Development of Multiple Time Point Stability Indicating Assay Method and Validation of Nabumetone by RP-HPLC

 

Gude Sai Sushmitha¹, Dr. Girish Pai², Dr. Muddu Krishna¹, Dr. S G Vasantharaju¹*

¹Department of Pharmaceutical Quality Assurance, Manipal Academy of Higher Education,

Manipal- 576104, India.

²Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal- 576 104, India.

 

ABSTRACT:

Nabumetone is a Non-steroidal anti-inflammatory drug (NSAID) which belongs to a drug class which provide antipyretic and analgesic effects in lower doses and anti-inflammatory effects in higher doses. A simple, highly sensitive, accurate and isocratic RP-HPLC stability-indicating method was developed and validated for determination nabumetone by using Cosmosil C₁₈(150x4.6mm, 5μm) column using Acetonitrile: water pH-5 (55:45 %v/v) as mobile phase at a flow rate of 0.8ml/min. The detection was done by using PDA(Photo diode array) detector at 229nm. Calibration curves for all drugs were in the range of 0.125-8 μg/ml and the linear regression coefficients were more than 0.999. Recovery rate was in the range 98.8% to 100.5%. The limits of detection was found to be 0.035μg/ ml. Also, the limits of quantification was 0.107 μg/ml. Repeatability and Intermediate precision was done and the % RSD was found to be less than 1%, by changing few method parameters robustness was evaluated which has shown %RSD of less than 2%. Study aimed to conduct forced degradation studies and develop a simple, effective method which can effectively separate drug from its degradation products. This study was conducted at multiple time points to estimate the rate of degradation. Stress testing was done for acidic degradation, basic degradation, neutral hydrolytic degradation, oxidative degradation, dry heat degradation and Photo stability study. The order of stability for Nabumetone was found to be H₂O₂ <acid < alkali < wet heat<photolysis <dry heat. This developed method was applied on tablet formulation to calculate % assay and it was found to be 101.9%.

 

 

 

INTRODUCTION:

Nabumetone is a non-acid pro drug as it is converted into 6-methoxy-2-naphthyl acetic acid (6-MNA) upon hepatic biotransformation, it is referred as non-steroidal anti-inflammatory drug(NSAID), it is used for treating osteoarthritis, rheumatoid arthritis (RA) and synovitis of osteoarthritis.

 

 

 

 

Nabumetone blocks cyclo-oxygenase 1 and 2 which in turn decreases the formation of prostaglandins and thromboxanes by decreasing the level of its precursors (prostaglandin H2 and thromboxane A2) which leads to prevention of inflammation, pain and platelet aggregation.^1,2

 

 

Figure 1: Structure of nabumetone

 

A stability indicating assay method is a validated quantitative analytical method used to detect change in drug’s stability with time. It measures the changes in active ingredients concentration without any interventions from other degradation products. The RP-HPLC was used as analytical tool for separation and quantifying the impurities and it is most frequently coupled with a PDA detector. To determine the susceptibility of the drug to different degradation mechanisms was done by selecting the suitable condition, extent of the test (exposure period) and the test was terminated based upon the extent of decomposition. After exposing the drug to required time it was analysed by using stability indicating assay method^4. Current Good manufacturing practises (c GMP) says that all pharmaceutical drug substances need to be tested with a stability-indicating assay method as it helps to identify possible degradation products along with its pathway and intrinsic stability. The character of the stress testing will depend upon each drug substance and type of drug product. Nabumetone is susceptible under variety of conditions. So, it is important to develop a method that separates the drug from its degradation products. Literature review revealed that there are only few reported methods about nabumetone and its stability, they failed to develop a effective stability indicating method which could effectively separate drug from its degradation product^5,6,10,11,12. So, this study aimed to conduct forced degradation studies and develop an simple, effective method which can effectively separate drug from its degradation products. This study was conducted at multiple time points to estimate the rate of degradation and aimed at 5-20% amount of degradation.

 

MATERIALS AND METHODS:

Reagents and chemicals:

HPLC grade acetonitrile was obtained from Merck life scienes Pvt. Ltd. Analytical grade Sodium hydroxide; Hydrochloric acid and hydrogen peroxide were obtained from Finar Ltd, Himedia laboratories Pvt. Ltd and Merck life sciences Pvt. Ltd respectively. Nabumetone was obtained as gift sample from Micro labs Pvt. Ltd, Hosur, Tamil Nadu.

