Development and validation of spectrophotometric

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Jul 8, 2013 - Development and validation of spectrophotometric and HPTLC meth- .... general procedures for each method described under construction.

http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, 2014; 40(9): 1190–1198 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.810634

RESEARCH ARTICLE

Development and validation of spectrophotometric and HPTLC methods for simultaneous determination of rosiglitazone maleate and metformin hydrochloride in the presence of interfering matrix excipients Hoda Mahgoub1,2, Rasha M. Youssef2, Mohamed A. Korany2, Essam F. Khamis2, and Miranda F. Kamal3 1

Faculty of Pharmacy, Department of Pharmaceutical Chemistry, King Abdulaziz University, Jeddah, Saudi Arabia, 2Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria, Egypt, and 3Faculty of Pharmacy and Drug Manufacturing, Department of Analytical and Pharmaceutical Chemistry, Pharos University in Alexandria, Somouha, Alexandria, Egypt Abstract

Keywords

Two simple methods have been developed and validated for the simultaneous determination of rosiglitazone maleate (ROS) and metformin hydrochloride (MET) in synthetic mixtures and coated tablets in a ratio of 1:250 (ROS:MET). The first method was a spectrophotometric one. The minor component, ROS was determined by measuring the values of absorbance at max 312 nm and the D1 amplitudes at 331 nm where MET shows no absorption contribution. However, absorbance interferences from tablet excipients were successfully corrected by D1 at 331 nm zero-crossing technique. Study of spectral interference from tablet excipients was included in the text. Standard curves for Amax and D1 methods were in the concentration range 20.0–80.0 mg mL1. The major component, MET was determined both in binary mixtures and tablets by measuring its Amax at 236 nm. Extensive dilution eliminated any absorption contribution from the coexisting ROS or tablet matrix. Standard curves showed linearity in the concentration range 4.0–12.8 mg mL1. The second method was based on high performance thin layer chromatography (HPTLC) separation of the two drugs followed by densitometric measurements of their spots at 230 nm. The separation was carried out on Merck HPTLC aluminium sheets of silica gel 60 F254 using methanol:water:NH4Cl 1% w/v (5:4:1 v/v/v) as the mobile phase. Linear calibration graphs of peak area values were obtained versus concentrations in the range of 0.4–2.0 mg band1 and 20.0–100.0 mg band1 for ROS and MET, respectively. According to International Conference on Harmonisation (ICH) guidelines, different validation parameters were verified for the two methods and presented.

Coated tablets, HPTLC, metformine hydrochloride, rosiglitazone maleate, spectrophotometry

Introduction Type 2 diabetes disorder is characterized by disturbed insulin production leading to high blood glucose level. In addition, insulin resistance (i.e. reduced responsiveness to normal circulating insulin concentrations) is a common feature of this disorder. To provide an efficient control, combination therapy is often used. Rosiglitazone maleate (ROS) and metformin hydrochloride (MET) are widely used in a combination to treat type 2 diabetes patients. ROS belongs to the family of thiazolidinediones while MET is a biguanide. Both are potent oral antihyperglycemic agents. ROS works as an insulin sensitizer, by binding to the peroxisome proliferator-activated receptors in fat cells and making the cells more responsive to insulin. MET is the first-line drug of

Address for correspondence: Hoda Mahgoub, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, King Abdulaziz University, Jeddah 21589, P.O. Box 80260, Saudi Arabia. Tel: 203 4871317. Fax: 203 4873273. E-mail: [email protected]

