alprazolam and sertraline in combined dosage forms

DRUG FORMULATIONS AND CLINICAL METHODS

Development of a Stability-Indicating High-Performance LiquidChromatographic Method for the Simultaneous Determination ofAlprazolam and Sertraline in Combined Dosage Forms

ASHUTOSH PATHAK and SADHANA J. RAJPUT

The Maharaja Sayajirao University of Baroda, Centre of Relevance and Excellence in Novel Drug Delivery Systems,

Pharmacy Department, Quality Assurance Laboratory, G.H. Patel Building, Donor’s Plaza, Fatehgunj, Vadodara, Gujarat,

India–390 002

The objective of the current study was to develop a

validated stability-indicating high-performance

liquid chromatographic method for alprazolam and

sertraline in combined dosage forms. The method

was validated by subjecting the drugs to forced

decomposition under hydrolysis, oxidation,

photolysis, and thermal stress conditions

prescribed by the International Conference on

Harmonization. The drugs were successfully

separated from major and minor degradation

products on a reversed-phase C18 column by

using 75 mM potassium dihydrogen phosphate

buffer (pH 4.3)–acetonitrile–methanol (50 + 45 + 5,

v/v/v) as the mobile phase with determination at

227 nm. The flow rate was 0.9 mL/min. The method

was validated with respect to linearity, precision,

accuracy, system suitability, and robustness. The

responses were linear over the ranges of 1–80 and

5–200 �g/mL for alprazolam and sertraline,

respectively. The recoveries of both drugs from a

mixture of degradation products were in the range

of 97–101%. The utility of the procedure was

verified by its application to marketed formulations

that were subjected to accelerated stability studies.

The method distinctly separated the drugs and

degradation products, even in actual samples. The

products formed in marketed tablets were similar

to those formed during stress studies.

Stability studies of drug substances via acid hydrolysis,

base hydrolysis, water hydrolysis, oxidation, and

thermal and photolytic stress testing are a part of

development strategy under the International Conference on

Harmonization (ICH) requirements (1). These studies provide

information on a drug’s inherent stability and help to

validate analytical methods to be used for evaluation of

stability. Stability-indicating assays are currently being

developed (2, 3) by using the stress-testing approach of the

ICH guidelines, Q1A [R2] (1). The approach has been further

extended to stress tests of drug combinations (4, 5). These

tests allow accurate and precise quantification of drugs, their

degradation products, and their interaction products.

Alprazolam (ALP), 8-chloro-1-methyl-6-phenyl-4H-s-

triazolo[4,3-a][1, 4]benzodiazepine, is a benzodiazepine

derivative that is currently used in the treatment of generalized

anxiety, panic attacks with or without agoraphobia, and

depression (6); sertraline (SERT), (1S-cis)-4-(3,4-dichloro-

phenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthaleneamine, is

a new antidepressant, a selective serotonin reuptake inhibitor

used in the treatment of psychic deviations like obsessive-

compulsive, panic, and post-traumatic stress disorders (7).

Coadministration of SERT and ALP is common in the

treatment of panic disorders (8).

Several high-performance liquid chromatographic (HPLC)

methods have been reported for the determination of ALP

(9–19) and SERT (20–24) individually. To our knowledge, no

stability-indicating HPLC assay method has been reported for

the simultaneous determination of ALP and SERT in tablets in

the presence of their degradants by using the ICH approach of

stress testing. The focus of the present study was to develop a

simple, rapid, precise, and accurate isocratic reversed-phase

stability-indicating HPLC method for the simultaneous

determination of ALP and SERT in tablet dosage form.