 

Instrument and Analytical conditions:

A Shimadzu LC 20 AD equipped with Pump: LC-10 ADVP, Auto injector: SIL-10 ADVP, Column oven: CTO-10 ASVP, System controller: SCL- 10A VP and detector: Photo diode array detector: SPD M-10A VP was used. The chromatographic separation was done by using Cosmosil C₁₈ (150x4.6mm, 5μm)column using Acetonitrile: water pH-5 (55:45 %v/v) as mobile phase at a flow rate of 0.8ml/min. The detection was done by using PDA (Photo diode array) detector at 229nm. The column temperature was set at 25⁰C and 10 μl volume of 1μg/ml solution was injected into the HPLC system and the chromatograms were recorded.

 

Preparation of standard:

Primary stock solution- 10mg of standard nabumetone was weighed and taken into a clean and dry 10ml volumetric flask and made up to the mark with acetonitrile to give concentration of 1000μg/ml. Secondary stock solution- 1ml of 1000μg/ml solution was taken into another 10ml volumetric flask and made up to volume with acetonitrile to give 100 μg/ml concentration. Working stock solution-1ml of 100μg/ml was transferred into a similar flask and made up to 10ml volume with acetonitrile to give 10μg/ml concentration solution. Standard preparation-Finally,1ml was taken in a 10ml volumetric flask from 10 μg/ml solution and made up to the mark with acetonitrile to give 1 μg/ml solution which is injected into HPLC system.

 

Preparation of test solution:

20 tablets were weighed individually and the average weight of 20 tablets was 607.5 mg. Tablets were crushed and 1000 µg/ml of test solution was weighed by taking 10mg equivalent weight of crushed tablet powder. As 500 mg of drug was present in 607.5mg and 10 mg equivalent weight was 12.15 mg. So, 12.15 mg was weighed and made up to 10ml and the concentration was equivalent to 1000 µg/ml the solution was sonicated to dissolve excipients and filtered. 1ml was taken from filterate and made upto 10ml to give 100µg/ml and later 1 ml was taken from that solution to give 10µg/ml. Finally 1ml was taken to 10ml and 1 µg/ml solution was made to inject into HPLC solution

 

Forced Degradation study conditions:

The stress studies include acid, alkali, neutral hydrolysis which reveals the effect pH on drug degradation and oxidative, thermal and photolysis.

 

pH degradation (Acidic, Basic and neutral)^[3]:

1N Hydrochloric acid and 1N Sodium hydroxide were considered as acidifying and alkaline reagent respectively, drug was dissolved in 25ml and refluxed. Aliquots were withdrawn at different time intervals such as 0 hr(before reflux), 4 hr, 8hr, 12 hr at 80⁰C and later neutralised to pH 7 and diluted to 1μg/ml. Study was aimed to observe 10-20% degradation which was considered as significant change. Similarly neutral degradation was performed by dissolving drug in purified water of pH 7.0±0.05.

 

Oxidative degradation:

Nabumetone was exposed to oxidizing medium to see its degradation. 2.5 ml of drug from stock solution was taken into 25 ml of 3% hydrogen peroxide solution and kept in a dark place at room temperature for about 8 hours. Aliquots were withdrawn at different time intervals such as 0 hr (before reflux), 2 hr, 4 hr and 8 hr,it was diluted to 1μg/ml to inject into HPLC system. Study was aimed to observe 10-20% degradation which was considered as significant change.

Dry heat degradation:

It is a part of thermal stress degradation study where effect of high temperature on the drug was observed. 1 gram of drug was taken in a crucible and kept at 80⁰C for about 8 hours. The % degradation was found to be less than 10%. So, the time of the study was increased to 12 hours.