History Received 6 January 2013 Revised 14 April 2013 Accepted 28 May 2013 Published online 8 July 2013

choice for the treatment of type 2 diabetes, in particular, in overweight and obese people and those with normal kidney function. Metformin works by suppressing glucose production by the liver. Metformin activates AMP-activated protein kinase (AMPK), an enzyme that plays an important role in insulin signaling, whole body energy balance and the metabolism of glucose and fat1. ROS and MET are combined in a ratio of 1:250 in tablets indicated for treatment of patients suffering from type 2 diabetes. Few methods have been reported for the determination of their binary mixture in its pharmaceutical preparation including RP-HPLC2, HPLC3,4, spectrophotometry4 and CZE5. Furthermore, this mixture has been determined in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization6, by gradient LC with UV detection7 and by HPLC-ESI-MS8. In the present work, two simple methods have been developed and validated for the simultaneous determination of ROS and MET in synthetic mixtures and tablet form. The objective of the work is to overcome two problems; the extremely high

Simultaneous determination of rosiglitazone and metformin

DOI: 10.3109/03639045.2013.810634

concentration ratio of the two drugs in tablets and the interference from the tablet excipients particularly on the minor component (ROS). The first method is a spectrophotometric one, which involves simple measurements of absorbance and derivative zerocrossing values relying on the capability of the latter to overcome the spectral overlap from coated tablet matrix. The second method is a chromatographic one based on high performance thin layer chromatography (HPTLC) separation of the two drugs followed by densitometric measurements. Validation of the proposed methods according to US Pharmacopeial Convention (USP)9 validation elements and International Conference on Harmonisation (ICH)10 guidelines is presented.

Experimental Apparatus A Thermo-Spectronic UV-Vis spectrophotometer connected to Harvest computer system was used. The absorption spectra were measured in 1-cm quartz cells. The absorbance data were processed using Excel software. HPTLC plates (20  10 cm, aluminum plates with 250 mm thickness precoated with silica gel 60 (F254) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100 mL Camag microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Camag Linomat IV (Switzerland) applicator. The slit dimension was kept at 6  0.2 mm and 20 mm s1 scanning speed was employed. Ascending development of the mobile phase was carried out in 20  10 cm twin trough glass chamber (Camag, Switzerland). Densitometric scanning was performed at 230 nm on a Camag TLC scanner III operated in the reflectance absorbance mode and controlled by CATS software (V 3.15, Camag). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm.

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Ammonium chloride (El-Nasr Chemical Ind. Co., Suez, Egypt) prepared as 1% w/v aqueous solution, 1 g of ammonium chloride in 100 mL distilled water, sonicate 2 min to dissolve. Methanol (Aldrich, Munich, Germany). Standard solutions Standard solutions of 0.25 mg mL1 of ROS and 10 mg mL1 of MET were prepared in methanol. Chromatographic conditions For optimal sensitivity of HPTLC method, solutions of testing samples and standard were applied to TLC plates as bands rather than spots. Bands were 6 mm long and a 20 -mL sample was applied to each band. The bands were separated by a distance of 10 mm apart and 15 mm from the bottom of the plate. The chromatographic tank was saturated with the mobile phase in a usual mode for at least 30 min. The plate was developed in the ascending way with methanol:water:NH4Cl 1% w/v (5:4:1 v/v/v) as the mobile phase. After developing over a distance of 11 cm, the plate was air-dried and scanned for the two drugs at 230 nm, under the following conditions (mode, absorbance; lamp, deuterium; band width, 6 mm; scanning speed, 20 mm/s; slit dimensions, 5.00  0.45 mm). Construction of calibration graphs Spectrophotometric methods

Reagents and materials

Amax and D1 methods for ROS determination. Various aliquots from ROS standard solution were diluted with methanol to give working standard solutions with final concentration range stated in Table 1. The first derivative spectra (D1) of each working standard solution were scanned in a range of 200–400 nm against methanol as blank. The values of absorbance at max 312 nm and the D1 amplitudes at 331 nm (zero-crossing of tablet excipients) were measured and found proportional to the concentration of ROS.