Experimental

Apparatus

Chromatography was performed with a Shimadzu

(Shimadzu Corp., Kyoto, Japan) chromatographic system

equipped with an isocratic HPLC pump (Shimadzu LC-20AT),

a UV-visible detector (Shimadzu SPD-20AV), and a manual

fixed-loop (20 �L) injector with a Rheodyne syringe-loading

sample injector (Model 7725). The LC separations were

performed at 32 ± 2�C on a Phenomenex Luna C18 (250 �

4.6 mm, 5 �m) column. Spinchrom (CFR Version 2.4.1.93)

software was used for LC peak integration. The mobile phase

was degassed by sonication with an Ultrasonics Selec (DTC

503; Vetra, Italy) ultrasonic bath. The standard substances were

weighed on a Precisa analytical balance (205 ASCS Swiss

Quality, Zurich, Switzerland). Stability studies were carried out

1344 PATHAK & RAJPUT: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 6, 2008

Received October 13, 2007. Accepted by SW January 28, 2008.Corresponding author's e-mail: [email protected]

in a photostability chamber (NEC-103R Newtronic, Mumbai,

India) which was set at 25 ± 1�C. The photostability chamber

was equipped with an illumination bank on the inside top, as

defined under Option 2 in ICH guideline Q1B (25). The light

bank consisted of a combination of one black light UV lamp set

at UV 200 W/h/m2 and 4 white fluorescent lamps set at

1.2 million lux hrs. The samples were placed at a distance of

9 in. from the light bank. Both fluorescent and UV lamps were

turned on simultaneously. The samples were exposed for a total

of 15 days.

Peak purity analysis was carried out with another HPLC

system (all equipment from Waters Corp., Milford, MA),

equipped with a 2996 photodiode array (PDA) detector. The

thermal stability study was carried out in a dry-air oven

(Sedko Laboratory Equipment, Ahmedabad, India).

Reagents and Materials

Pure ALP and SERT were gift samples from Torrent

Research Center (Gandhinagar, India) with 99.94 and 99.92%

purity, respectively. Combination products, ALPRAX PLUS

(label claim: 0.5 mg ALP and 25 mg SERT per tablet, Torrent

Pharmaceutical, Ahmedabad, India) and ALPRAX FORTE

(label claim: 0.5 mg ALP and 50 mg SERT per tablet, Torrent

Pharmaceutical), were purchased from a local shop.

Acetonitrile, methanol, and water (HPLC grade) were

purchased from Spectrochem Pvt. Ltd (Mumbai, India).

Potassium dihydrogen phosphate, hydrochloric acid, sodium

hydroxide pellets, and hydrogen peroxide solution (all

analytical reagent grade) were purchased from Rankem

(Mumbai, India); orthophosphoric acid was purchased from

Qualigens Fine Chemicals (Mumbai, India).

Preparation of Standard Solutions

Individual standard stock solutions of ALP (100 �g/mL)

and SERT (500 �g/mL) were prepared in methanol. For the

calibration plot of ALP, various dilutions were prepared from

the stock solution in the range of 1–80 �g/mL, with SERT kept

constant (50 �g/mL) throughout in the binary mixture

(Figure 1). Similarly, for the calibration curve of SERT,

various dilutions were made from the SERT stock solution in

the range of 5–200 �g/mL by keeping ALP constant

(10 �g/mL) throughout in the binary mixture (Figure 2). All

dilutions of the stock solutions were made with mobile phase.

Forced Degradation Studies of Standard Drug

Solutions and Their Binary Mixtures

Standard stock solutions of ALP (1 mg/mL) and SERT

(5 mg/mL) were individually prepared by dissolving 25 mg

standard ALP and 125 mg standard SERT each in 25 mL

methanol; 2 mL ALP stock solution and 1 mL SERT stock

solution were diluted separately to 10 mL with 3% H2O2,

distilled water, 0.1 M HCl, and 0.1 M NaOH, to obtain

concentrations of 200 and 500 �g/mL for ALP and SERT,

respectively. Binary mixtures of these drugs were prepared

from the above stock solutions by combining 2 mL ALP stock

solution and 1 mL SERT stock solution and diluting to 10 mL

with 3% H2O2, distilled water, 0.1 M HCl, and 0.1 M NaOH,

to obtain concentrations of 200 and 500 �g/mL for ALP and

SERT, respectively, in the binary mixture.

