Stability evaluation of tramadol enantiomers using a chiral stability-indicating capillary electrophoresis method and its application to pharmaceutical analysis

Research Article

Stability evaluation of tramadol enantiomersusing a chiral stability-indicating capillaryelectrophoresis method and its applicationto pharmaceutical analysis

In this study, a chiral stability-indicating CE assay was developed for the stability

evaluation of tramadol (TR) enantiomers in commercial tablets using maltodextrin as

chiral selector. To investigate the stability-indicating power of the analytical method as

well as stability evaluation of TR enantiomers, active pharmaceutical ingredient and TR

tablets were subjected to photolysis, heat, oxidation and hydrolysis to conduct stress

testing. Best separation for the TR enantiomers was achieved on an uncoated fused-silica

capillary at 201C using borate buffer (50 mM, pH 10.2) containing 10% m/v maltodextrin.

All determinations were performed by a UV detector at 214 nm. A constant voltage of

20 kV was applied to obtain the separation. The range of quantitation for both enantio-

mers was 5–100 mg/mL (R40.996). Intra- and inter-day RSD (n 5 6) were less than 10%.

The percent relevant errors were obtained to be less than 4.0 for both enantiomers. The

limits of quantitation and detection for both enantiomers were 5 and 1.5 mg/mL,

respectively. Degradation products resulting from the stress studies were the same for

both enantiomers and did not interfere with the detection of the enantiomers.

Keywords: Electrophoresis / Enantiomer / Maltodextrin / Stability evaluation /TramadolDOI 10.1002/jssc.201100021

1 Introduction

Tramadol (TR) (Fig. 1), (7)-trans-2-[(dimethylamino)

methyl]-1-(3-methoxyphenyl) cyclohexanol [1], is an opioid

analgesic with noradrenergic and serotonergic properties

that may contribute to its analgesic activity. It is used for

moderate to severe pain [2]. TR is formulated as a racemic

mixture where (1)-TR preferentially inhibits serotonin re-

uptake, whereas (�)-TR mainly inhibits noradrenalin re-

uptake [3–5]. Enantiomeric forms of a drug can differ in

potency, toxicity and behavior in biological systems [6]. The

(1)-TR exhibits a tenfold higher analgesic potency than the

(�)-TR [7]. Chiral discrimination between enantiomers is

becoming one of the most important fields in analytical

chemistry, especially for pharmaceutical industry, clinical

analysis, food analysis and forensic analysis [8].

Recently, CE has been shown to be an efficient separa-

tion technique for enantiomer separation and drug analysis

[9–19]. Some advantages including high efficiency and

resolution, low analysis cost, relatively short analysis time

and small volume of sample make CE an alternative tech-

nique to HPLC [20]. Several analytical CE techniques have

been published in the literature for the separation and

determination of (1)-TR and (�)-TR in pharmaceutical

dosage forms and in biological fluids [21–30]. Stability is

important from a quality-control perspective in the phar-

maceutical industry and therefore any analytical method

developed should preferably be stability indicating. Regula-

tory guidance provided in international conference on

harmonization (ICH) requires the development and valida-

tion of stability-indicating procedures for the analysis of

drugs and drug products [31, 32].

Maltodextrins (MDs) are complex malto-oligo and

polysaccharides mixtures formed by hydrolysis of starch,

with dextrose equivalent (DE) lower than 20. MDs are a type

of very powerful chiral selective substances among the

chiral selectors such as crown ethers, CDs, proteins

and macrocyclic antibiotics. In CE, these compounds

enable highly efficient chiral separations of a broad range of

basic and acidic drugs [33–39]. In the present study, a

Ali Mohammadi1,2

Saeed Nojavan3�

Mohammadreza Rouini4

Ali Reza Fakhari3

1Department of Drug & FoodControl, Faculty of Pharmacy,Tehran University of MedicalSciences, Tehran, Iran

2Pharmaceutical QualityAssurance Research Centre,Faculty of Pharmacy, TehranUniversity of Medical Sciences,Tehran, Iran

