Stability-indicating HPTLC method for simultaneous determination of nateglinide and metformin hydrochloride in pharmaceutical dosage form

Abstract

A stability indicating high performance thin layer chromatography (HPTLC) method was developed and validated for determination of two anti-diabetic drugs, nateglinide and metformin hydrochloride in co-formulations. Study was performed on pre-coated silica gel HPTLC plates using chloroform:ethyl acetate:acetic acid (4:6:0.1 v/v/v) as the mobile phase. A TLC scanner set at 216 nm was used for direct evaluation of the chromatograms in the reflectance/absorbance mode. Method was validated according to ICH guidelines. The correlation coefficients of calibration curves were found to be 0.996 and 0.995 in the concentration range of 200–2400 and 500–3000 ng band⁻¹ for nateglinide and metformin, respectively. The method had an accuracy of 99.72% for nateglinide and 100.08% for metformin hydrochloride. The method had the potential to determine these drugs simultaneously from dosage forms without any interference of the tablets excipients. Nateglinide and metformin hydrochloride were also subjected to acid, base, oxidation, wet, heat and photo-degradation studies. The degradation products obtained were well resolved from the pure drugs with significantly different R_f values. As the method could effectively separate the drugs from its degradation products, it can be used for stability-indicating analysis.

1. Introduction

Diabetes mellitus is difficult to control with a single oral hypoglycaemic agent and the rate of mono therapy failure is high. Hence combination therapy with complementary classes of drugs that act on different aspects of glycemic control would be expected to be an effective strategy for the control of diabetes. Nateglinide (Fig. 1a) is an amino-acid derivative which lowers the blood glucose levels and stimulates insulin secretion. Chemically, it is formulated as (−)-N-[(trans-4-isopropylcyclohexyl) carbonyl-d-phenylalanine salt and is used in the treatment of type two diabetes mellitus (USP, 2010). Metformin hydrochloride (Fig. 1b) is a biguanide hypoglycaemic agent used in the treatment of non-insulin-dependent diabetes mellitus. It is effective in glycemic control and decreases the intestinal absorption of glucose. It is chemically formulated of N,N-dimethylimidodicarbonimidicdiamide hydrochloride (IP, 2010; BP, 2010). These two drugs are formulated as a tablet in a combined formulation.

Figure 1.

Chemical structure of nateglinide (a) and metformin hydrochloride (b).

Validated assays have been reported for each drug individually. For analysis of nateglinide from human plasma, high performance liquid chromatographic analysis with UV detection (Steffen et al., 2003), LC/MS (Varanasi et al., 2008), coumarin-type fluorescent detection (Malli et al., 2007), and HPTLC/TLC determination (Gumieniczek et al., 2009) was reported. Spectrophotometric techniques have also been reported for determination of nateglinide in bulk and tablet dosage forms (Jain et al., 2009; Rastogi et al., 2009). High performance liquid chromatography for metformin individually in human plasma (Amini et al., 2005; Yuen and Peh, 1998; Cheng and Chou, 2001) and in combination with other drugs in plasma and pharmaceutical dosage form (Önal, 2009; Zhang et al., 2007; Lakshmi et al., 2009), LC/MS analysis (Mistri et al., 2007), UV spectrophotometric quantitation methods in bulk and pharmaceutical formulation (Arayne et al., 2009) and HPTLC methods (Ghassempour et al., 2006) have been reported.

There are no reported assay methods that permit the simultaneous quantification of nateglinide and metformin hydrochloride in combined dosage form. The aim of the present work was to develop and validate a new simple, rapid, selective, cost effective and stability indicating HPTLC method for simultaneous determination of nateglinide and metformin hydrochloride in pharmaceutical formulation.