The above solutions in water, 0.1 M HCl, and 0.1 M NaOH

were heated at 80�C for 12 and 24 h, respectively. For

oxidative degradation, drugs in 3% H2O2 were stored at room

temperature for 24 h. Degradation was also carried out in the

solid state by exposing pure drugs and drug product to dry

heat at 80�C for 48 h. For photolytic studies, drug solutions in

water, 0.1 M HCl, and 0.1 M NaOH were exposed in a

photostability chamber for 15 days. Also, solid drugs at 1 mm

thickness were spread on a Petri plate and exposed in the

photostability chamber for the same time period. Suitable

controls were maintained under dark conditions. Samples

were withdrawn periodically and diluted with mobile phase to

yield concentrations of both ALP (20–40 �g/mL) and SERT

(50–100 �g/mL) for assay.

Analysis of the Marketed Formulation

Twenty tablets from each brand of one batch were

accurately weighed, their mean weight was determined, and

the tablets were powdered in a glass mortar. An amount of the

powder equivalent to 2 tablets was dissolved in 20 mL

methanol, and the mixture was sonicated for 15 min. The

resulting mixture was filtered by using a 0.45 �m nylon filter

paper in a 25 mL volumetric flask, and the filtrate was diluted

to volume with methanol. Solutions of ALP (40 �g/mL) and

SERT (2 and 4 mg/mL) were prepared. From these solutions

suitable dilutions were made to obtain working solutions of

ALP (20–40 �g/mL) and SERT (50–100 �g/mL) for analysis,

and the possibility of interference from excipients during

analysis was studied.

Forced Degradation Studies of Tablets

Contents of 4 tablets were dissolved in 25 mL methanol to

obtain concentrations of 80 �g/mL for ALP and 4000 �g/mL

for SERT.

For degradation induced by acid hydrolysis, base

hydrolysis, neutral (water) hydrolysis, and oxidation, 5 mL

0.1 M HCl, 5 mL 0.1 M NaOH, 5 mL water, and 5 mL 3.0%

H2O2 were separately added to 5 mL aliquots of drug solution.

Acid, base, and water mixtures were refluxed for 12 and 24 h

at 80�C and then cooled to room temperature. The mixture for

oxidation was kept at room temperature for 24 h. Suitable

dilutions of degraded samples were made with mobile phase

to obtain the concentrations of ALP (20 �g/mL) and SERT

(100 �g/mL) for chromatographic analysis.

The tablets were also subjected to thermal stress at 80�C

for 48 h. An amount of the degraded tablet powder equivalent

to 4 tablets was dissolved, and the solution was transferred to a

25 mL volumetric flask and diluted to volume with methanol.

Further dilutions were made with mobile phase to obtain

working concentrations of ALP (20–40 �g/mL) and SERT

(50–100 �g/mL) for analysis.

The photostability of the drug was also studied by

exposing the tablets and their solutions in water, 0.1 M HCl,

and 0.1 M NaOH in a photostability chamber for 15 days and

then continuing as indicated for dry heat degradation. The

PATHAK & RAJPUT: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 6, 2008 1345

resulting solutions were analyzed as degraded samples by

using the same chromatographic conditions.

Chromatographic Separations

HPLC studies were carried out with all the reaction

solutions individually, and with a mixture of the solutions in

which decomposition was observed. The stressed samples

were initially analyzed by HPLC using a reversed-phase C18

column and the mobile phase buffer–acetonitrile (55 + 50,

v/v). Because the separation and peak shape were not good,

methanol was added as an organic modifier, and further trials

were carried out by varying the pH and simultaneously

optimizing the ratio of the buffer and solvents. Eventually, a

mobile phase composition of potassium dihydrogen

phosphate buffer (75 mM, pH 4.3, adjusted with 0.05%

orthophosphoric acid)–acetonitrile–methanol (55 + 45 + 5,

v/v/v) gave the best results. During these studies, the injection

volume was 20 �L, and the mobile phase flow rate was

constant at 0.9 mL/min; the analytical wavelength was

227 nm.

Method Validation

Linearity was established by triplicate injections of

solutions containing standard ALP and SERT in the

concentration ranges of 1–80 and 5–200 �g/mL, respectively.