ROS was obtained from Apex Pharma (New Cairo, Egypt) while MET was provided by Pharaonia Pharmaceuticals (Alexandria, Egypt). Avandamet tablets (GlaxoSmithKline, Egypt) contain 2 mg ROS and 500 mg MET per tablet. The tablet core contains sodium starch glycollate, hypromellose (E464), microcrystalline cellulose (E460), lactose monohydrate, povidone (E1201) and magnesium stearate while the film coating contains hypromellose (E464), titanium dioxide (E171), macrogol and iron oxide red (E172).

Amax method for MET determination. An appropriate volume from MET standard solution was diluted with methanol to obtain intermediate standard solution 40 mg mL1 of MET. Various aliquots from the intermediate standard solution were diluted with distilled water to give working standard solutions with final concentration range stated in Table 1. The values of absorbance at max 236 nm were measured against water as blank and found proportional to the concentration of MET.

Table 1. Characteristic parameters for the regression equations of the proposed methods for the simultaneous determination of ROS and MET. Spectrophotometry

HPTLC

ROS Parameters 1

Linearity range (mg mL ) LOQ (mg mL1) LOD (mg mL1) Intercept (a) Slope (b) Correlation coefficient (r) Sa Sb Sy/x Sb2 Sb%

(Both scanned at 230 nm)

Amax (312 nm)

D1 (331 nm)

MET Amax (236 nm)

ROS

MET

[20, 37.5, 45, 55 & 80] 20.00 6.66 4.30  103 1.10  102 0.9994 1.10  102 2.10  104 1.00  102 4.50  108 1.91

[20, 37.5, 45, 55 & 80] 20.00 6.66 9.30  104 1.10  103 0.9996 8.90  104 2.10  105 5.50  104 4.40  1010 1.91

[4, 8, 10, 12 & 12.8] 4.00 1.30 3.70  103 7.26  102 0.9998 6.70  103 6.80  104 4.80  103 4.60  107 9.30  102

[0.4, 0.6, 1, 1.5 & 2]* 0.40* 0.13* 825.57 4191.15 0.9988 141.54 120.63 164.23 14 553.20 2.88%

[20, 40, 60, 80 & 100]* 20.00* 6.67* 33 884.87 1094.19 0.9991 1271.65 18.75 1185.82 351.54 1.71%

*Concentration in mg band1. Sa is standard deviation of intercept, Sb is standard deviation of slope and Sy/x is standard deviation of residuals.

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HPTLC method for ROS and MET determination

Results and discussion

A 20-mL volume of each standard solution was applied to TLC plate. The plate was developed in the previously described mobile phase (‘‘Chromatographic conditions’’ section). The peak area values for each drug were plotted against the corresponding concentrations (Table 1). The concentrations of ROS and MET were computed from the corresponding calibration graphs.

Spectrophotometric methods

Analysis of tablets Twenty tablets containing ROS and MET as active ingredients were weighed and finely powdered. Portions of the powder equivalent to 2 mg of ROS and 500 mg of MET were weighed and accurately transferred into a 100-mLvolumetric flask using about 70 mL of methanol. The sample solution was sonicated for 30 min and diluted to volume with methanol. The solution was filtered and the filtrate was centrifuged2,3 twice, each at 5000 rpm for 10 min. Suitable dilution for the supernatant solution was carried out with methanol for the spectrophotometric determination of ROS and the HPTLC determination of ROS and MET. Further dilution (500-fold) of the diluted supernatant was carried out with water for the spectrophotometric determination of MET. The general procedures for each method described under construction of calibration graphs were followed and the concentrations of each of ROS and MET were calculated.

