and Discussion
bandwidth was 2 nm.
PRO is administered intravenously after surgery to patients
who are unable to take oral medication. A commercial vial of
PRO reconstituted immediately before use can be intra-
venously administered in 5% glucose or 0.9% saline solu-
tion. In vivo and in vitro studies have shown that PRO is
quickly and quantitatively hydrolyzed into PA: 1 g of PRO
generates 500 mg of PA.
The stability of PRO in reconstituted solutions dissolved
in 100 ml of either 5% glucose or 0.9% saline solution at
4 °C and 25 °C was studied following the experimental pro-
cedures previously described. The pH values at the beginning
of the tests were 3.85 and 3.70 for saline and glucose solu-
tions, respectively. During the stability study a decrease in
The absorbance spectra of the test and reference solutions were recorded
in 1-cm quartz cells over the range of 225—270 nm. The main instrumental
parameter affecting the shape of derivative spectra is Dl. This parameter
needs to be optimized to provide a good sensitivity and an adequate signal-
to-noise ratio. Various values of Dl were tested and a value of Dlꢁ8 nm
was found optimal in connection with both slit width and wavelength inter-
val. Under these conditions, smoothing function was not necessary. Apart
from this, no specific expedients were found necessary to optimize the pro-
cedure.
A digital Crison GLP-22 pH-meter was used to determine the pH of all
the solutions assayed, since this parameter could play a major role in the
prevalence of local skin irritation.18,19)
Standard Solutions The analytic technique used to determine quantita-
tively PRO and PA in the tests solutions was the first-derivative spectropho-
tometry. The method used was that proposed by Ródenas et al.,17) slightly
modified by us to measure the amplitudes (nm) at 248.7 nm for PRO and the pH values was observed, with final pH values of approxi-
245.7 nm for PA.
mately 2, for all the solutions and temperatures assayed.
Tables 1 and 2 show the mean values of the kinetic rate con-
stants calculated for PRO and PA in both media and esti-
Method validation was performed according to the Proceedings of the In-
ternational Conference on Harmonization (ICH).20) Validations were run on
three consecutive days and included calibration curves processed in tripli-
mated after fitting to zero-, first-, and second-order kinetics
the experimental data obtained when performing the stability
tests at 25 °C and 4 °C, respectively. The tables also include
the correlation coefficients (calculated from the mean values)
as well as the ranges of the rate constants obtained in each
individual test.
Tests Performed in 5% Glucose Solution The average
loss of PRO after 60 h (final sampling time at 25 °C) was ap-
proximately 64% when the stability studies were performed
at 25 °C. At that time, the percentage was much lower,
cate for each drug. The standard stock solutions of PRO (1 mg/ml) and PA
(1 mg/ml) were prepared by dissolving 25 mg of PRO or PA in 25 ml of ab-
solute ethanol. Different volumes of the standard stock solutions of PRO and
PA were diluted with ethanol to give a final concentration ranging from
2.5—20 mg/l for PRO and from 2.5—15 mg/l for PA. Calibration curves for
both PRO and PA were linear over the range examined and coefficients of
determination (r2) were consistently greater than 0.999. The detection limits
achieved for PRO and PA were 0.41 and 0.25 mg/l, respectively.
To investigate the effects of PRO and PA on their simultaneous determina-
tion, calibration curves for each drug were obtained with samples at a con-
stant concentration of one of the components and variable concentrations of
the other. The concentrations tested were 2.5, 10, and 20 mg/l for PRO with
a constant of 15 mg/l for PA or, in the other case, 2.5, 10 and 15 mg/l for PA approximately 28%, when the tests were carried out at 4 °C.
with a constant concentration of 20 mg/l for PA. The results were not altered
by the simultaneous presence of PA and PRO.
Between- and within-assay reproducibility was assessed by quantifying
six replicates of three standard samples on 3 consecutive days. The concen-
After fitting to zero-, first-, and second-order kinetics the
experimental data obtained for PRO in the stability tests per-
formed at 25 °C and 4 °C, the best results were obtained
when data were fitted to second-order kinetics (Fig. 1).