The limit of detection (LOD) and limit of quantification

(LOQ) values were calculated from the calibration curves as k

SD/b where k = 3 for LOD and k = 10 for LOQ, SD is the

standard deviation of the responses, and b is the slope of the

calibration curve (26). The intraday precision was established

by making 3 injections of the lowest, middle, and highest

concentrations in the above ranges for ALP (10, 40, and

80 �g/mL) and SERT (20, 80, and 160 �g/mL) on the same

day. These injections were also repeated on 3 different days to

determine interday precision. Intermediate precision was

established through separation studies on 2 different columns.

Accuracy was evaluated by fortifying a mixture of degraded

solutions with 3 standard solutions containing known

concentrations of ALP (20, 25, and 30 �g/mL) and SERT (40,

50, and 60 �g/mL), and percent recoveries of the added drugs

were determined. The specificity of the method was

established through study of resolution factors of the drug

peaks from the nearest peak, and also from all other peaks.

The specificity of the method toward the drugs was also

established through determination of the purity of ALP and

SERT peaks from HPLC analysis of a mixture of stressed

samples through study of purity plots by using a PDAdetector.

Robustness of the method was determined by deliberately

varying certain parameters like flow rate (mL/min),

concentration of acetonitrile (mL) in the mobile phase, and

manufacturer of acetonitrile. Each parameter was studied at

3 levels (–1, 0, and 1) except for acetonitrile, for which

2 different manufacturers were studied. One factor at a time

was changed to estimate the effect. The assay was carried out

in triplicate (n = 3) at 3 different concentration levels, i.e., 10,

40, and 80 �g/mL for ALP and 20, 80, and 160 �g/mL for

SERT. In the system suitability tests, 6 replicate injections of

freshly prepared working standard solutions of ALP and

SERT (50 �g/mL each) and 2 injections of the solutions

prepared for the specificity procedure were injected into the

chromatograph, and the relative standard deviation (RSD)

values of the peak areas, resolution factors, tailing factors, and

number of theoretical plates were determined.

Results and Discussion

Forced Degradation Studies

Conditions used for forced degradation were attenuated to

achieve degradation in the range of 20–80%. The following

degradation behavior of the drugs was observed during the

HPLC studies:

Acidic conditions.—The individual drugs and their

combination were heated in 0.1 N HCl for 12 h. Compared

with SERT, ALP was more susceptible to the degradation

process. About 15–20% degradation of ALP was observed,


Figure 1. Chromatograms showing the separation of alprazolam (ALP; 1–80 �g/mL) and sertraline (SERT;

50 �g/mL) in the synthetic mixture.


Figure 4. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in astressed sample of the synthetic mixture subjected to alkali hydrolysis.

Figure 3. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in astressed sample of the synthetic mixture subjected to acid hydrolysis.

Figure 2. Chromatograms showing the separation of alprazolam (ALP; 10 �g/mL) and sertraline (SERT;

5–200 �g/mL) in the synthetic mixture.


Figure 7. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in astressed sample of the synthetic mixture subjected to thermal degradation.

Figure 6. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in astressed sample of the synthetic mixture subjected to oxidative hydrolysis.

Figure 5. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in astressed sample of the synthetic mixture subjected to water hydrolysis.


Figure 9. Chromatogram showing the separation of alprazolam (ALP; 20 �g/mL) and sertraline (SERT; 1000 �g/mL)in a degraded formulation: (I) formed under acidic, alkali, oxidative, thermal, and photolytic conditions; (II) formedunder neutral, oxidative, and photolytic conditions; (III) formed under acidic, alkali, neutral, oxidative, thermal, andphotolytic conditions; (IV) formed under acidic, oxidative, and photolytic conditions; (V) formed under alkali,neutral, oxidative, thermal, and photolytic conditions; (VI) formed under photolytic conditions; and (VII) formedunder neutral, oxidative, and thermal conditions.

Figure 10. Chromatogram showing the separation of alprazolam (ALP; 2 �g/mL) and sertraline (SERT; 100 �g/mL)in a degraded formulation (I)–(VII). See Figure 9 legend.

Figure 8. Chromatographic separation of alprazolam (ALP; 40 �g/mL) and sertraline (SERT; 100 �g/mL) in anacidic stressed sample of the synthetic mixture subjected to photolytic degradation.

whereas a minute degradation of SERT was seen (Figure 3).