2. Experimental

2.1. Materials

Analytical pure samples of nateglinide (C.C. Lab., Torrent Pharmaceutical Ltd., Indrad, India) and metformin hydrochloride (USV Limited, Baddi, Himachal Pradesh, India) were used in the study. The pharmaceutical dosage form used in this study was Glinate-MF (Glenmark Pharmaceutical Limited, Baddi, Himachal Pradesh, India) procured from the local market and labelled to contain 60 mg of nateglinide and 500 mg of metformin hydrochloride per tablet. The solvents and chemicals used in the study were of AR grade (Research Lab., Nashik, Maharastra, India).

2.2. Instrumentation

Microsyringe (Linomat syringe 659.004, Hamilton-Bonaduz Schweiz, Camag, Switzerland), pre-coated silica gel 60 F-254 aluminium plates (10 × 10 cm, 250 μm thickness; Merck, Germany), Linomat 5 applicator (Camag, Muttenz, Switzerland), twin trough chamber (20 × 10 cm; Camag, Muttenz, Switzerland), saturation pad (Camag, Muttenz, Switzerland), UV chamber (Camag, Muttenz, Switzerland), TLC scanner III (Camag, Muttenz, Switzerland), winCATS version 1.4.0 software (Camag, Muttenz, Switzerland) were used in this study. Microsoft excel was also used to treat data statistically.

2.3. Preparation of standard solutions

Standard stock solutions were prepared by dissolving separately 10 mg of nateglinide and metformin hydrochloride each in 10 mL of methanol to obtain a concentration of 1000 μg mL⁻¹. The standard stock solutions were suitably diluted methanol to obtain the working standard solutions of both nateglinide and metformin hydrochloride.

2.4. Preparation of sample solutions

Twenty tablets (Glinate-MF, labelled to contain 60 mg of nateglinide and 500 mg of metformin hydrochloride per tablet, Glenmark Pharmaceutical Limited, Baddi, Himachal Pradesh, India) were weighed and crushed to fine powder. An accurately weighed powder sample equivalent to 100 mg of metformin hydrochloride was weighed, transferred to a 100 mL volumetric flask and volume made up to about 70 mL with methanol. The solution was sonicated for about 30 min, then diluted to volume with the same solvent and filtered through Whatman filter paper No. 42. Working sample solutions were freshly prepared by diluting suitable volumes of the stock sample solution with methanol.

2.5. Optimized chromatographic conditions

Suitable volumes of standard and sample solutions (μL) were applied to the HPTLC plates, 10 mm from the bottom and 10 mm from the side edges in the form of bands or streaks with band length of 6 mm. The mobile phase consisting of chloroform:ethyl acetate:acetic acid (4:6:0.1 v/v/v) was used in each chromatographic run. Ascending development technique was carried out in twin trough chambers. The optimized chamber saturation time for the mobile phase was 20 min at room temperature (25 ± 2 °C) that was assisted by saturation pads. The distance covered by the solvent front was 8 cm, which took about 15 min. The spots were scanned using the TLC scanner 3 in the reflectance/absorbance mode at 216 nm and all measurements were operated by winCATS software. Concentrations of the separated compounds were determined from the intensity of reflected light and peak areas were used for evaluation.

2.6. Analysis of marketed formulation

The tablet sample solutions were prepared as discussed above. Suitable working sample solutions (2.5 μL) containing metformin hydrochloride and nateglinide in the concentration ratio of 1:8.33 (2500 ng:300 ng of metformin hydrochloride and nateglinide, respectively) were prepared, applied on HPTLC plates and analysed under the optimized chromatographic conditions.

2.7. Method validation

The method was validated in compliance with ICH guidelines (ICH, 1994, 1996). The following parameters were used for validation of the developed method.

2.7.1. Linearity

Linear relationship between peak area and concentration of the drugs was evaluated over the concentration range expressed in ng band⁻¹ by making five replicate measurements in the concentrations range of 200–2400 ng band⁻¹ for nateglinide and 500–3000 ng band⁻¹ for metformin hydrochloride, respectively.