Amax and D1 methods for ROS determination As can be seen from Figure 1(A), the minor component, ROS, can be assayed by applying the conventional Amax method at 312 nm without any interference from the major component, MET. Analysis of ROS in synthetic mixtures with MET gave successful results. However, measuring absorbance at 312 nm for ROS determination in tablets, gave positive erroneous result. Such high recovery revealed an irrelevant absorption from the formulation matrix (‘‘Reagents and materials’’ section). Accordingly, a study of each excipient individually in this film coated tablet was

(A) 1.9 1.7 1.5 1.3 1.1 A

0.9 0.7 0.5 0.3 0.1 -0.1 200

220

240

260

280

300

320

340

360

380

400

Wavelength (nm) (B)

1 0.9 0.8 0.7 0.6 0.5

A

Figure 1. UV absorption spectra of (A) 20 mg mL1 ROS (black line) and 5000 mg mL1 MET (gray line) in methanol and (B) 0.04 mg mL1 ROS and 10 mg mL1 MET in distilled water.

ROS and MET are combined in tablets formulation in a ratio of 1: 250. The UV absorption spectra for 20 mg mL1 ROS and 5000 mg mL1 MET solutions in methanol were scanned in the range of 200–400 nm (Figure 1A). The spectra showed high overlap between the two drugs in the region of 200–280 nm. At 312 nm, ROS gives maximum absorption band, while MET showed no absorption above 280 nm. The two drug solutions in Figure 1(A) were 500-fold diluted and rescanned in the same wavelength range (Figure 1B). This figure revealed an absorption maximum for MET at 236 nm. The extensive dilution of ROS solution eliminated its absorption over the whole wavelength range.

0.4 0.3 0.2 0.1 0 -0.1 200

220

240

260

280 300 320 Wavelength (nm)

ROS

340

MET

360

380

400

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carried out. All of these excipients are non-absorbing in the UV-Vis region except povidone which absorbs below 225 nm, while titanium dioxide and iron oxide red absorb above 300 and 400 nm, respectively11. Figure 2(A) shows the absorption spectra of ROS and simulated tablet excipient solutions in methanol. It explains the spectral interference of the excipient matrix in the region of ROS measurement (280–350 nm). To overcome such spectral overlap, the derivative spectrophotometric technique12,13 was employed. The main instrumental parameter affecting the shape of derivative spectra is the wavelength increment over which the derivative spectrum is obtained (D). This parameter needs to be optimized to obtain a well-resolved large peak, good selectivity and increased sensitivity for the determination14. Generally, the noise level decreases with an increase in D, thus decreasing the fluctuation in the derivative spectrum. However, if the value of D is too highly increased, the spectral resolution is decreased. Therefore, the optimum value of D should be determined by taking into account the noise level and the resolution of the spectrum. The D parameter was optimized to give a well-resolved peak and to eliminate the present interference. Several values of D were tested and D ¼ 3 nm was found optimal. Figure 2(B) shows the corresponding D1 spectra for ROS and tablet excipient solutions in methanol. As can be seen, ROS could be quantified by measuring its D1 amplitude at 331 nm where the excipients showed no contribution (zero-crossing point).

Amax method for MET determination The major component, MET has been successfully determined both in binary mixtures and tablets by measuring its absorbance at max 236 nm (Figure 1B). Methanolic solutions of the synthetic mixtures or the tablet, after measurement of ROS, were 500-fold diluted using distilled water for MET measurement. Methanol was used to dissolve high concentration of MET. Although the stock was prepared in methanol, the step dilution was made with distilled water for economic purposes. Extensive dilution eliminated any absorption contribution from the coexisting ROS or tablet matrix. HPTLC method The experimental conditions for HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible compact, flat, dark fluorescence quenched bands against a bright green background for the simultaneous determination of ROS and MET in mixtures with high concentration ratio (1:250). Both pure drugs were spotted on TLC silica plates. Different solvent systems were tried for their separation. The greatest differences between the Rf values of the two compounds (0.7  0.05 for ROS and 0.4  0.02 for MET), with minimum tailing, were obtained by using a mobile phase consisting of methanol:water:NH4Cl 1% w/v (5:4:1 v/v/v). Figure 3 shows that the two compounds could be separated with good resolution with sharp and symmetrical peaks. Well-defined bands for both drugs were obtained when the chamber was saturated with the mobile

(A) 0.5

0.4

A

0.3

0.2

0.1

0 280

290

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310

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330

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370

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390

400

Wavelength (nm) (B) 0.02 0.01 0 300

310

320

330

-0.01 D1

Figure 2. Zero-order (A) and first derivative (B) spectra of 20 mg mL1 standard ROS (—) and simulated tablet excipients (- - -) in methanol.