On further heating up to 24 h, there was a slight rise in the

proportion of the peaks of the degradation products of ALP.

The major degradation products formed from ALP in the

combination product were at retention times (RTs) of 4.6, 5.4,

and 5.5 min, whereas for SERT, a small degradation peak was

found at the RT of 6.4 min.

Degradation in alkali.—ALP underwent alkali hydrolysis,

but the rate of hydrolysis was slower than that under acidic

conditions. It took 24 h for the drug to decompose by 10%.

However, SERT the degradation pattern of SERT was similar

to that seen under acidic conditions. Degradation peaks for

ALP were at 4.6 and 5.3 min, and for SERT, at 6.9 min

(Figure 4).

Neutral (water) conditions.—About 8–10% of the ALP

degraded when the drug combination was refluxed in water at

80�C for 24 h, whereas mild degradation of SERT was seen at

6.9 min. Degradation peaks for ALP were observed at 4.8, 5.2,

and 15.1 min (Figure 5).

Oxidative conditions.—Both drugs were highly labile

during oxidative hydrolysis in 3% H2O2, when they were kept

at room temperature for 24 h. SERT was comparatively more

labile than ALP. Around 10–15% degradation was observed

in the case of SERT, whereas the degradation of ALP was

about 5–10%. The major degradation products of ALP were at

RTs of 4.6, 5.3, 5.6, 6.1, and 15.7 min, whereas for SERT, a

major degradation peak was found at 4.9 min, and a minute

degradation was observed at 7.1 min (Figure 6).

Solid-state study (thermal degradation).—Both drugs were

relatively stable when exposed to dry heat at 80�C for 48 h.

The percentages of both drugs remaining after 48 h of

exposure to dry heat were in the range of 95–98%.

Degradation peaks of ALP were at 4.5, 5.4, and 15.1 min,

whereas that of SERT was at 7.0 min (Figure 7).

Photolytic conditions.—ALP was highly susceptible to

photolytic degradation in comparison with SERT. ALP was

more susceptible under acidic photolytic conditions (about

30–35% degradation) in comparison with alkaline and neutral

conditions. The major degradation peaks for ALP were at 2.5,

4.3, 4.6, 5.8, and 6.1 min, whereas for SERT they were at 5.0,

7.0, and 9.1 min (Figure 8).


Table 1. Results from regression analysis of the calibration curves for the determination of alprazolam (ALP) and

sertraline (SERT) by the proposed HPLC method

SD

Drug Range, �g/mL Regression equation ra Slope Intercept LOD, �g/mL LOQ, �g/mL

ALP 1–80 AALP = 142.86CALP – 46.23 0.9996 1.123 0.274 0.032 0.107

SERT 5–200 ASERT = 55.528CSERT + 62.418 0.9999 1.179 0.819 0.065 0.216

a r = Regression coefficient.

Table 2. Intraday and interday precision studies for the determination of alprazolam (ALP) and sertraline (SERT) by

the proposed HPLC method

Intraday precisiona Interday precisiona

Drug Added, �g/mL Measured ± SD, �g/mL (RSD, %) Standard error Measured ± SD, �g/mL (RSD, %) Standard error

ALP 10 9.709 ± 0.21 (1.16) 0.094 9.51 ± 0.15 (1.56) 0.106

40 39.89 ± 0.35 (0.87) 0.157 38.85 ± 0.36 (0.92) 0.252

80 79.84 ± 0.37 (0.46) 0.166 78.93 ± 0.34 (0.43) 0.241

SERT 20 19.97 ± 0.16 (0.79) 0.071 19.18 ± 0.15 (0.78) 0.105

80 79.34 ± 0.42 (0.53) 0.188 78.64 ± 0.50 (0.63) 0.355

160 158.24 ± 0.76 (0.48) 0.341 158.40 ± 0.78 (0.49) 0.552

a n = 6.

Table 3. Intermediate precision studies for the

determination of alprazolam (ALP) and sertraline (SERT)

by the proposed HPLC method

Retention time ± SD, min

Column SERTa ALPa

Phenomenex Luna C18 8.35 ± 0.037 13.24 ± 0.095

Waters C18 8.43 ± 0.154 13.40 ± 0.226

a n = 3.

