Development and Validation of A Stability-Indicating RP-HPLC Method for the Determination of Sitagliptin Phosphate and Simvastatin in the Presence of their Degradation Products in Bulk and Binary Mixture

 

Lobhe Gayatri A¹*, Shah Amol², Singhvi Indrajeet³

¹Department of Pharmaceutical Chemistry, VJSM's Vishal Institute of Pharmaceutical Education and Research, Ale, Pune-412411, (Maharashtra) India.

²Sitabai Thite College of Pharmacy, Shirur, Pune-412210, (Maharashtra) India

³Pacific Academy of Higher Education and Research University, Udaipur-313024 (Rajasthan) India

 

ABSTRACT:

A simple, selective, and precise stability-indicating reversed-phase liquid chromatographic method has been developed and validated for the determination of sitagliptin phosphate monohydrate (STG); and simvastatin (SIM) in the presence of acid and alkali degradation products of STG, and the hydrolytic degradation product of SIM. The method was based on gradient elution on a reversed phase C18 column (150 mm × 4.6 mm,5 μm)— Cosmosil using a mobile phase consisting of acetonitrile and 10mM potassium dihydrogen orthophosphate (pH 3.0; 0.01M) (85:15% v/v)  and  flow rate 1.0ml/min. Quantitation was achieved using UV detection at 225 nm for SIT and SIM and their degradation product was achieved. Linearity, accuracy, and precision were found tobe acceptable over the concentration ranges of 20-100 μg mL−1 and 0.4–20 μg mL−1 for STG and SIM, respectively. Limits of detection and quantitation for SIT were found to be 5.65 ng and 17.12 ng, respectively, while for SIM were found to be 2.72 ng and 8.25 ng, respectively. The proposed method was validated as per ICH guideline.

 

 

INTRODUCTION:

Sitagliptin phosphate monohydrate (STG), 7-[(3R)-3-amino-1-oxo-4-(2,4,5- trifluorophenyl) butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3a] pyrazine phosphate monohydrate (Figure 1a), is a novel oral hypoglycemic drug that belongs to dipeptidyl-peptidase-4 inhibitors which stimulate glucose-dependent insulin release.[1,2] Simvastatin (SIM), (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-Hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-Tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyl 2,2-dimethyl-butanoate (Figure 1b), is an oral antihyperlipidemic drug that belongs to statins that lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase. This enzyme catalyzes the conversion of (HMG-CoA) to mevalonate, an early and rate limiting step in cholesterol

biosynthesis.[3,4] It is a member of the statin class of pharmaceuticals and is a synthetic derivate of a fermentation product of Aspergillus terreus. SIM can be obtained in various synthesis pathways. During synthesis apart from the main reaction, unwanted side reactions are one of the major causes of impurities. The possible degradation pathways of SIM are provided in Fig.2.

 

 

Fig. 1: Structure of Sitagliptin Phosphate and Simvastatin

 

 

Fig. 2: Chemical structures of (A) Sitagliptin alkali degradation product, (B) sitagliptin acid degradation product, and (C) simvastatin hydrolytic degradation product.

 

A detailed survey of analytical literature for STG revealed that only few methods have been described for the determination of STG in pharmaceutical preparations or biological fluids including spectrophotometry,[5,6] HPLC,[7–9] and spectrofluorometry.[10] Similarly, a survey of analytical literature for the determination of SIM either in biological samples or pharmaceutical preparations revealed methods based on HPLC,[11–14] LC/MS/ MS,[15–18] and spectrophotometry.[19–21] Besides, STG and SIM were simultaneously determined in bulk and dosage form by RP-HPLC[22] and in human plasma by LC-MS/MS.[23] An analytical method has been reported for the determination of STG in the presence of its alkali degradation product[9,10] and other reported stability-indicating methods of SIM in single form or in other combinations.[24–26]

 

Although a number of methods are available for evaluating STG, a common method for separation if its possible impurities with good efficiency remains still in research. With the objective of developing a method for rapid separation with shorter runtimes, a simple, precise, accurate stability indicating RP-HPLC coupled with a UV absorbance detector method was developed for the quantitative determination of impurity in API substance. Also modification done due to the high lipophillic nature of SIM compared to STG, Cosmosil C18 column is recommended in this procedure to reduce the relative retention of SIM to the required retention of STG. Ultimately, it have been reduced the retention of SIM (at 6.43 ± 0.02 min) at the maximum by using C18 column and suitable buffer without affecting the specificity for STG at lower retention time (2.91 ± 0.02 min). Thus, the aim of the present work was to develop a more sensitive and selective stability-indicating LC method for the simultaneous determination of STG and SIM in the presence of both acidic and alkaline degradation products of STG and the hydrolytic degradation product of SIM. The chemical structures of these degradants have been investigated and elucidated (Figure 2). The separation on the wide spread and conventional reversed phase columns was of interest.