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-0.02 -0.03 -0.04 -0.05 Wavelength (nm)

340

350

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Figure 3. A typical HPTLC chromatogram of 0.4 mg band1 ROS and 100 mg band1 MET in their mixture using 20-mL band volume.

Figure 4. Three-dimensional overlay of chromatograms obtained from (tracks 1–8, starting from left side) MET standards (in concentration range 20–100 mg band1), followed by (tracks 9–16) ROS standards (in concentration range 0.4–2.0 mg band1), then (tracks 17, 18) their synthetic mixtures in the ratio 250:1, respectively, and finally (track 19) tablet. All tracks were scanned at 230 nm.

phase for at least 30 min at room temperature. Different scanning wavelengths were tried and 230 nm was chosen as a common wavelength to match the high concentration ratio of the drugs present in the formulation. The optimum band width chosen, taking into consideration the range of concentrations applied and number of tracks, was 6 mm. Figure 4 shows a three-dimensional overlay for typical HPTLC chromatograms obtained from standard MET (the major component), standard ROS (the minor one), their synthetic mixtures (MET:ROS in a ratio 250:1) and pharmaceutical preparation. All tracks were scanned efficiently at the same wavelength (230 nm).

According to ICH, at least five concentrations must be used. Under the described experimental conditions, the graphs obtained by plotting Amax and D1 values for ROS and Amax values for MET and peak areas of both drugs versus concentrations (in the ranges stated in Table 1) showed linear relationships. Using the method of least squares, regression equations, correlation coefficients and variances around the slopes (Sb2 ) for different calibration data were calculated and presented in Table 1. The intercepts for linear equations of HPTLC method are not zero, as expected, compared with spectrophotometric procedures.

Validation of the proposed procedures

Limits of detection (LOD) and limits of quantitation (LOQ)

The methods were validated according to USP9 validation elements and ICH10 guidelines.

For spectrophotometric methods, LOD and LOQ were accurately calculated. For HPTLC, LOD is considered as the concentration which has a signal-to-noise ratio of 3:1. LOQ is the concentration which has a signal-to-noise ratio of 10:1. Using the proposed methods, LOD and LOQ for each compound were calculated and are presented in Table 1. Their values indicate good accuracy and high sensitivity.

Linearity and range The linearity of the proposed methods was evaluated by analyzing series of different concentrations of each of ROS and MET.

Simultaneous determination of rosiglitazone and metformin

DOI: 10.3109/03639045.2013.810634

Accuracy and precision In order to assess the accuracy and precision of the proposed methods for assaying each drug in laboratory-made mixtures with different proportions, five determinations (each in triplicate) were carried out. Satisfactory recoveries with small relative errors were obtained (Table 2), which indicates high accuracy of the proposed methods. The values of RSD% were less than 2, indicating good precision of the proposed methods (Table 2). Selectivity Method selectivity was checked by analyzing synthetic mixtures containing different ratios of both drugs, where good percentage recoveries were obtained indicating that they did not interfere with each other (Table 2). In addition, the application of the proposed methods for the simultaneous determination of the two drugs in dosage form, without interference from the excipients, clearly demonstrates the selectivity of these methods (Table 3). Specificity The specificity15–17 of the HPTLC method was ascertained by analyzing each of the two drugs in the standard and sample solutions. The spots for ROS and MET in the sample were confirmed by comparing the Rf and spectra of the spots with those