2.7.2. Precision

Precision of the developed method was studied by performing repeatability and intermediate precision studies. The sample application and measurement of peak area was determined by performing six replicate measurements of the same band using a sample solution containing 2500 ng band⁻¹ of metformin hydrochloride and 300 ng band⁻¹ of nateglinide each.

2.7.3. Recovery studies

Recovery studies were carried out by spiking three different known amounts of the standard substances to the drug product (standard addition method). Hence, 240, 300 and 360 ng band⁻¹ of nateglinide and 2000, 2500 and 3000 ng band⁻¹ of metformin hydrochloride were spiked to the dosage form that contained 300 and 2500 ng band⁻¹ of nateglinide and metformin hydrochloride, respectively, after sample dilution.

2.7.4. Limit of detection (LOD) and limit of quantitation (LOQ)

The limits of detection and quantification of the developed method were calculated from the standard deviation of the y-intercepts and slope of the calibration curves of nateglinide and metformin hydrochloride using the formulae as given below.

$$\begin{array}{cc}\hfill & \text{Limits of Detection}=3\alpha /S\hfill \\ \hfill & \text{Limits of Quantification}=10\alpha /S\hfill \end{array}$$

where α is the standard deviation of the y-intercepts and S is the slope of the calibration curve.

2.7.5. Robustness

The effect of deliberate variations in method parameters like the composition of the mobile phase, volume of the mobile phase, time from spotting to development and time from development to scanning were evaluated in this study. The effect of these changes on both the R_f values and peak areas was evaluated by calculating the relative standard deviations (RSD) for each parameter.

2.7.6. Specificity

Peak purity of both nateglinide and metformin hydrochloride was assessed to evaluate the specificity of the method. The sample and standard bands were scanned at three different levels, i.e., peak start (S), peak apex (M), and peak end (E) positions. The specificity of the method was also determined by performing forced degradation studies. Standard stock solutions (1000 μg mL⁻¹) of nateglinide and metformin hydrochloride were employed in the study.

2.8. Stability studies

To evaluate the stability indicating properties of the developed HPTLC method, forced degradation studies were carried out in accordance to the ICH guidelines. The standard drugs were subjected to acid, base, oxidation, wet heat, dry heat and photo-degradation studies.

2.8.1. Acid-induced degradation study

HCl (0.001 M, 5 mL) was added separately to 5 mL methanolic stock solutions of nateglinide and metformin hydrochloride in 25 mL volumetric flasks. The mixtures were refluxed at 40 °C for 30 min and the volume was made up with methanol (200 μg mL⁻¹). The forced degradation was performed in the dark to exclude the possible degradation effect of light. The resulting solutions (12.5 μL, 2500 ng band⁻¹ for metformin hydrochloride and 1.5 μL, 300 ng band⁻¹ for nateglinide) were applied to TLC plates and the chromatograms were run as described above.

2.8.2. Base-induced degradation study

For the base degradation study, NaOH (0.001 M, 5 mL) was added separately to 5 mL methanolic stock solutions of nateglinide and metformin hydrochloride in a 25 mL volumetric flask. The mixtures were refluxed at 40 °C for 30 min and the volume was made up with methanol (200 μg mL⁻¹). The samples were then applied and analysed as described in the acid induced degradation study.

2.8.3. Hydrogen peroxide-induced (oxidation) degradation study

H₂O₂ (1.5%, 5 mL) was added separately to 5 mL methanolic stock solutions of nateglinide and metformin hydrochloride in 25 mL volumetric flasks and the volume was made up with methanol (200 μg mL⁻¹). The sample solutions were refluxed at 40 °C for 30 min. The samples were then applied and analysed as described in acid induced degradation study.

2.8.4. Wet degradation study

For wet heat degradation study, 5 mL stock solutions of each drug were transferred to 25 mL volumetric flasks separately. To each 5 mL methanol was added and the samples were refluxed at 40 °C for 30 min. The volume was made up with methanol (200 μg mL⁻¹) and then applied and analysed under the optimized chromatographic conditions.