The six replicates prepared for each sample were statisti-
cally equal after ANCOVA analysis, that is, the regression
lines from the different batches had a common slope and a
common time-zero intercept (aꢁ0.05). Therefore all the
experimental data obtained from all the batches were com-
bined to estimate the mean values. In this case, the mean val-
ues and ranges of the degradation rate constants were
2.77ꢂ10ꢀ3 ml/mg · h (2.67ꢂ10ꢀ3—2.96ꢂ10ꢀ3 ml/mg · h) at
25 °C and 6.1ꢂ10ꢀ4 ml/mg · h (5.33ꢂ10ꢀ4—6.78ꢂ10ꢀ4 ml/
trations of PRO used were 2.5, 10, and 17.5 mg/l and those of PA were 2.5,
7.5, and 12.5 mg/l. When analyzed using two-way analysis of variance, the
within-assay coefficient of variation (CV) was 1.7% for PA and 1.88% for
PRO, and the between-assay CV was 2.3% for PA and 3.5% for PRO.
Stability Studies The study of the degradation kinetics of PRO was per-
formed at 4 °C and 25 °C in 0.9% saline and 5% glucose solutions. The tests
solutions were prepared by dissolving 250 mg of PRO (obtained from Pro-
efferalgan vials) in 25 ml of 0.9% saline or 5% glucose sterilized physiologic
solutions. Different aliquots of the previous solutions were kept at 4 °C (re-
frigerator) and 25 °C (stability oven) for 132 h and 60 h, respectively, with
independence of the medium used in each case. At preestablished times, the
samples were diluted with absolute ethanol. The first-order derivative spec-
trum of these solutions was recorded under the same instrumental conditions mg · h) at 4 °C (Tables 1, 2).
described above. Each sample was prepared and tested six times.
The values of t90% for PRO (the time it takes for the sub-
Analysis of Kinetic Data The in vitro degradation kinetics of PRO at
both temperatures and media assayed was studied after fitting the experi-
mental data obtained to zero-, one-, and second-order kinetics. The Stat-
graphics Plus 4.0 (John Wiley and Sons, New York) computer program was
utilized to estimate the values of the rate degradation constants.
Before pooling the data from several batches to estimate t90%, a prelimi-
nary statistical study was performed to determine whether the regression
lines from different batches had a common slope and a common time-zero
intercept. Analysis of covariance (ANCOVA) was used, where time is con-
sidered the covariate, to determine the differences in slopes and
intercepts of the regression lines among batches. Although the ICH21) uses a
significance level of 0.25 to compensate for the expected low power of the
design due to the relatively limited sample size in a typical formal stability
study, in our case, since we used six batches, the significance level used was
0.05. The Statgraphics Plus 4.0 computer program was used for this statisti-
stance to degrade by 10%) were 3.17 h at 25 °C and 13.42 h
at 4 °C, and the six batches were statistically equal. At these
times, the reduction obtained for the pH values of the solu-
tions was of approximately 1 pH unit, which is worth consid-
ering because the pH of the solution is important in local
intolerance to parenteral administration. Despite the low pH
of the solutions, Depré et al.8) reported that the local and sys-
temic tolerability of 1 g and 2 g of PRO given intravenously
was excellent.
The experimental data obtained for PA were fitted to zero-,
first-, and second-order kinetics with the best fit obtained
with zero-order kinetics (Fig. 2) if goodness of fit is used as a
cal analysis. If the tests for equality of slopes and equality of intercepts do selecting factor for the reaction kinetics.
not result in rejection at the significance level of 0.05 (i.e., there is no signif-
icant difference in slope and intercepts among batches), the data from all
batches can be combined.
The ANCOVA (aꢁ0.05) performed showed that there was
no significant difference in slope and intercept among the
batches which permitted combining all the experimental
data. In this case, the mean values and ranges of the appear-

March 2005
279
Fig. 1. Mean Profiles (ꢃS.D.) Obtained after Fitting to Second-Order Kinetics the Experimental Data Obtained for PRO
Fig. 2. Mean Profiles (ꢃS.D.) Obtained after Fitting to Zero-Order Kinetics the Experimental Data Obtained for PA
ance rate constants were 4.45ꢂ10ꢀ2 mg/ml · h (3.92ꢂ10ꢀ2—
(percentage of recovery corresponding to the maximum pos-
sible) at 25 °C was obtained whereas a percentage of 31%
was recovered when the tests were realized at 4 °C.
Tests Performed in 0.9% Saline Solution The average
loss of PRO after 60 h was approximately 64% when the tests
were performed at 25 °C. This percentage was much lower,
approximately 29%, when the tests were carried out at 4 °C.