 

EXPERIMENTAL:

Instrumentation and Chromatographic Conditions:

The main aim of present research work was to separate STG and SIM and its DPs. An Cosmosil C-18 column (4.6 × 150 mm, 5 μm) was found to be suitable for this analysis after having tried with different columns. During the optimization process on above-mentioned column, several conditions with various mobile phases like methanol/water and acetonitrile/water in different proportionalities were tried in an isocratic mode. The peaks corresponding to DPs did not resolve completely and tailing was observed. To get acceptable separation between the drug and its DPs, potassium dihydrogen ortho-phosphate buffer was used. Further, studies were carried out using varied proportions of acetonitrile, potassium dihydrogen ortho-phosphate buffer. The pH of the buffer, flow rate and composition of the mobile phase were systematically varied to optimize the method. To detect drug and DPs with sufficient peak intensity, the wavelength at 225 nm was chosen. Finally, a mobile phase consisting of acetonitrile, potassium dihydrogen ortho-phosphate buffer (pH 3.0; 0.01M) (85:15% v/v) at a flow rate of 1.0 ml/min using, in an isocratic mode gave good separation of drugs and its DPs. The advantage of the method was validated, robust, cost-effective methodologies for routine drug estimations is the urgent need of the pharmaceutical RandDs. Quality is an essential attribute in any pharmaceutical product and impurity profiling offers a broad scope with the changed perspectives of the research scenario outlined in ICH guideline Q2A (R1).

 

Equipment Used High Performance Liquid Chromatography Equipped with Auto Sampler and DAD or UV Detector

Column: Cosmosil C18 (4.6 X 150 mm, 5 μm, Make: XTerra)

Flow Rate: 1 mL/min

Wavelength: 225 nm

Injection Volume: 20μL

Column Oven: Ambient

Mode of operation: Isocratic

Detector: UV 730 D and SF930 D

Soft ware: Autochro-3000

MFD By: Younglin ( S.K)

Elma S100 ultrasonic processor model KBK 4200 (Germany) was used.

 

Reagents and Reference Samples:

Pharmaceutical grade of STG (certified to contain 99.80%) and SIM (certified to contain 99.93%) were kindly supplied by Watson Pharmaceutical Pvt Ltd, Goa; India, HPLC grade Methanols, Acetonitrile, potassium dihydrogen phosphate were used as solvents to prepare the mobile phase. Methanol and Acetonitrile used were of HPLC grade (Qualigens Fine Chemicals) and sodium hydroxide, hydrochloric acid, hydrogen peroxide, o-phosphoric acid and  potassium dihydrogen phosphate were of Analytical reagent grade (LOBA Chemicals Ltd., Mumbai) used without further purification.

 

Preparation of Phosphate buffer:

The Buffer Solution was prepared by weighing 7.0 grams of KH₂PO₄ into a 1000 ml beaker, dissolved and diluted to 1000 ml of water [HPLC grade]. Then the pH was adjusted to 3 with o-phosphoric acid.

 

Preparation of mobile phase:

The Mobile Phase was prepared by mixing a mixture of 850 ml of Acetonitrile HPLC (85%) and above buffer150 ml (15 %) and degassed in ultrasonic water bath for 5 minutes. Then it was filtered through 0.45 μ filter under vacuum filtration.

 

Diluent Preparation: The same Mobile Phase was used as Diluent.

 

Standard Stock Solution Preparation:

The Standard Stock Solution of the drug was prepared by weighing accurately and transferred 10 mg STG and 10 mg of SIM working standard into a 10 ml and 100 ml clean dry volumetric flask respectively. About 70 ml of Diluent were added and sonicated to dissolve it completely and the volume was made up to the mark with the same solvent. Further from the above prepared Stock Solution pipette out 2 ml of Sitagliptin and Simvastatin into a 10 ml volumetric flask and diluted up to the mark with diluent.

 

Preparation of Acid and Alkali Degradation Products of STG:

The acid degradation product of STG was prepared by dissolving an amount of 1 g STG bulk powder in 250 mL of 6 N aqueous hydrochloric acid. The solution was then heated for 8 hr on a boiling water bath, cooled and neutralized by 6 N aqueous sodium hydroxide. The formed precipitate was filtered, washed several times, and dried.