2.8.5. Heat degradation study

For dry heat degradation study, the standard powder drugs were placed in an oven at 50 °C for 24 h. Appropriate dilutions were prepared in methanol and then analyzed under the optimized chromatographic conditions.

2.8.6. Photo-degradation study

For the photo-degradation study, the standard powder drugs were exposed to UV light in a photo-stability chamber for 24 h. Appropriate dilutions were prepared in methanol and then analysed under the optimized chromatographic conditions.

3. Results and discussion

3.1. HPTLC method optimization

For the selection of appropriate mobile phase for the effective separation of nateglinide and metformin hydrochloride, several runs were made by using mobile phases containing solvents of varying polarity, at different concentration levels. Different mobile phase systems like toluene:methanol, chloroform:methanol, chloroform:diethyl ether:ethyl acetate, chloroform:ethyl acetate:acetic acid at different concentration levels were tried. Among the different mobile phase combinations employed, the mobile phase consisting of chloroform:ethyl acetate:acetic acid in the ratio of 4:6:0.1 v/v/v gave the best resolution with sharp well defined peaks with R_f values of 0.80 ± 0.02 and 0.17 ± 0.02 for nateglinide and metformin hydrochloride, respectively. The use of acetic acid was found to be necessary for the elution of metformin from the plate due to the fact that it is a weak basic drug, thereby interacting with the unreacted silanol groups on silica stationary phases (Ghassempour et al., 2006). However, increasing the ratio of acetic acid was found to be deleterious and affected the peak shape of nateglinide. For the selection of analytical wavelength for the quantification of the drugs, the standard spots applied on silica gel were scanned and their overlain spectra were obtained on the HPTLC instrument. From the overlain spectra (Fig. 2), it was observed that both nateglinide and metformin hydrochloride exhibited strong absorbance at about 216 nm which was selected as the analytical wavelength for further analysis. The densitogram of the blank mobile phase (Fig. 3) also showed no peak confirming the purity of the standard peaks obtained using the proposed mobile phase.

Figure 2.

UV spectra comparison of the spots of the standards (1) and dosage forms (2) for nateglinide (N) and metformin hydrochloride (M).

Figure 3.

Typical densitogram of blank mobile phase.

3.2. Analysis of marketed formulation

The marketed formulation, Glinate-MF was analysed using the developed method. The chromatogram of tablet sample showed only two peaks at R_f value of 0.80 and 0.17 for nateglinide and metformin hydrochloride, respectively, indicating that there is no interference of the excipients present in the tablet formulation. The content of nateglinide and metformin hydrochloride was calculated by comparing peak areas of sample with that of the standard (Table 1). The densitogram of tablet formulation is shown in Fig. 4.

Table 1.

Assay results of the pharmaceutical dosage form.

Component	Amount present (mg per tablet)	% Amount founda	SD	% RSDa
Nateglinide	60	100.48	0.606	0.603
Metformin hydrochloride	500	100.10	0.369	0.368

Tablet used for analysis – Glinate-MF (Glenmark Pharmaceutical Limited, Baddi, Himachal Pradesh, India).

^aDenotes average of six estimation.

Figure 4.

Typical densitogram of nateglinide (1) and metformin hydrochloride (2) in pharmaceutical dosage form.

3.3. Method validation

3.3.1. Linearity

Peak areas were found to have better linear relationship with the concentration than the peak heights. For nateglinide, the r² was found to be 0.996, and for metformin hydrochloride the r² was 0.995. Calibration graphs were constructed in the concentration range of 200–2400 ng band⁻¹ for nateglinide and 500–3000 ng band⁻¹ for metformin hydrochloride. The correlation coefficients, y-intercepts and slopes of the regression lines of the two drugs were calculated and are presented in Table 2.

Table 2.

Summary of linear regression and validation data.