These values did not differ significantly from the results
obtained in 5% glucose solution.
After fitting the experimental data to zero-, first-, and sec-
ond-order kinetics obtained for PRO in the tests performed at
25 °C and 4 °C, the best results were obtained when data
were fitted to second-order kinetics (Fig. 1). In this case, the
mean values and ranges of the degradation rate constant were
2.60ꢂ10ꢀ3 ml/mg · h (2.34ꢂ10ꢀ3—2.77ꢂ10ꢀ3 ml/mg · h) at
25 °C and 5.47ꢂ10ꢀ4 ml/mg · h (5.12ꢂ10ꢀ4—5.64ꢂ10ꢀ4
ml/mg · h) at 4 °C (Tables 1, 2). The t90% values for PRO (all
six batches were statistically equal) were 3.61 h and 12.36 h
at 25 °C and 4 °C, respectively.
5.15ꢂ10ꢀ2 mg/ml · h) at 25 °C and 1.29ꢂ10ꢀ2 mg/ml · h
(1.13ꢂ10ꢀ2—1.46ꢂ10ꢀ2 mg/ml · h) at 4 °C (Tables 1, 2).
PRO hydrolyzes as previously indicated in a second-order
kinetic process and PA is the only degradation product found
for PRO. Therefore both reaction mechanisms should be the
same. However, the use of the goodness of fit for the selec-
tion of the reaction model can be problematic in some cases.
Moreover, in our case, the differences found in the r values
between second-order and zero-order models are probably
not sufficiently large to assume strongly that PA exhibits a
different reaction than PRO.
It should also be pointed out that the differences found
between observed and estimated values for PA correspond to
the first experimental data, which are close to the quantifica-
tion limit in both fitting models (second- and zero-order reac-
tions).
The free PA content increased with time as PRO
hydrolyzed, and the PA released at every sampling time was
in accordance with the quantity of PRO hydrolyzed. After
60 h of the stability test, a PA recovery of approximately 64%
The experimental data obtained for PA were fitted to zero-,
first-, and second-order kinetics with the best fit obtained to

280
Vol. 53, No. 3
Table 1. Mean Values and Ranges of the Kinetic Rate Constants Calculated for PRO and PA at 25 °C
Propacetamol
Paracetamol
Glucose solution Saline solution
Glucose solution
Saline solution
K (zero-order)
(mg/ml · h)
K (ꢂ102)
10.54
[9.90—11.16]
ꢀ0.966
10.53
[9.94—11.02]
ꢀ0.952
4.45
[3.92—5.15]
0.991
4.01
[3.67—4.41]
0.991
r
K (first-order)
K (ꢂ102)
1.64
[1.58—1.76]
ꢀ0.987
1.61
[1.49—1.69]
ꢀ0.981
2.18
[1.78—2.64]
0.962
2.11
[1.92—2.27]
0.963
(hꢀ1
)
r
K (second-order)
(ml/mg · h)
K (ꢂ103)
2.77
[2.67—2.96]
0.989
2.60
[2.34—2.77]
0.989
13.08
[15.32—11.40]
ꢀ0.907
12.24
[10.93—13.67]
ꢀ0.910
r
Table 2. Mean Values and Ranges of the Kinetic Rate Constants Calculated for PRO and PA at 4 °C
Propacetamol
Paracetamol
Glucose solution
Saline solution
Glucose solution
Saline solution
K (zero-order)
(mg/ml · h)
K (ꢂ102)
3.46
[3.07—3.70]
ꢀ0.982
3.14
[3.06—3.22]
ꢀ0.972
1.29
[1.13—1.46]
0.994
1.30
[1.09—1.45]
0.995
r
K (first-order)
K (ꢂ103)
2.00
[1.74—2.15]
ꢀ0.992
1.78
[1.70—1.82]
ꢀ0.985
3.57
[3.07—3.91]
0.979
8.72
[6.72—11.50]
0.977
(hꢀ1
)
r
K (second-order)
(ml/mg · h)
K (ꢂ104)
6.12
[5.33—6.78]
0.995
5.47
[5.12—5.64]
0.993
5.49
[4.60—6.47]
ꢀ0.948
6.40
[4.26—10.87]
ꢀ0.930
r
zero-order kinetics (Fig. 2). In this case, the mean values and when the tests were performed in 5% glucose and 0.9%
ranges of the appearance rate constant were 4.01ꢂ10ꢀ2 saline solutions, respectively.
mg/ml · h (3.67ꢂ10ꢀ2—4.41ꢂ10ꢀ2 mg/ml · h) at 25 °C and
References
1.30ꢂ10ꢀ2 mg/ml · h (1.09ꢂ10ꢀ2—1.45ꢂ10ꢀ2 mg/ml · h) at
1) Farhat F., Savoyen M. C., Jayr C., Cahiers d’LAnesthesiologie, 43,
4 °C (Tables 1, 2). The same comments apply as indicated for
the tests performed in 5% glucose.