3Department of Chemistry,Faculty of Sciences, ShahidBeheshti University, G. C.,Tehran, Iran

4Department of Pharmaceutics,Faculty of Pharmacy, TehranUniversity of Medical Sciences,Tehran, Iran

Received January 11, 2011Revised April 8, 2011Accepted April 12, 2011

Abbreviations: API, active pharmaceutical ingredient; DE,dextrose equivalent; MD, maltodextrin; TR, tramadol

�Additional correspondence: Dr. Saeed Nojavan

E-mail: [email protected]

Correspondence: Dr. Ali Mohammadi, Department of Drug &Food Control, Faculty of Pharmacy, Tehran University of MedicalSciences, P.O. Box 14155-6451, Tehran 14174, IranE-mail: [email protected]: 198-21-66461178

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 1613–1620 1613

stability-indicating CE method was developed and validated

for the assay and stability evaluation of TR enantiomers in

commercial tablets. The use of MD as a neutral chiral

selector was investigated and the electrophoretic conditions

for the assay were optimized.

2 Materials and methods

2.1 Reagents and chemicals

Drug standard powders as hydrochloride salt were obtained

from the Ministry of Health and Medical Education

(Tehran, Iran) and used without further purification. MD

with a DE of 16.5–19.5 was purchased from Aldrich.

Analytical grade H3BO3, NaOH, borax decahydrate, hydro-

gen peroxide and HCl were purchased from Merck

(Darmstadt, Germany). Biomadols50 tablets, each tablet

containing 50 mg TR hydrochloride, were obtained from a

local pharmacy (Tehran, Iran). HPLC-grade water was

obtained through a Milli-Qs system (Millipore, Milford,

MA, USA) and was used for preparation of all solutions.

2.2 CE equipment

CE was carried out using a Lumex Capel 105 (Ohiolumex,

Twinsburg, Russia) equipped with a UV detector operated at

214 nm. The electrophoretic experiments were performed in

an uncoated fused-silica capillary (Ohiolumex, Twinsburg,

Russia) 60 cm� 75 mm id (50 cm effective length). Throughout

the studies, CE was performed at 201C, at a constant potential

of 20 kV. Separations were performed using 50 mM borate

buffer at pH of 10.2 containing 10% m/v MD. Typical current

levels for this separation were approximately 80 mA throughout

the runs. Before use, the capillary was conditioned for 20 min

with 0.5 M HCl, 5 min with water, 30 min with 0.5 M NaOH

and 5 min with water. Additionally, the capillary was washed

for 2 min with 0.5 M NaOH, 1 min with water and 2 min with

the running buffer with positive pressure applied at the

injection end before each run. Acquisition of electrophero-

grams was computer-controlled by Chrom&Spec software

version 1.5. The analytes were injected at the anodic end by

applying pressure (30 mbar� 5 s). The pH measurements and

preparation of the buffer solutions were made using a digital

pH/mV/Ion Cyberscan model 2500.

2.3 Stock and standard solutions

Stock solutions of racemic TR (250 mg/mL for each

enantiomer) were prepared in water. The stock solutions

were protected from light using aluminum foil and stored

for a week at 41C with no evidence of decomposition. The

stock solutions were further diluted with water before use to

yield final concentrations of 5, 10, 25, 50 and 100 mg/mL for

each enantiomer.

2.4 Preparation of tablets for assay

Twenty tablets were weighed, powdered and mixed well. An

amount of powder equivalent to the weight of one tablet was

accurately weighed into each of the six 50-mL volumetric

flasks and 45 mL of water was added to each flask. The

volumetric flasks were sonicated for 20 min to effect

complete dissolution of the TR and the solutions were then

made up to volume with water. Aliquots of the solution were

filtered through a 0.45-mm nylon filter and 1 mL of the

filtered solution was transferred to a 20-mL volumetric flask

and made up to volume with water, to yield starting

concentration of 25 mg/mL for each enantiomer.