Establishment of a Stability-Indicating Method for a

Mixture of Stressed Solutions

The mobile phase potassium dihydrogen phosphate

(75 mM; pH 4.3 adjusted with orthophosphoric

acid)–acetonitrile–methanol (55 + 40 + 5, v/v/v) was first used

to analyze individual standard drug samples for the linearity

study (Figures 1 and 2). It was then applied to stressed

samples of individual drugs and subsequently used to analyze

synthetic mixtures, which showed recognizable degradation

(Figures 3–8). The method was then successfully applied to

degradation studies of formulations (Figures 9 and 10).

Validation of the Proposed Method

The method was validated with respect to the following

parameters given below:

Linearity.—Linear calibration plots for the above method

were obtained in the calibration ranges of 1–80 and

5–200 �g/mL for ALP and SERT, respectively, and the

correlation coefficients obtained were >0.999 (Table 1). The

results show a good correlation between peak area and analyte

concentration.

LOD and LOQ.—The LOD values for ALP and SERT

were 0.032 and 0.065 �g/mL, respectively, and the LOQ

values for ALP and SERT were 0.107 and 0.216 �g/mL,

respectively (Table 1).

Precision.—Data obtained from analysis of the samples on

the same day (n = 3) and on consecutive days (n = 3) are given

in Table 2. The RSD values obtained were well below 2%

(i.e., in the ranges of 0.482–1.162 and 0.432–1.569% for

intra- and interday precision, respectively). The RSD values

indicate that the method is sufficiently precise. The

intermediate precision established for the method showed that

similar resolution was obtained when the experiment was

conducted with 2 different reversed-phase HPLC columns

(Table 3).

Accuracy.—Percent recoveries were calculated from the

differences between the peak areas obtained for the fortified

and the unfortified solutions. Good recoveries were obtained

for each fortification level (Table 4), indicating that the

method is accurate.

Specificity.—The specificity of the HPLC method was

shown by the complete separation of ALP and SERT from their

degradation products. The degradation products of ALP and

SERT were found to be similar for both tablets and active

pharmaceutical ingredient (API) standards. Typical

chromatograms obtained from the assays of pure ALP and

SERT and stressed samples are shown in Figure 1 and 2 and

Figures 3–8, respectively. The resolution factor (Rs) values for

the acidic, alkaline, neutral, oxidative, and thermal degradation

products were always >1.9, which ensured the complete

separation of ALP and SERT from their degradation products.

Studies using PDA detection to determine the purity of

ALP and SERT peaks showed purity angle (PA) values of

0.296 and 0.180 and purity threshold (TH) values of 0.345 and

0.284 for ALP and SERT, respectively. The PA value was less

than the TH values (as evident from the purity plots in


Table 4. Recovery studies for the determination of

alprazolam (ALP) and sertraline (SERT) by the

proposed HPLC method

Drug Added, �g/mLRecovery ± SD, %

(RSD, %) Standard error

ALP 20 101.93 ± 0.13 (0.63) 0.091

25 100.30 ± 0.14 (0.54) 0.095

30 100.68 ± 0.22 (0.73) 0.155

SERT 40 98.03 ± 0.304 (0.77) 0.214

50 97.25 ± 0.54 (1.109) 0.381

60 98.46 ± 0.70 (1.881) 0.203

Figure 12. Purity plot for SERT.

Figure 11. Purity plot for ALP.

Figures 11 and 12)], thereby indicating that the ALP and

SERT peaks were free from any coeluting peaks.

Robustness.—The method remained robust even with

small variations in flow rate (±0.1 mL/min) and concentration

of acetonitrile (±5 mL) in the mobile phase. There was no

significant difference in peak area and retention time. It was

also found that acetonitrile from a different manufacturer had

no significant influence on the determination. Insignificant

differences in peak areas and less variability in retention times

were observed (Table 5).