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of both standards, respectively. The peak purity of each of MET and ROS was assessed by comparing their spectra at three different levels; peak start (S), peak apex (M) and peak end (E) positions of the spots, i.e. r2 (S, M) ¼ 0.9999 and r2 (M, E) ¼ 0.9999 for both drugs. Good correlations (r ¼ 0.9999 and 0.9998) were obtained between the standard and the sample for ROS and MET, respectively (Figure 5). Robustness The robustness of the proposed methods was evaluated by analyzing ROS and MET at three concentration levels as cited in Table 4. For spectrophotometric methods, the parameters studied were different models of spectrophotometers and different lots of the used solvent (methanol). However for HPTLC method, the parameters studied were mobile phase composition, mobile phase volume, duration of saturation and time from chromatography to scanning. It was found that variation in the above parameters had no significant influence on the determination of ROS and MET using the proposed methods. The low values of RSD% of Amax and D1 values (for spectrophotometric methods), and the low RSD% of peak areas along with nearly unchanged retardation factor (Rf) (for HPTLC method) obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed methods (Table 4).

Table 2. Evaluation of the accuracy and precision of the proposed methods for the determination of ROS and MET in synthetic mixtures. Spectrophotometric method ROS Mean% recovery  SD

a

MET b

RSD%

ROS:MET (mg mL1)

Amax (312 nm)

20:5000 25:5000 20:2500 30:5000 40:2500

99.90  1.12 98.90  1.10 101.00  0.03 100.50  1.05 99.80  0.84 98.90  0.85 100.60  1.00 99.60  1.00 98.70  1.00 99.00  0.90

Er%

c

Amax (236 nm)

Amax (312 nm) D1 (331 nm) Amax (312 nm) D1 (331 nm) Mean% recovery  SDa RSD%b

D1 (331 nm)

1.12 0.03 0.84 0.99 1.01

1.11 1.05 0.86 1.00 0.91

0.10 1.00 0.20 0.60 1.30

1.10 0.50 1.10 0.40 1.00

99.70  1.09 101.60  0.50 101.10  0.78 100.20  0.86 98.60  0.90

1.09 0.49 0.77 0.86 0.91

Er%c 0.30 1.60 1.10 0.20 1.40

HPTLC method ROS:MET (mg band1)

ROS Mean% recovery  SD

a

c

RSD%

Er%

1.08 0.92 0.89 1.21 0.88

0.98 1.80 1.80 1.30 0.10

100.98  1.09 101.80  0.94 98.02  0.87 98.70  1.19 100.10  0.88

0.4:100 0.4:50 0.6:20 0.7:50 2.0:100

MET b

Mean% recovery  SDa RSD%b 100.70  0.88 100.60  0.91 101.10  0.89 99.20  0.86 99.60  0.90

0.87 0.91 0.88 0.87 0.90

Er%c 0.70 0.60 1.10 0.80 0.40

a

Mean  standard deviation of five determinations. Percentage relative standard deviation. c Percentage relative error. b

Table 3. Assay results for the determination of ROS and MET in tablets using the proposed methods. Pharmaceutical preparation

Recovery%  SDa ROS D1 (331 nm)

AvandametÕ tablets tc Value Fc Value a

b

100.30  0.25 1.06 1.30

MET HPTLC

Amax (236 nm)

101.50  0.89

100.10  0.40

HPTLC 100.60  0.98 1.90 3.38

Mean  standard deviation of five determinations. Avandamet tablets labeled to contain 2 mg ROS and 500 mg MET per tablet (GlaxoSmithKline.-the Batch No. is 091699A). Theoretical values of t and F at p ¼ 0.05 are 2.31 and 6.39, respectively.

b c

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Figure 5. Spectra illustrating peak purity of (A) MET and (B) ROS, each is obtained from corresponding standards and tablet.