2.5 Forced degradation studies

To assess that the electromigration technique is stability

indicating and also for the determination of enantiomers

stability, TR tablets and TR active pharmaceutical ingredient

(API) powder were stressed under thermolytic, photolytic,

hydrolytic and oxidative stress conditions. After stressing

under stress conditions, all solutions and solid samples were

prepared to yield starting concentration of 25 mg/mL for

each enantiomer. Photodecomposition and thermolytic

degradation were carried out in both solid and solution

states.

Solutions for oxidation stress studies were prepared in

hydrogen peroxide (5% v/v), protected from light and stored

at room temperature for 16 h. During the initial forced

degradation studies, it was observed that acid hydrolysis and

basic hydrolysis were slow reactions at room temperature

with degradation of not more than 5% when the resultant

solution was analyzed 8 h after preparation. Thus, in later

experiments, the temperature was increased up to 901C.

Solutions for acid and alkali degradation studies were

prepared in 1 M hydrochloric acid and 1 M sodium hydrox-

ide, respectively. Both solutions were protected from light

and placed in a water bath with gentle shaking at 901C

for 8 h. Solution for neutral degradation studies was

prepared in water, protected from light and heated on a

water bath at 901C for 48 h prior to analysis. For conducting

thermolytic degradation, tablets and API were exposed to

dry heat (901C) in an oven for 72 h. Then, tablets and API

were removed and prepared for analysis as previously

described.

Figure 1. Chemical structure of (7)-trans-TR (MW: 263.38). Theasterisk denotes the chiral centre.

J. Sep. Sci. 2011, 34, 1613–16201614 A. Mohammadi et al.


For photostability studies, about 20 mg of API powder

was spread in a thin layer of less than 2 mm on a glass dish.

Solutions of the API were prepared in water. Tablets were

prepared for exposure in the same way. All samples were

exposed to light for 48 h resulting in an overall illumination

of Z200 W h/m2 at 251C with UV radiation of between 320

and 400 nm. Concurrently, control samples, which consis-

ted of all preparations previously described but protected

with aluminium foil, were also exposed to light. Finally, all

samples for analysis were prepared as previously described.

3 Results and discussion

3.1 Method development and optimization

3.1.1 Choice of the chiral selector

Neutral and charged CDs were previously investigated by

some authors for enantioseparation of TR [21–30]. For

example, Rudaz et al. [30] found that neutral CDs cannot

resolve the TR enantiomers but carboxymethylated-b-CD

(CMB) as a charged CD is capable of separation of the

mentioned enantiomers. Thus, in this work, an attempt was

made to establish separation of TR enantiomers using MD

(DE 5 16.5–19.5) as a neutral chiral selector in the

concentration range of 1–20% (m/v) using phosphate and

borate buffer (25, 50, 75 mM) with the pH range of 2.0–11.0.

Best separation for the TR enantiomers from each other and

from their degradation products was achieved on an

uncoated fused-silica capillary 60 cm� 75 mm id column

(50 cm effective length) at 201C using borate running buffer

(50 mM, pH 10.2) containing 10% m/v MD. All determina-

tions were performed by a UV detector at 214 nm.

According to the previous published works, since MD with

lower DE values have longer oligomeric chains, they should

have more binding sites than those with higher DE values.

This issue mainly improves resolution and is much less

effective on the migration time. However, in our work, good

separation of the enantiomers was easily obtained using MD

with higher DE values. A typical electropherogram of a

mixture of standard solution of TR enantiomers (25 mg/mL)

under described separation conditions can be observed in

Fig. 2. Different interactions such as hydrogen-bonding,

ionic and hydrophobic interactions between chiral analytes

with the helical structure of the MD emerge as the basis of

the enantioselectivity [40, 41]. The change in conformation

from a flexible coil to a helix in the presence of chiral

analytes and buffer salts may play an important role in

selective interactions. The helical structure of MD mimics

the cavity responsible for chiral recognition by CDs [36–38].

Figure 3 shows the effect of the chiral selector concentration

on the resolution of TR enantiomers. As the MD

concentration increased from 1 to 20% m/v, an increase

in the resolution and migration times was observed.