Parameters⁎	Nateglinide	Metformin hydrochloride
Linearity range	200–2400 ng band−1	500–3000 ng band−1
Linear regression equation	y = 119.3x + 51.01	y = 174.0x + 2191
Slope ± SD	119.3 ± 0.263	174.0 ± 0.524
Intercept ± SD	51.01 ± 0.713	2191 ± 1.329
Correlation coefficient (r2)	0.996	0.995
Limit of detection (LOD)	0.020	0.022
Limit of quantification (LOQ)	0.060	0.066
Repeatability (RSD)	0.725	0.730
Intra-day (RSD)	0.710	0.361
Inter-day (RSD)	0.790	0.837

^⁎Denotes average of six estimations.

3.3.2. Precision

Repeatability and intermediate precision of the developed method were expressed in terms of relative standard deviation (RSD) of the peak area. The results showed that the repeatability, intra- and inter-day variation of the results at concentration of 300 ng band⁻¹ for nateglinide and 2500 ng band⁻¹ for metformin hydrochloride were within the acceptable range. The coefficients of variation for both the inter-day and intra-day precision of the method was found to be less than 1% for both drugs (Table 2).

3.3.3. Accuracy/recovery studies

The recovery studies were carried out at 80%, 100% and 120% of the test concentration as per ICH guidelines. The percentage recovery of nateglinide and metformin hydrochloride at all the three levels was found to be satisfactory (Table 3). For nateglinide, the % recovery was found between 98.83% and 100.33% and for metformin hydrochloride between 99.92% and 100.37%, respectively.

Table 3.

Recovery study of the method (using the standard addition method) for nateglinide and metformin hydrochloride.

Drug	Recovery level (%)	Initial amount (ng band−1)	Amount added (ng band−1)	% Recovery⁎	% RSD⁎
Nateglinide	80	300	240	98.83	0.550
	100	300	300	100.33	0.667
	120	300	360	100	0.835
Metformin hydrochloride	80	2500	2000	100.37	0.276
	100	2500	2500	99.92	0.639
	120	2500	3000	99.95	0.420

^⁎Denotes average of three estimations at each level of recovery.

3.3.4. Limit of detection (LOD) and limit of quantitation (LOQ)

The limits of detection and quantitation were found to be 0.020 and 0.060 ng band⁻¹ for nateglinide and 0.022 and 0.066 ng band⁻¹ for metformin hydrochloride, respectively, indicating the sensitivity of the developed method.

3.3.5. Robustness of the method

The robustness of the method evaluated by assessing the effect of variations in method parameters on peak areas showed low RSD values (less than 1.0%) indicating robustness of the method (Table 4).

Table 4.

Robustness study for the developed method.

Parameters	Drug	% SD⁎	% RSD⁎
Mobile phase composition (±0.1 mL)	Nateglinide	0.297	0.296
	Metformin hydrochloride	0.543	0.543
Amount of mobile phase (±5%)	Nateglinide	0.984	0.982
	Metformin hydrochloride	0.982	0.741
Time from spotting to chromatography (±10 min)	Nateglinide	0.539	0.534
	Metformin hydrochloride	0.951	0.947
Time from chromatography to scanning (±10 min)	Nateglinide	0.654	0.656
	Metformin hydrochloride	0.493	0.490

^⁎Denotes average of three estimations at each level.

3.3.6. Specificity

The peak purity test of nateglinide and metformin hydrochloride spots were assessed by comparing their respective spectra at peak start, peak apex and peak end positions of the spot and their spectra were overlaid to assess spectral matching.

3.4. Stability studies

The results of the forced degradation study of nateglinide and metformin hydrochloride using chloroform:ethyl acetate:acetic acid [4:6:0.1 (v/v/v)] as the mobile phase system are summarized in Table 5.

Table 5.

Stability studies for the developed method.