After 60 h of the stability test, approximately 64% of PA
was recovered at 25 °C (percentage of recovery correspond-
ing to the maximum possible), whereas 29% was recovered
when the tests were performed at 4 °C.
When comparing the results obtained in both media (5%
glucose and 0.9% saline solutions), it was found that the
hydrolysis kinetics of PRO conversion into PA does not
depend on the medium. These results are not in accordance
with the ones published by Ródenas et al.17) that reported a
negligible hydrolysis of PRO in saline solution after 3 h and a
75.4% degradation in 5% glucose solution after 80 min of
initiating the stability test. It should be noted that the authors
of the latter paper did not take into consideration the influ-
ence that temperature has on the stability of PRO, which we
have pointed out in our work.
351—356 (1995).
2) Beaulieu P., Anesthesia, 49, 739—740 (1994).
3) Pettersson H., Owall A., Jakobsson J., Acta Anaesthesiol. Scand., 48,
867—870 (2004).
4) “Martindale 31 ed.,” E.F. Reynolds, London, 1996, pp. 81—85.
5) “Goodman and Gilman’s: The Pharmacological Basis of Therapeu-
tics,” 10 th ed., McGrawHill, New York, 2001, pp. 624—663.
6) Care pain relief and palliative care. Technical Report Series 804,
WHO, 1990.
7) Forrest J. A. H., Clements J. A., Prescott L. F., Clin. Pharmacokinet.,
7, 93—107 (1982).
8) Depré M., Van Hecken A., Verbesselt R., Tjandra-Maga T. B., Gerin
M., Schepper P. J., Fund. Clin. Pharmacol., 6, 259—262 (1992).
9) Flouvat B., Leveneu A., Fitoussi S., Delhotal-Landes B., Gendron A.,
Int. J. Clin. Pharmacol. Ther., 42, 505—507 (2004).
10) Oborilova A., Mayer J., Pospisil Z., Koristek Z., J. Pain Symptom Man-
age., 24, 608—615 (2002).
11) Granry J. C., Paediatr. Anaesth., 7, 445—449 (1997).
12) White P. F., Anesth. Analg., 92, 1569—1575 (2001).
13) Peduto V. A., Anaesthesiol. Scand., 42, 293—298 (1998).
14) Bannwarth B., Netter P., Lapicque F., Gillet P., Pere P., Boccard E.,
Royer R. J., Gaucher A., Br. J. Clin. Pharmacol., 34, 79—81 (1992).
15) Hassan E. M., J. Pharm. Biomed. Anal., 21, 1183—1189 (2000).
16) Morelli B., J. Pharm. Biomed. Anal., 33, 423—433 (2003).
17) Ródenas V., García M. S., Sánchez-Pedreño C., Albero M. I., Talanta,
52, 517—523 (2000).
18) Lewis G. B. H., Hecker J. F., Br. J. Anaesth., 57, 220—233 (1985).
19) Fransson J., Espander-Jansson A., J. Pharm. Pharmacol., 48, 1012—
1015 (1996).
20) International Conference on Harmonization (ICH). “Validation of An-
alytical Procedures: Methodology,” ICH Q2B, 1996.
21) International Conference on Harmonization (ICH). “Evaluation of Sta-
tistical Data,” ICH Q1E, 2002.
Conciusions
The degradation process of PRO can be best fitted to sec-
ond-order kinetics independent of the medium used (saline
or glucose solution). The hydrolysis kinetics of PRO conver-
sion into PA depends on the temperature but not on the assay
medium (saline or glucose solution). The degradation rate
constants obtained for PRO were approximately 4.5 times
higher at 25 °C than at 4 °C. The values of t90% for PRO were
3.17 h and 3.61 h at 25 °C, and 13.42 h and 12.36 h at 4 °C