 

Photo stability studies:^[8][9]

The photo stability testing of drug substances must be evaluated to demonstrate that a light exposure does not result in unacceptable change. A Photo stability chamber was designed specifically to perform stability of a drug substance or drug product under near UV or visible light testing as per ICH Q1B Guidelines. It contains two lamps with electrical connections in a 30cm x 30cm box covered with lid and secured using lock and key for better control. 10-Watt LED Lamp (Havells A60 -Cool White) was used as light source which was validated using Quinine Chemical Actinometry. Two small petri dishes were taken with some amount of drug powder spread to thickness of 3mm, in which one was covered with aluminium foil which was considered as drug control and the other without foil was directly exposed to light. The illumination period was calculated to be 120 hours. After the exposure period, 10 mg drug sample was weighed separately from two petri dishes in 10ml of acetonitrile to give 1000μg/ml of stock solution; it was diluted to 1μg/ml and injected into HPLC system. Study was aimed to observe minimum of 10-20% degradation which was considered as significant change.

 

METHOD VALIDATION:^[7]

1 Specificity:

a) Blank interference:-

Performed by using blank and standard solution.Identification of active ingredient peaks and diluents.

b) Forced degradation:-

Performed by subjecting the drug and blank to stress conditions like acid or base hydrolysis, oxidation, etc. Identification of active ingredient peak and impurity/ degradant peak by retention time.

 

2 System suitability:

Six replicates of 100% concentration injected and Plate count, tailing factor, resolution and reproducibility (% RSD of retention time and peak area) are determined from those replicate injections of standard solution.

 

3 Linearity:

The plotting mean chromato­graphic peak area against the concentration of each compound was made. Each solution was injected in triplicate and the mean peak area value was observed within the concentration range of   0.125-8 μg/ ml

 

4 Accuracy:

The accuracy of HPLC method was tested by calculating the recovery of certain amounts of each of the sartans added separately at three different concentrations (0.8, 1 and 1.2 μg/ml) to samples representing the average weight of the corresponding sample concentrations. The recoveries were also confirmed by determination of these drugs in samples containing 80, 100 and 120 %

 

5 Precision:

It was performed at two different levels Repeatability and Intermediate precision by injecting six replicates of 100% concentration. The % RSD was determined.

 

6 Limiting values:

LOD- The detection limit of an individual analytical method is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

LOQ- The quantitation limit of an individual analytical method is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products.

 

7 Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Method parameters included flow rate of mobile phase (±10%), mobile phase composition (± 2 units) and detector wavelength (±2nm)

 

RESULTS AND DISCUSSION:

Optimised analytical conditions:

The log P of nabumetone is 3.08, which indicates that the drug is lipophilic having affinity towards non-polar stationary phase. Hence reverse phase HPLC was used for separation. GraceSmart (250×4.6mm, 5μ) was used for initial trials. However, Cosmosil C₁₈ (150×4.6mm, 5μm) was selected as the final stationary phase due to reduced tailing and system pressure. Both the methanol and acetonitrile were used. It was seen that in case of methanol the back pressure of column was high, slightly broader peaks were observed and considerable base line shifts when compared to acetonitrile. Hence acetonitrile was selected as organic mobile phase. To avoid the tedious process of preparing buffer, Ultra clear water was considered as aqueous phase as separation was achieved by adjusted its pH to 5 ±0.05 with 0.1% acetic acid. Flow rate of 0.8ml/min was optimized due to good symmetrical peak shapes with the retention time of the analyte 7.0 minutes.

 

 

Figure 2: Chromatogram of nabumetone at optimised conditions

 

Forced degradation studies:

In case of hydrolytic stress degradation, 1N HCl and 1N NaOH were used as acidic and basic reagents respectively and water was used as a medium in neutral hydrolysis. Drug was refluxed with acid, alkali and water for about 12 hours at 80⁰C aiming 5-20% degradation. In acidic hydrolytic degradation, two degradation peaks were foundat 2.3 R_t (D1) and 3.6R_t  (D2) where as a subsequent decrease in D1 peak area was observed in time with increase in D2 peak’s area at the end of 12 hours 46% degradation was observed. In base hydrolytic degradation, At the end of 12hours two merged degradation product peaks were formed at 2.1 R_t (D1) and 2.3 R_t (D2). The area of D2 (at 2.2 Rt) was increased and area of drug peak decreased with time by showing about 44% degradation. In neutral hydrolytic degradation separate degradant peaks were not observed but decrease in drug peak’s area was seen with time, at the end of 12 hours almost 40% degradation was observed. In oxidative degradation studies 3% v/v hydrogen peroxide was used as oxidising agent in which drug solution was added and stored at room temperature, at the end of 8 hours two small degradation peaks were observed at 1.3 R_t (D1) and 4.9 R_t (D2) showing decrease in drug peak’s area with 47% degradation. In dry heat thermal studies, drug powder was exposed to 8 hours initially where sufficient degradation was not observed, later the degradation time was increased to 12 hours where 10% degradation was seen. In photolytic degradation, drug was exposed to 10 Watt LED bulb which illuminates 600 lumens in turn which is equal to 10,000 lux, according to ICH Q1b specifications the drug should be exposed to 1.2 x 10⁶ lux hours which is equal to 120 hours (5 days). So, the drug powder was exposed to 120 hours in a stability chamber along with a control and the % degradation was found to be 20%. In all the degradation studies, the peak purity index for the drug was greater than single point threshold which indicates that there was no merging of impurity peak with the analyte peak and hence the peak purity passed. The stability indicating method was applied on tablet formulation (in house) of nabumetone and the % assay was found to be 101.9%.