The alkali degradation product of STG was prepared according to the reported method.[9] An amount of 1 g STG bulk powder was dissolved in 250 mL of 5 N aqueous sodium hydroxide then the solution was refluxed for 6 hr on a boiling water bath, cooled, and neutralized by 5 N aqueous hydrochloric acid.

 

Preparation of the Hydrolytic Degradation Product of SIM:

SIM (200 mg) was dissolved in 0.5 M NaOH [27] The solution was stirred and followed up by injection onto the column using the same chromatographic conditions till complete disappearance of SIM peak (2 hr). The solution was acidified using 2 M HCl. The organic layer was separated and the aqueous layer was washed with methanol. The combined organic layer and washings were evaporated to dryness.

 

Calibration Curves:

Accurately measured aliquots of working standard solutions equivalent to 20-100 μg/ml of STG and equivalent to 0.4–20.0 μg/ml of SIM were transferred into two sets of 10 mL volumetric flasks. Each flask was completed to volume with acetonitrile– Phosphate Buffer pH 3 (85:15, v/v). A volume of 50 μL of each solution was injected in triplicates into the chromatograph. The conditions including the mobile phase, the flow rate, and detection program, previously mentioned in Section 2.3, were then applied. A calibration curve was obtained for each drug by plotting area under the peak (AUP) against concentration (C).

 

Assay of STG and SIM in Laboratory Prepared Mixtures:

The procedure mentioned in Section 2.8.1 was repeated to prepare concentrations equivalent to 20.0–100.0 μg mL⁻¹ of STG and 0.4–20.0 μg mL⁻¹ of SIM in laboratory prepared mixtures. The concentrations of STG and SIM were calculated using calibration equations.

 

Method Development:

The aim of the present work was to develop a simultaneous and sufficiently selective analytical method to discriminate STG and SIM from their corresponding hydrolysis degradation products, with detection and quantitation sensitivity for the cited drugs in their binary mixture, much higher than that of the reported LC method.[22] This was achieved using a simple RP-LC with UV detection. Regarding the relative difference between the physicochemical properties of the cited molecules, the use of conventional HPLC columns available in most quality control laboratories was the challenge. During the optimization cycle, several chromatographic conditions were attempted either by using various mobile phase compositions of buffers, methanol, and acetonitrile, or in different proportions and pH values. Operating an isocratic elution was tried and showed satisfactory analysis time or good resolution between peaks, and it was found that at least 85% of acetonitrile was needed to elute STG and SIM and its degradation product in reasonable analysis time. Thus program was found to be the best one to achieve a reasonable run time analysis with good resolution between the eluted peaks, high sensitivity, and good peaks shape. The buffer pH 3 was found optimum for peak shape and resolution, while changing column temperature had no effect on eluted peak shapes. The lower pKa value of SIM makes it more non-ionized at the acidic pH of the mobile phase and hence more retained on the column. A flow rate of 1mL min⁻¹ was applied. a programmed detection have maximum absorbance at 225 nm for high sensitivity of eluted peaks (Figures 3 and 4).

 

 

Fig. 3: HPLC chromatogram of a 20 µL injection of a synthetic binary mixture of STG (40 µg/mL) and SIM (8µg/mL).

 

System Suitability Tests:

System suitability tests are an integral part of liquid chromatographic methods in the course of optimizing the conditions of the proposed method.[30] They are used to verify that the resolution and reproducibility were adequate for the analysis performed. The parameters of (10 μg/mL), simvastatin hydrolytic degradation product (2 μg/mL), and SIM (20 μg/mL). these tests are column efficiency (number of theoretical plates), tailing of chromatographic peak, repeatability as % RSD of peak area for six injections and reproducibility of retention as % RSD of retention time of a solution of a 40 μg mL⁻¹ STG and 8 μg mL⁻¹ SIM (100% concentration). The results of these tests for the proposed method are listed (Table 1).

 

TABLE 1: System Suitability Tests for the Proposed LC Method

Parameters	STG	SIM
Theoretical plates per column	3465.8	6828.2
USP tailing factor	1.25	1.20
%RSD (n=6) peak area	0.15	0.11
%RSD(n=6) Rt	0.17	0.14

^a n =6

Fig. 4: HPLC chromatogram of 20 μL injection of a mixture solution containing sitagliptin alkaline degradation product (20 μg/mL), SIT (40 μg/mL), sitagliptin acidic degradation product

 

Method Validation:

Linearity:

In this study, 11 concentrations were chosen for each of the cited drugs. Each concentration was analyzed three times and linearity was studied for STG and SIM. A linear relationship between AUP and concentrations (C) was obtained and the regression equation for each drug was also computed (Table 2). The linearity of the calibration curves was validated by the high value of correlation coefficients. The analytical data of the calibration curves including standard deviations for the slope and intercept are summarized (Table 2).