Maximum separation of the enantiomers with optimal

migration times was obtained at 10% m/v MD in the run

buffer. In comparison to the Rudaz et al. study [30],

optimized method in this work is quite simple. MDs as

chiral selectors are less expensive than CDs and especially

charged CDs. Although migration times of enantiomers in

this work were lengthy (more than 20 min).

3.1.2 Voltage effects

At higher voltages, Joule heating occurs and alters electro-

osmotic flow velocity and analyte mobility. Voltage effect on

the separation efficiency of TR enantiomers was investi-

gated over the 15–22 kV range. After each run, resolution

factor (Rs) was calculated and the optimal voltage for the

analysis was determined. At voltages of 15, 18, 20 and 22 kV,

resolution values of 1.9, 1.9, 2.8 and 2.5, respectively, were

obtained. An applied voltage of 20 kV was thus selected for

further analyses in order to avoid analytical difficulties

associated with Joule heating.

3.1.3 Buffer concentration effects

Peak resolution can be affected by changes in buffer

concentration. For the investigation of buffer concentration

Migration time (min)

Abs

orba

nce

(mA

U)

(+)-TR (-)-TR

Figure 2. A typical electropherogram of a mixture of standardsolution of TR enantiomers (25 mg/mL). Experimental conditions:capillary: 60 cm (50 cm effective length)�75 mm id; detection:214 nm; applied voltage: 20 kV; temperature: 201C; injection:hydrodynamically at 30 mbar for 5 s; buffer solution: boratebuffer (50 mM, pH 10.2) containing 10% m/v maltodextrin.

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20Maltodextrin concentration (%m/v)

Res

olut

ion

Figure 3. Influence of the MD concentration on the separation ofTR enantiomers. Experimental conditions are the same as inFig. 2.

J. Sep. Sci. 2011, 34, 1613–1620 Other Techniques 1615


on the separation efficiency of TR enantiomers, several

investigatory electrophoretic runs were performed using

borate buffer (pH 10.2) at 25, 50 and 75 mM, containing

10% m/v MD as chiral selector. At buffer concentrations

of 25, 50 and 75 mM, resolution values of 2.6, 2.8 and

2.7, respectively, were obtained. Higher concentration

buffer can decrease resolution significantly, probably due

to high current generated inside the capillary. At buffer

concentration of 75 mM, migration times of both enantio-

mers were more than 28 min. As a result, an optimum

buffer concentration of 50 mM was selected for further

analyses.

3.1.4 Capillary temperature effects

Buffer viscosity, resistance and dielectric constant are

temperature dependent. Also the host–guest complexation

mechanism is a kinetically and thermodynamically driven

process [42, 43]. Thus, the main effect of temperature is

observed on migration velocity and the efficiency of the

separation. To determine the optimum temperature for the

separation, several electrophoretic runs at different cartridge

temperatures (15, 20, 25, 30 and 351C) were performed and

the separation of the enantiomers examined. Control of

capillary temperature is extremely important for assay

repeatability. In this study, a circulating coolant containing

water was used to maintain the constant temperature inside

the capillary cartridge. Both resolution and migration times

were decreased from 2.8 to 0.9 and from 31.35 to 14.96 min

with an increase in temperature from 15 to 351C,

respectively. A complete resolution with reasonable migra-

tion times was obtained at 201C.

3.1.5 pH effects

In CZE, pH of buffer plays an important role in the

enantioseparation of acidic or basic analytes because it

determines the ionization extent of each individual analyte

and the ionic state of capillary column wall when bare

column is used [44]. Therefore, variation of buffer pH

usually becomes a key strategy to optimize a separation.

Thus, in this work, the effect of buffer pH on the

enantioseparation of TR was investigated. Also, electro-

osmotic flow is pH dependent. Thus, control of this

parameter can improve resolution and selectivity. Several

runs at pH range of 2.0–11.0 (low pH range with phosphate

buffer and high pH range with borate buffer) were

performed, the resolution factor (Rs) was calculated and

the optimal pH for the analysis was determined. With a

decrease in pH from 11.0 to 9.0, resolution decreased from

2.8 to 1.3 and migration times increased from 21.90 to

25.41 min. Both TR and chiral selector are probably neutral

compounds in the pH range of 9.4–11.0. However,

resolution increased with an increase in pH, probably as a

result of higher complexation of TR with the chiral selector.