 

Table. 1: Summary of Forced Degradation studies

Stress study	Conditions used	% Degradation
Acid hydrolytic degradation	1N HCl	At 0 hr	15.3%
		At 4 hr	30.2 %
		At 8 hr	38 %
		At 12 hr	46%
Base hydrolytic degradation	1N NaOH	At 0 hr	14%
		At 4 hr	20.5 %
		At 8 hr	42.2 %
		At 12 hr	44.8 %
Neutral hydrolytic degradation	Water	At 0 hr	2.4 %
		At 4 hr	13.6 %
		At 8 hr	16.5 %
		At 12 hr	40.4 %
Oxidative degradation	3% v/v Hydrogen peroxide	At 0 hr	3.9 %
		At 2 hr	8.7 %
		At 4 hr	23.5%
		At 8  hr	47.4 %
Dry heat degradation	80⁰C (In hot air oven)	At 12 hr – 10 %
Photolytic degradation	Exposed for 120hr (equivalent to 1.2 x 106 lux hours) under 10 W LED bulb	At 120hr – 20 %

 

 

Figure 3: Rate of degradation  (acid, alkali, neutral and oxidative studies)

 

 

Figure 4: Chromatogram of nabumetone in 1N HCl at 12 hr at 80⁰C

 

Figure 5: Chromatogram of nabumetone in 1N NaOH at 12 hr at 80⁰C

 

 

Figure .6: Chromatogram of nabumetone in water at 12 hr at 80⁰C

 

 

Figure 7: Chromatogram of nabumetone in 3% v/v hydrogen peroxide at 12 hr

 

 

Figure.8: Chromatogram of nabumetone after exposing it to light for 120 hours

 

Figure 9: Chromatogram of nabumetone after exposing it to dry heat at 80⁰C

 

METHOD VALIDATION:

System suitability:

System suitability was assessed by injecting six (6) replicates of 1 µg/ml into system     for evaluating the suitability of the proposed optimised method.

 

Table2: The summary (average readings) of system suitability parameters considering n=6

PARAMETER	OBSERVATION (average reading)	%RSD
Retention time	7.008±0.12	0.09
Area	232724±440	0.10
Theoretical Plate count	2856.27	0.31
Tailing Factor	1.07	0.56
Capacity Factor(k1)	4.04	--

 

Linearity:

A series of standard solutions of nabumetone were prepared. The linearity range was considered as 0.125-8 µg/ml. Each standard solution was injected in triplicate into HPLC system and chromatograms were recorded by considering optimised conditions.

 

Table 3: Linearity results

 

 

Figure 9: Linearity Plot

Specificity:

Blank i.e., diluents without sample and 1μg/ml standard solution containing nabumetone were injected into chromatographic system and chromatograms were recorded

Interference of blank:

Blank chromatogram should not show any peak at drug’s retention time (7.0min)

 

 

Figure 10: Blank chromatogram for specificity

 

Peak purity index:

 

Figure 11: Peak Purity Curve

 

Limiting values:

Limit of detection of an individual analytical method is the lowest amount of detectable analyte in the sample. The quantification limit of an individual analytical method is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

 

It is determined based on slope and standard deviation of the response.