System suitability.—The parameters, retention time,

resolution factor, tailing factor, and number of theoretical

plates were evaluated. The results (Table 6) obtained from the

system suitability tests are in agreement with the requirements

of The United States Pharmacopeia (26). The variation in

retention times among 6 replicate injections of ALP and SERT

standard solutions was very low, with RSD values of 0.741

and 0.480%, respectively.

Applicability of the Developed Method to Marketed

Formulations

A clear separation of the drugs and degradation products

was obtained for tablets with no interference from excipients

(Figures 9 and 10). This result indicated that the method could

be extended to the study of the available drug content in

commercial products (Table 7). Overall, ALP was found to be

more prone to degradation, because assay results were lower

than the label claim for the tablets. In comparison, SERT was

less affected by degradation.

Conclusions

The combination of ALP and SERT has recently gained

popularity for the treatment of anxiety disorders. The major

constraint in the determination of these drugs in combination

is the ratio in which they are incorporated in tablets

(ALP:SERT = 1:50 or 1:100). The present study examined the


Table 6. System suitability parameters for the

determination of alprazolam (ALP) and sertraline (SERT)

by the proposed HPLC method

Parameter SERT ALP

RT ± SD, min 8.31 ± 0.013 13.1 ± 0.017

Resolution factor — 4.79

Tailing factor ± SD 1.021 ± 0.016 1.124 ± 0.033

No. of theoretical plates ± SD 52613 ± 0.297 73391 ± 0.268

RSD, % 0.480 0.741

Table 7. Assay results obtained for the combined

dosage form by using the proposed HPLC method

Tableta

Drug,b

mg/tabletDrug

found ± SD, %Standard errorof estimation

ALPRAX PLUS ALP, 0.5 mg 97.54 ± 1.16 1.156

SERT, 25 mg 99.01 ± 0.15 0.107

ALPRAX FORTE ALP, 0.5 mg 96.43 ± 1.16 1.152

SERT, 50 mg 99.81 ± 0.70 0.496

a n = 3.b ALP = Alprazolam; SERT = sertraline.

Table 5. Robustness studies for the determination of alprazolam (ALP) and sertraline (SERT) by the proposed HPLC

method

Mean retention time ± SD, mina Mean asymmetric factor ± SDa

Factor Level SERT ALP SERT ALP

Flow rate, mL/min

0.8 –1 8.39 ± 0.017 13.27 ± 0.056 1.026 ± 0.003 1.243 ± 0.011

0.9 0 8.32 ± 0.013 13.19 ± 0.090 1.025 ± 0.002 1.229 ± 0.003

1 1 8.31 ± 0.013 13.08 ± 0.012 1.026 ± 0.002 1.237 ± 0.011

ACNb in mobile phase, %

40 –1 8.37 ± 0.031 13.21 ± 0.032 1.027 ± 0.013 1.245 ± 0.012

45 0 8.32 ± 0.013 13.19 ± 0.090 1.022 ± 0.012 1.219 ± 0.0012

55 1 8.29 ± 0.017 13.10 ± 0.013 1.021 ± 0.002 1.234 ± 0.024

ACN manufacturer

RANKEM 8.28 ± 0.012 13.08 ± 0.017 1.025 ± 0.003 1.235 ± 0.0003

SPECTROCHEM 8.31 ± 0.017 13.33 ± 0.140 1.024 ± 0.012 1.227 ± 0.004

a n = 4.b ACN = Acetonitrile.

stability behavior of ALP and SERT individually and in

combination according to ICH guidelines. ALP was found to

be more susceptible under stress conditions in comparison

with SERT.

The work described in this paper has shown that the

developed method is precise, accurate, linear, and stability

indicating. The method was found to be specific to the drugs,

because the peaks of the degradation products did not interfere

with the drug peaks. Application of this method to the

determination of ALP and SERT in tablet dosage form shows

that neither the degradation products nor the excipients

interfere with the analysis. This finding indicates that the

proposed method could be used as a stability-indicating

method for the simultaneous determination of ALP and SERT

either in the bulk drug or in pharmaceutical formulations.

Acknowledgments

We are thankful to the Analytical Department of Alembic

Ltd (Vadodara, India) for providing the facilities for the

photostability studies used in our research. Thanks are also

extended to Torrent Pharmaceutical (Gandhinagar, India) for

supplying gift samples of ALP and SERT.