Migration times decreased with an increase in pH, probably

as a result of higher electroosmotic flow.

TR has a pKa of 9.4 and is positively charged

in buffers of pH below 9.4 due to the protonation of

its side chain amino group [29]. Thus, electromigration

velocities of TR enantiomers were high in pH lower

than 9.4 and this can cause a decrease in the resolution

and migration of TR enantiomers. Although in very

low pH (pHo4) migration times of TR enantiomers

increased up to 50 min, enantioseparation did not

occur because in acidic buffers electroosmotic flow is

relatively low. Thus, a complete resolution with reason-

able migration times was obtained at pH 10.2 (borate

buffer). Figure 4 shows influence of buffer pH on the

separation of TR enantiomers in the pH range of 9.0–11.0

(borate buffer).

3.2 Validation of the method

Validation was performed with respect to parameters

including linearity, limit of detection (LOD), limit of

quantification (LOQ), precision, accuracy, selectivity, recov-

ery and robustness.

3.2.1 Linearity

The equations of the calibration plots were established by

linear regression of the peak area versus enantiomer

concentration for both enantiomers. The calibration plots

for both enantiomers were linear from 5 to 100 mg/mL

(Table 1). Linear regression analysis resulted in correlation

coefficients (R) of 0.998 and 0.999 for (1)-TR and (�)-TR,

respectively. The RSD values for slopes and intercepts were

lower than 0.7 and 0.2, respectively.

3.2.2 Limits of detection and quantification

The LOD was obtained from a signal-to-noise ratio of 3 to 1

and was determined to be 1.5 mg/mL for both enantiomers.

The LOQ of 5.0 mg/mL (S/N 5 10) was estimated (Table 1)

with an RSD value less than 8.0% for both enantiomers,

which is lower than the normal acceptance criterion

of 10.0% [45].

0

0.5

1

1.5

2

2.5

3

8.5 9 9.5 10 10.5 11

Res

olut

ion

pH

Figure 4. Influence of the buffer pH on the separation of TRenantiomers. Experimental conditions are the same as in Fig. 2.



3.2.3 Precision and accuracy

The intra- and inter-day precision and accuracy data for

both enantiomers were assessed by using standard solutions

prepared to produce solutions of three different concentra-

tions (5, 25 and 100 mg/mL). Repeatability or intra-day

precision was investigated by injecting six replications

of each concentration and inter-day precision was assessed

by injecting the same samples over five consecutive days.

Intra- and inter-day precision data varied between 0.9 and

10.0% for both enantiomers (Table 2). Accuracy of the

method was determined by interpolation of replicate (n 5 6)

peak areas of the same three standard solutions from

calibration plots prepared as previously described. In each

case, the percent relevant error and accuracy were calculated

and found to be less than 4.0% for both enantiomers

(Table 3).

3.2.4 Selectivity

The results of stress testing studies indicated a high degree

of selectivity. Figure 5 depicts resultant electropherograms

following storage of TR tablet powder under stress

conditions. The degradation behavior of TR was similar in

both tablets and API powder. Selectivity was also checked by

monitoring co-injection of acetaminophen (ACT) and cis-TR

into analysis system (Fig. 6). Combination of acetamino-

phen and TR is formulated in tablet dosage form.

3.2.5 Recovery

Known amounts of standard powder were added to samples

of tablets, which were then dissolved, diluted and analyzed

as previously described. The recovery of (1)-TR and (�)-TR

were found to be in the range of 98.7–102.0%. These results

show the proposed method is accurate. Completed data are

shown in Table 4.

3.2.6 Robustness

The robustness of the method was investigated under a

variety of conditions including changes of buffer pH, buffer

concentration, voltage, capillary temperature and chiral

selector concentration. The resolution values obtained as a

result of small deliberate variations in the method

parameters and by changing analytical operators have

proven that the method is robust and the data are

summarized in Table 5.