 

Table4: LOD and LOQ values

Drug	LOD (µg/ml)	LOQ (µg/ml)
Nabumetone	0.035	0.107

 

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value accepted either as a conventional true value or an accepted reference value and the value found.  The accuracy was done by recovery studies. The known amount of standard drug was spiked to the pre analyzed standard samples and the recovery of drug was calculated. Accuracy was performed at 3 levels of 80, 100 and 120% of standard concentration.

 

Table 5: Accuracy results at three different levels

Spiked level	Area of spiked solution	Area of un spiked  solution	% recovery
80%	438224	198172	100.2
100%	488006	247078	100.5
120%	533993	297259	98.8
Mean % recovery	-	-	99.8

 

Precision:

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra –assay precision. The peak area of nabumetone standard solution (1.00µg/mL) was analyzed on the same day six times. The % RSD was calculated for the resultant peak area.

Table 6: Precision results at 100% level

S.No	Retention Time	Area
1.	7.01	231999
2.	7.00	231774
3.	6.99	231883
4.	7	231536
5.	7.01	231759
6.	7	231336
Mean	7.00	231714.5
±SD	0.01	240.82
%RSD	0.11	0.10

 

Intermediate precision:

Intermediate precision expresses with in laboratory variations: Different days, different analyst and different equipment, different columns. To assess the degree of reproducibility of the above method, nabumetone standard solution (1.00 µg/mL) was analyzed on different days with six different samples. The chromatogram was recorded and % RSD was calculated for the resultant peak area.

 

Table 7: Method Precision and intermediate precision results

Method precision			Intermediate precision
	Retention Time	Peak area		Retention time	Peak area
	7.01	232586		6.73	241682
	7	233912		6.73	243150
	7.02	231475		6.71	242802
	7.01	233694		6.70	248487
	6.99	232147		6.69	244711
	7	233486		6.69	245125
Mean	7.01	232883.3	Mean	6.69	244326.2
± SD	0.01	968.76	± SD	0.02	2399.86
%RSD	0.12	0.41	%RSD	0.274	0.98

 

Robustness:

Robustness of an analytical method is measure of its capacity to remain unaffected small for but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

 

A.     Influence of change in flow rate (±10% units):

The flow rate of the mobile phase was changed by ±10% of optimized flow rate i.e., 0.72ml/min and 0.88ml/min. The standard solution of Nabumetone (1.00µg/ml) was injected six times and the % RSD was calculated for the resultant peak area.

 

B.     Influence of variations of mobile phase composition (±2 units):

The mobile phase composition was changed by ±2.0 units. The standard solution of Nabumetone (1.00µg/ml) was injected six times and the % RSD was calculated for the resultant peak area.

 

C.       Influence of change in wavelength(±2 units):

The wavelength was changed by ±2.0 nm

1)227nm

2)231nm

 

 

 

Table 8: Robustness results of flow rate change

 

Table 9: Robustness results of wavelength change

 

Table 10: Robustness results of Mobile phase composition change

 

ASSAY:

Table 11: Assay results of tablet formulation

S.No	Name	Area of the peak
1	Nabumetone standard	234854
2	Nabumetone test	239547

             

               Area of unknown x Concentration of standard

% Assay = -------------------------------------------------- x 100

                 Area of standard x Concentration of unknown

 

    =239547/234854*10/10 x 100

    = 101.9%

 

CONCLUSION:

A simple, sensitive and effective stability indicating assay method was developed and validated. Degradation product concentrations increased as time passed with decrease in parent drug concentration and the degraded product peaks were well resolved from the parent drug peak. Based on the study done it was concluded that Nabumetone is susceptible to many stress conditions. Nabumetone is more susceptible to acidic, basic hydrolysis and oxidative stress experimental conditions with more than 40% within 12 hours. Photo stability study has shown about 20% degradation after exposing it under LED light for appropriate amount of time. Most of the drug content was degraded under oxidative conditions i.e., 47.43% (in12hr).Dry heat studies has shown minimum degradation among all forced conditions i.e., 10% (in 12hr).The order of stability for Nabumetone was found to be H₂O₂ <acid < alkali < wet heat<photolysis <dry heat. This developed method can be applied on tablet formulation to calculate % assay.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of MCOPS, MAHE for the facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

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Accepted on 04.10.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 4813-4820.