 

Accuracy:

Accuracy of the results was calculated by % recovery of six different concentrations (injected in triplicates) of STG and SIM combined in the laboratory prepared binary mixture. The results obtained including the mean of the recovery are displayed (Table 2).

 

Precision:

The repeatability of the method was assessed by six determinations for each of the three concentrations representing 80%, 100%, and 120%, respectively. The values of the precision (% RSD) of repeatability for STG and SIM peak area were found to be less than 1% in the three concentrations (Table 3).

 

TABLE 2: Results Obtained by the Proposed LC Method for the Simultaneous Determination of STG and SIM

Parameters	STG	SIM
Linearity range (μg/ml)	20-100	0.4–20
Wavelength of detection (nm)	225	225
Retention time	2.91	6.43
Regression coefficient	Area = 157.9x -5.923	Area = 132.3x + 16.02
LOD (μg/ml)	5.65	2.72
LOQ (μg/ml)	17.12	8.25
SD of slope	29.03	5.72
SD of Y-Intercept	0.984	0.468
Confidence limit of the slope	157.9 ± 0.56	132.3 ± 0.96
Confidence limit of the Y intercept	5.923  ± 73.41	16.02  ± 11.77
Standard error of the Estimation	65.83	10.88
Method precision
%RSD of interday Precision of  binary mixture	0.57- 1.23	0.47- 1.11
%RSD of intraday Precision of  binary mixture	0.09 -0.99	0.59-1.72
Accuracy of Binary Mixture (%recovered)	99.46  ± 1.06	100.19  ± 0.94

^a n =6

 

TABLE 3: Results for the Determination of Repeatability for STG and SIM in Laboratory Prepared Binary Mixture by the Proposed LC Method

^a n =6

 

Specificity:

Specificity is the ability of the analytical method to measure the analyte response in the presence of interferences including degradation products and related substances. In the present work, no chromatographic interference from any of the eluted degradation products was found at the retention time of the examined drugs. Also, the chromatograms of the samples were checked for the appearance of any excipients peaks (Figure 5). In addition, the chromatogram of each drug in the sample solution was found identical to the chromatogram received by the standard solution at the wavelengths applied (Figures 3 and 4). These results demonstrate the absence of interference from other materials in the pharmaceutical formulations, and therefore confirm the specificity of the proposed method.

 

 

 

 

Fig. 5: Calibration Curve of Sitagliptin Phosphate Monohydrate

 

 

Fig. 6: Calibration Curve of Simvastatin

 

Limit of Detection and Limit of Quantification:

Limit of detection (LOD) which represents the concentration of analyte at S/N ratio of 3.3 and limit of quantification (LOQ) at which S/N ratio is 10 were determined experimentally for the proposed methods (Table 2).

 

Robustness:

Robustness was performed by deliberately changing the chromatographic conditions. The flow rate of the mobile phase was changed from 0.8 mL min⁻¹ to 1.2 mL min⁻¹. The organic strength was varied, as the acetonitrile proportion from 90% to 80%, meanwhile the buffer was changed also from 10% to 20%. Also, a change of the phosphate buffer pH value was done from (pH 2.9) to (pH 3.1). Only one factor was changed at a time, while the others were kept constant. The studied parameter was the resolution factor (calculated with half-width method) between the intact drug peak and the nearest eluted peak. These variations did not have significant effect on chromatographic resolution by the proposed LC method for STG alkaline degradation product, STG intact drug, STG acidic degradation product, SIM alkaline degradation product, and SIM indicating good robustness of the proposed LC method (Table 4).