3.3 Stability studies

The degradation behavior of TR was similar in both tablets

and API powder. TR was found to be more stable under

thermolytic and photolytic stress condition in solid state in

both tablets and API powder, resulting in less than 8%

decomposition. Degradation under thermolytic stress condi-

tion in solution resulted in 8–13% decomposition for all

stressed samples. This drug was found to be sensitive to

oxidative, acid and alkali stress conditions and all stressed

samples were decomposed in the range of 25–32%. More

degradation occurred under photolytic stress condition in

both tablets and API solution resulting in decomposition in

the range of 32–35%. The complete stability data are

summarized in Table 6. The stability of stock solution was

determined by quantitation of drug in solution relative to

the response obtained for freshly prepared standard

solution. In all cases, no significant changes (o2.5%) were

observed.

3.4 Assay

The proposed method was successfully applied to

the determination of TR enantiomers in commercial

Table 2. Intra- and inter-day precision data of TR enantiomers

Added concentration

(mg/mL)

Measured concentration

(mean7SD), RSD (%)

(1)-TR (�)-TR

Intra-day (n 5 6)a) 5 4.970.41, 8.4 4.970.42, 8.6

25 25.170.56, 2.2 25.170.61, 2.4

100 99.970.93, 0.9 99.971.1, 1.1

Inter-day (n 5 5)b) 5 4.970.48, 9.8 4.870.48, 10.0

25 25.270.67, 2.7 25.070.71, 2.8

100 100.271.11, 1.1 100.271.23, 1.2

a) n 5 Injection number.

b) n 5 Day number.

Table 3. Accuracy data of TR enantiomers

Added concentration

(mg/mL)

Interpolated concentration

(mean7SD), RSD (%), REa) (%)

(1)-TR (�)-TR

5 4.970.21, 4.3, �2.0 4.870.25, 5.2, �4.0

25 25.070.35, 1.4, 0.0 24.070.29, 1.2, �4.0

100 100.770.56, 0.6, 10.7 100.670.45, 0.5, 10.6

a) RE, relative error.

Table 1. Validation parameters

Enantiomer Linear equationa) R LOQb) LODb) Linearityb)

(1)-TR Y 5 131X13.3 0.998 5 1.5 5–100

(�)-TR Y 5 128X15.4 0.999 5 1.5 5–100

a) In the equation, X shows the concentration (mg/mL) of analyte

and Y shows the peak area.

b) Concentration is based on mg/mL.











A E

F

G

H

B

C

D

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

Abs

orba

nce

(mA

U)

(+)-TR (-)-TR

Figure 5. Typical electropherograms of (A) untreated tablet, (B) thermal degraded tablet in solid state (1.7 and 1.5% degradation for (1)-TR and (�)-TR, respectively), (C) photodegraded tablet in solid state (2.7 and 2.9% degradation for (1)-TR and (�)-TR, respectively),(D) acid hydrolysis-degraded tablet (27.2 and 27.7% degradation for (1)-TR and (�)-TR, respectively); (E) base hydrolysis-degraded tablet(25.7 and 26.2% degradation for (1)-TR and (�)-TR, respectively), (F) oxidative degraded tablet (29.3 and 29.8% degradation for (1)-TR and(�)-TR, respectively), (G) photodegraded tablet in solution (34.3 and 34.5% degradation for (1)-TR and (�)-TR, respectively) and(H) neutral-hydrolysis degraded tablet (8.3 and 8.9% degradation for (1)-TR and (�)-TR, respectively). Degradation percentagecorresponding to each enantiomer is presented in parentheses. Experimental conditions are the same as in Fig. 2.



tablets. The results of the assay (n 5 6) yielded 96.4%

(RSD 5 3.7) and 95.8% (RSD 5 3.8) of label claim for

(1)-TR and (�)-TR, respectively. The migration time

(n 5 8) of (1)-TR and (�)-TR were 22.870.84 and

23.370.81 (mean7SD) min, respectively. The results

of the assay indicated that the method is selective for

the analysis of both enantiomers without interference

from the excipients used in manufacturing of tablet

formulations.