Stability in solutions The stability of ROS and MET in their solutions during the analytical procedures was studied. Solutions of the two drugs were prepared and stored at room temperature for 0.5, 1.0 and 2.0 h then they were analyzed using the proposed methods. Since no significant changes, in D1 and A values were obtained and no additional peaks were found in the HPTLC chromatograms, throughout the analysis time, thus the two drugs are stable in solutions at room temperature (25  C) for at least 4 h. The prepared stocks are stable for five days, on condition of storage in refrigerator (3  C).

Furthermore, for HPTLC, spot stability is very important. The time for which the sample is left to stand prior to chromatographic development can influence the stability of separated spots and is required to be investigated for validation18,19. Two-dimensional chromatography, using the same solvent system, was used to find out any decomposition occurring during spotting and development. If the decomposition occurred during development, peak(s) of decomposition product(s) would be obtained for the analyte both in the first and second directions of the run. Since no decomposition was observed during spotting and development using the proposed conditions, this indicated the stability of drugs in solutions.

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Table 4. Evaluation of the robustness of the proposed methods. Spectrophotometrica methods

Parameter tested

SD (RSD%) ROS

- Spectrophotometer of different models - Methanol of different lots

b

MET

Amax (312 nm)

D1 (331 nm)

Amax (236 nm)

0.91 (0.90) 1.01 (1.60)

1.40 (1.00) 1.50 (1.10)

0.92 (0.91) 0.56 (0.60)

HPTLCc method ROS

- Mobile phase composition Methanol:water:NH4Cl 1% w/v (4.5:4.5:1, 5:4:1 & 5.5:3.5:1 v/v/v) - Mobile phase volume (25, 30 & 35 mL) - Duration of saturation (20, 30 & 40 min) - Time from chromatography to scan (5, 10 & 20 min)

MET

SD (RSD%) of peak areas

Rf  SD

SD (RSD%) of peak areas

Rf  SD

113 (1.60)

0.70  0.04

1400 (1.70)

0.40  0.01

150 (1.80)

0.70  0.13

1295 (1.30)

0.40  0.12

101 (1.10)

0.70  0.09

1340 (1.20)

0.40  0.08

123 (0.50)

0.70  0.02

1276 (1.00)

0.40  0.04

Average of three concentrations 20, 40, 60 mg mL1 and 5, 10, 12 mg mL1 for ROS and MET, respectively. A ThermoSpectronic Helios Alpha (UK) UV–vis spectrophotometer connected to Harvest computer system and UV-1800 Shimadzu Spectrophotometer, Shimadzu Co, Kyoto, Japan. c Average of three concentrations 0.4, 0.8, 2.0 mg band1 and 20, 60, 100 mg band1 for ROS and MET, respectively. a

b

Analysis of tablets

References

The methods were applied to the determination of ROS and MET in AvandametÕ tablets. Satisfactory results (Table 3) were obtained for the recovery of both drugs and were in good agreement with the labeled claims. No significant differences were found between the results obtained by both procedures for the same batch at the 95% confidence level (Student’s t- and F-ratio tests).

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Conclusion The proposed methods provide accurate and reproducible results for the simultaneous determination of ROS and MET in binary mixture. Both methods have successfully overcome two major problems in the assay of this mixture. First of them, the ratio of concentrations of ROS:MET, that reaches up to 1:250 in their pharmaceutical preparation. The second one is the detected interference from the coated tablet excipients, mainly in the spectrophotometric region of the minor component (ROS) determination. The HPTLC method has proved to be more selective and rapid than the spectrophotometric method. This optimized HPTLC procedure is just a simple one, using minimum volume of solvents, compared to the other separation techniques. Furthermore, an extremely large number of samples can be analyzed at the same time. However, the spectrophotometric method retains the advantages of lower cost, higher environmental protection and easier applicability. The present methods have been successfully applied to the assay of commercial tablets and content uniformity test without any preliminary separation. Both methods have been completely validated and are, therefore, suitable for use in quality control laboratories where economy and time are essential.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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