Degradation condition	Number of degradation products (Rf values)	Area of degradation product (%)
Acid	4 (0.03, 0.13, 0.87, 0.90)	8.50, 2.88, 13.06, 7.09
Base	3 (0.06, 0.72, 0.92)	5.94, 6.15, 15.29
Oxidative	4 (0.04, 0.30, 0.59, 0.69)	7.86, 3.05, 11.46, 6.41
Wet	2 (0.05, 0.84)	7.74, 17.45
Heat	3 (0.16, 0.68, 0.90)	4.47, 8.71, 12.09
Photo	4 (0.05, 0.46, 0.64, 0.69)	4.37, 0.73, 8.05, 12.51

3.4.1. Acid induced degradation study

Nateglinide and metformin hydrochloride, both were found to undergo acid degradation very rapidly. The reaction in 0.001 M HCl at 40 °C under reflux for 30 min showed extensive degradation for nateglinide with additional peaks at R_f values of 0.87, 0.90 (about 13.06, 7.09% degradation), respectively. For metformin hydrochloride, additional peaks were observed with R_f values 0.03, 0.13 (about 8.50, 2.88% degradation), respectively (Fig. 5).

Figure 5.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in the acid degradation study.

3.4.2. Base induced degradation study

In base induced degradation study, nateglinide and metformin hydrochloride showed additional peaks at R_f values 0.72, 0.92 (about 6.15, 15.29% degradation) and 0.06 (about 5.94% degradation), respectively (Fig. 6).

Figure 6.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in base degradation study.

3.4.3. Oxidative induced degradation study

In the oxidative degradation study, it was found that both nateglinide and metformin hydrochloride were extremely liable to degradation. Nateglinide exhibited degradation peaks at R_f values 0.59, 0.69 (about 11.46, 6.41% degradation), respectively, and for metformin hydrochloride at R_f values 0.04, 0.30 (around 7.86, 3.05% degradation), respectively. The densitogram for the oxidative degradation study is shown in (Fig. 7).

Figure 7.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in oxidative degradation study.

3.4.4. Wet degradation study

The wet degradation studies suggested that both nateglinide and metformin hydrochloride were labile to wet degradation and showed additional peaks at R_f values of 0.84 (about 17.45% degradation) and 0.05 (about 7.74% degradation), respectively (Fig. 8).

Figure 8.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in wet degradation study.

3.4.5. Heat degradation study

In the heat degradation study, nateglinide and metformin hydrochloride showed additional peaks at R_f value at 0.68, 0.90 (about 8.71, 7.09% degradation) and 0.16 (about 4.47% degradation), respectively (Fig. 9).

Figure 9.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in heat degradation study.

3.4.6. Photo-degradation study

Nateglinide and metformin hydrochloride both showed additional peaks at R_f value 0.64, 0.69 (about 8.05, 12.51% degradation) and 0.05, 0.46 (about 4.37, 0.73% degradation), respectively, in the photo-degradation study (Fig. 10).

Figure 10.

Typical densitogram of nateglinide, metformin hydrochloride and degradation products in photo-degradation study.

4. Conclusion

A combination of nateglinide and metformin hydrochloride is currently available for the treatment of diabetes mellitus. As there are no reported methods for their simultaneous estimation, a stability indicating high performance thin layer chromatography (HPTLC) method was developed and validated for the determination of nateglinide and metformin hydrochloride in co-formulations on pre-coated silica gel HPTLC plates using chloroform:ethyl acetate:acetic acid (4:6:0.1 v/v/v) as the mobile phase with densitometric detection at 216 nm. The developed method was found to be simple, rapid, selective, sensitive and suitable for simultaneous determination of nateglinide and metformin hydrochloride. The HPTLC method offers several advantages over liquid chromatographic methods such as the possibility of simultaneous analysis of sample and standard on the same plate, short system equilibrium time, multiple/repeated scanning of chromatograms, higher mobile phase pH, large sample capacity, short run time, minimum solution consumption and no prior treatment for solvents like filtration and degassing. The stability indicating properties established following the recommendations of ICH guidelines also indicated that the drugs could be evaluated in presence of their degradation products and thereby can be employed for the simultaneous estimation of nateglinide and metformin hydrochloride and their degradation products in stability samples in the industry.