4 Concluding remarks

In this work, a stability-indicating CE method was developed

and validated for the separation and determination of TR

enantiomers in bulk and tablet dosage forms using MD as

chiral selector. The results of stress testing undertaken

according to the International Conference on Harmoniza-

tion guidelines reveal that the method is selective and

stability indicating, which shows the applicability of the

method for quality control. The degradation behaviors of TR

enantiomers were similar in both tablets and API powder.

TR was found to be more stable under thermolytic and

photolytic stress conditions in solid state rather than in

solution. TR solution was more stable under oxidative, acid

and alkali stress conditions compared to the photolytic

stress condition.

The authors have declared no conflict of interest.

5 References

[1] Neil, M. J., Smith, A., Heckelman, P. E., Budavari, S.,The Merck Index, An Encyclopedia of Chemicals, Drugsand Biologicals, 12th Edn., Merck & Co. Inc., WhiteHouse Station, New Jersey 1996, p. 1632.

[2] Sweetman. S. C., Martindale the Complete Drug Refer-ence, 35th Edn., Pharmaceutical Press, London 2006, p.115.

[3] Dayer, P., Desmeules, J., Collart, L., Drugs 1997, 53,18–24.

[4] Paar, W. D., Frankus, P., Dengler, H. G., Clin. Invest.1992, 70, 708–710.

[5] Raffa, R. B., Friderichs, E., Reimann, W., Shank, R. P.,Codd, E. E., Vaught, J. L., Jacoby, H. I., Selve, N., J.Pharmacol. Exp. Ther. 1993, 267, 331–340.

[6] Vermeulen, N. P. E., Koppele, J. M., in: Wainer, I. W.(Ed.), Drug Stereochemistry: Analytical Methods andPharmacology, 2nd Edn., Marcel Dekker, New York1993, p. 245.

trans-TR cis-TR

ACT


Abs

orba

nce

(mA

U)

Figure 6. Typical electropherogram of simultaneous injection ofcis-TR, trans-TR and acetaminophen (ACT). Experimental condi-tions are the same as in Fig. 2.

Table 4. Recovery data

Added concentration

of each enantiomer

(mg/mL), n 5 5

Obtained concentration

(mg/mL), (mean7SD)

Recovery (%)

(1)-TR (�)-TR (1)-TR (�)-TR

10 9.9170.84 9.8770.81 99.1 98.7

25 25.0370.73 25.0570.76 100.12 100.2

50 51.0270.57 50.9870.84 102.04 101.96

Table 5. Robustness testing of the method

Parameter Modification Resolution value

Buffer pH 9.5 1.50

10.2 2.76

10.9 2.21

Buffer concentration (mM) 25 2.61

50 2.76

75 2.71

Voltage (kV) 18 1.89

20 2.76

22 2.45

Capillary temperature (1C) 15 2.84

20 2.76

25 2.48

Chiral selector concentration (%w/v) 7 1.69

10 2.76

13 2.81

Table 6. Results of forced degradation studies, indicating

percentage degradation of TR enantiomers in both

tablets and active pharmaceutical ingredient

Stability condition/duration/state (%) Degrada-

tion in tablets

(%) Degrada-

tion in API

(1)-TR (�)-TR (1)-TR (�)-TR

Thermal/901C/72 h/solid 1.7 1.5 7.4 7.5

Photo/UV/48 h/ solid 2.7 2.9 8.2 7.8

Acidic/1 M HCL/8 h/901C/solution 27.2 27.7 30.2 30.3

Alkaline/1 M NaOH/8 h/901C/solution 25.7 26.2 28.6 28.2

Oxidative/5% H2O2/16 h/solution 29.3 29.8 27.3 27.6

Photo/UV/48 h/solution 34.3 34.5 31.3 31.5

Thermal/neutral/901C/48 h/solution 8.3 8.9 12.3 12.5



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