References

(1) ICH, Q1A (R2) (2003) Stability Testing of New Drug

Substances and Products, International Conference on

Harmonization, Geneva, Switzerland

(2) Singh, S., Singh, B., Bahuguna, R., Wadhwa, L., & Saxena,

R. (2006) J. Pharm. Biomed. Anal. 41, 1037–1040

(3) Rao, B.M., Chakraborty, A., Srinivasu, M.K., Devi, M.L.,

Kumar, P.R., Chandrasekhar, K.B., Srinivasan, A.K., Prasad,

A.S., & Ramanatham, J. (2006) J. Pharm. Biomed. Anal. 41,

676–681

(4) Grosa, G., Grosso, E.D., Russo, R., & Allegrone, G. (2006) J.

Pharm. Biomed. Anal. 41, 798–803

(5) Kumar, V., Bhutani, H., & Singh, S. (2007) J. Pharm.

Biomed. Anal. 43, 769–773

(6) Verster, J.C., & Volkerts, E.R. (2004) CNS Drug Rev. 10,

45–76

(7) Johnson, B.M., & Chang, P.-T.L. (1996) Anal. Profiles Drug

Subst. Excipients 24, 443–486

(8) Fisekovic, S., & Loga-Zec, S. (2005) Bosnian J. Basic Med.

Sci. 5, 78–81

(9) Perez-Lozano, P., Garcia-Montoya, E., Orriols, A., Minarro,

M., Tico, J.R., & Sune-Negre, J.M. (2004) J. Pharm. Biomed.

Anal. 34, 979–987

(10) Schmith, V.D., Cox, S.R., Zemaitis, M.A., & Kroboth, P.D.

(1991) J. Chromatogr. 568, 253–260

(11) Greenblatt, D.J., Javaid, J.I., Locniskar, A., Harmatz, J.S., &

Shader, R.I. (1990) J. Chromatogr. 534, 202–207

(12) Samanidou, V.F., Pechlivanidou, A.P., & Papadoyannis, I.N.

(2007) J. Sep. Sci. 30, 679–687

(13) Huidobro, A.L., Ruperez, F.J., & Barbas, C. (2007) J. Pharm.


(14) Saavedra, L., Huidobro, A.L., Garcia, A., Cabanelas, J.C.,

Gonzalez, M.G., & Barbas, C. (2006) Electrophoresis 27,

2360–2366

(15) Nudelman, N.S., & Gallardo Cabrera, C. (2002) J. Pharm.


(16) Nudelman, N.S., & Cabrera, C.G. (2002) J. Pharm. Sci. 91,

1274–1286

(17) Allen, L.V., Jr, & Erickson, M.A., 3rd (1998) Am. J. Health

Syst. Pharm. 55, 1915–1920

(18) Caira, M.R., Easter, B., Honiball, S., Horne, A., &

Nassimbeni, L.R. (1995) J. Pharm. Sci. 84, 1379–1384

(19) Hancu, G., Gaspar, A., & Gyeresi, A. (2007) J. Biochem.

Biophys. Methods 69, 251–252

(20) Adams, A.I., & Bergold, A.M. (2001) J. Pharm. Biomed.

Anal. 26, 505–508

(21) Chen, D., Jiang, S., Chen, Y., & Hu, Y. (2004) J. Pharm.


(22) He, L., Feng, F., & Wu, J. (2005) J. Chromatogr. Sci. 43,

532–535

(23) Patel, J., Spencer, E.P., & Flanagan, R.J. (1996) Biomed.

Chromatogr. 10, 351–354

(24) Chen, X., Duan, X., Dai, X., & Zhong, D. (2006) Rapid

Commun. Mass. Spectrom. 20, 2483–2489

(25) ICH, Q1B (1996) Stability Testing: Photostability Testing of

New Drug Substances and Products, International

Conference on Harmonization, Geneva, Switzerland

(26) The United States Pharmacopeia (2000) USP-24, NF-19,

United States Pharmacopeial Convention, Inc., Rockville,

MD, pp 2149–2151


alprazolam and sertraline in combined dosage forms

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