 

TABLE 4: Robustness Study of the Proposed RP-LC Method

factor	level	Retention Time		Asymmetry
		STG	SIM	STG	SIM
A: Flow rate (ml/min)
0.9	-1	2.96	7.00	1.22	1.32
1.0	0	2.93	6.96	1.20	1.62
1.1	+1	2.90	6.94	1.09	1.28
%RSD		1.62	0.69	1.32	0.97
B: % of Acetonitrile (ml)
83	-1	2.99	6.99	1.34	1.71
85	0	2.93	6.96	1.52	1.22
87	+1	2.89	6.91	1.06	1.85
%RSD		0.89	0.53	0.76	0.83
pH of Mobile Phase
2.8	-1	2.96	7.01	1.28	1.24
3	0	2.93	6.96	1.22	1.33
3.2	+1	2.88	6.93	1.06	1.02
%RSD		0.98	0.87	0.95	1.25

^a n =6

Statistical Analysis:

A reference method was applied for STG[9] and a pharmacopoeial method was applied for SIM.[30] A statistical analysis of the results obtained by the proposed method and the reference methods was carried out by “SPSS statistical package version 11”. The significant difference between groups was tested by one way analysis of various (ANOVA) (F-value) and student’s t-test (t-value) at p = 0.05 (Table 5). The test ascertained that there was no significant difference among the methods.

 

TABLE 5: Statistical Comparisons between the Recovery Results of the Proposed LC Methods and the Reference Methods for the Cited Drugs

Drug	Group	Mean%	SD±	t-value	F-valuea
STG	Proposed method	99.46	1.09	1.34	1.14
	Reference method	100.38	1.22
SIM	Proposed method	100.19	0.98	0.55	1.11
	Reference method	98.94	1.00

^a There is no significant difference between the proposed method and the reference methods by using the student’s t-test (tabulated = 2.228) and F-value (tabulated = 5.050) at p < 0.05.

 

RESULTS:

HPLC greatly reduces the analysis time and allows for the determination of many individual components in a mixture using one single procedure.[28] So, the development of a highly sensitive and selective RP-LC method for the simultaneous determination of STG and SIM using UV detection, in the presence of degradation products of both drugs, was of interest as no such method has been reported. STG alkali degradation product (Figure 1b) was prepared by the hydrolysis of the amide bond of the intact drug and complete degradation was confirmed using TLC plates. Meanwhile, STG acid degradation product was prepared by the hydrolysis of the amide bond by heating in 6 M aqueous hydrochloric acid till complete degradation, which was confirmed using TLC plates. and IR spectroscopy which revealed shift of the characteristic peak of the carbonyl group of the amide bond at 1639 cm⁻¹ to 1697 cm⁻¹ and the appearance of the characteristic broad peak of the carboxylic– hydroxyl group at 2400–3000 cm⁻¹). A reported method for the forced degradation of SIM [29] proved that the hydrolysis of intact SIM under both acidic and alkaline conditions yields the same degradation product (carboxylic acid derivative). In the present work, SIM hydrolytic degradation product (Figure 1c) was prepared by a very mild and rapid procedure for the efficient and selective alkaline hydrolysis of esters in non-aqueous conditions.[27]. Also, IR spectrum of SIM hydrolytic degradation product showed the presence of carboxylic–hydroxyl band at 2400–3000 cm^-1 and alcoholic–hydroxyl band at 3441 cm⁻¹ (Figure 2b) that added one more evidence for lactone hydrolysis and formation of β-hydroxy acid. Hence, the used hydrolytic degradation method represents a useful tool to assess SIM in the presence of its active metabolite (β-hydroxy SIM acid).

 

 

Fig. 2b: IR spectrum (a) of the hydrolytic degradation product of simvastatin.

 

DISCUSSION:

In order to achieve simultaneous estimation of the two components, initial trials were performed with the objective of selecting adequate and optimum chromatographic conditions. Parameters, such as ideal mobile phase and their proportions, detection wave length and concentrations of the standard solutions were carefully studied. Several solvents were tested in varying proportions. Finally, a mixture of acetonitrile: buffer (85:15 v/v) was selected as the optimum mobile phase. The optimized chromatographic conditions were selected based on sensitivity, retention times and peak shape. The method was validated in terms of linearity, accuracy, precision, LOD, LOQ, robustness and specificity as per ICH guidelines. The accuracy data shows that the method is accurate within desired range. The LOD and LOQ values were low which indicates that the method is sensitive. The method was robust as minor changes in the chromatographic parameters did not bring about any significant changes in peak area and retention times of STG and SIM.

 

CONCLUSION:

The proposed LC method has the advantages of simplicity, specific as both the peaks were well separated from their impurities and degraded components and convenience for the simultaneous separation and quantification of STG and SIM in laboratory prepared mixture. The impurities were well separated with good resolution and peak shape, good retention times finding applications in impurity profiling of related substances. The method has been found to be better due to use of a less economic and readily available mobile phase and lack of extraction procedure which makes the method specifically suitable for routine quality control analysis work and biological fluids.

 

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Received on 01.06.2016       Accepted on 30.06.2016     

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