A new Stability indicating RP-HPLC method for the determination of Apremilast-An Antirheumatic drug

 

Mukthinuthalapati Mathrusri Annapurna*, Debi Prasad Pradhan, Malineni Sushmitha

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

A novel and simple reverse phase liquid chromatographic method has been established for the determination of Apremilast in pharmaceutical dosage forms. Apremilast is used to treat psoriatic arthritis. The proposed work was performed on Shimadzu Model CBM-20A/20 Alite with Intersil ODS3 C₁₈ column (250 mm × 4.6 mm i.d., 5 µm particle size). A mixture of 0.1% acetic acid and acetonitrile was used as mobile phase in this method with flow rate 0.8 ml/min (UV detection at 203 nm) and the method was validated as per ICH guidelines. The linear regression equation was found to bey = 78597x – 14159 with correlation coefficient 0.9999. Forced degradation studies were performed by exposing the drug Apremilast to acidic, alkaline, oxidation and thermal stress degradations. The proposed RP-HPLC method was found to be robust and specific and this method is suitable for the assay of pharmaceutical dosage forms as well as kinetic studies.

 

 

 

INTRODUCTION:

Apremilast (APT) is used for the treatment of different types of psoriasis and psoriatic arthritis. It can also be used for the treatment of other immune system related inflammatory diseases¹. Apremilast (Fig 1) is chemically N-{2-[(1S)-1-(3-Ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide. Apremilast is an inhibitor of PDE4, an enzyme that breaks down cyclic adenosine monophosphate and it is the dominant enzyme responsible for this reaction in inflammatory cells. Only one spectrophotometric method² and one liquid chromatographicmethod³ were reported for the determination of Apremilast till now in literature.

 

In the present study the authors have developed a stability indicating⁴ RP-HPLC method for the determination of APT and the method was validated as per ICH guidelines⁵.

 

Fig 1. Chemical structure of Apremilast (APT)

MATERIALS AND METHODS:

Chemicals and reagents:

Apremilast is available as tablets with brand name OTEZLA (Celgene Corporation Ltd; Label claim: 10 mg and 20 mg of APT. Acetonitrile, sodium hydroxide, hydrochloric acid, acetic acid and hydrogen peroxide were purchased from Merck (India). All chemicals are of HPLC grade and used as received. 

 

Instrumentation:

Chromatographic separation was achieved by using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with Intersil ODS3 C18 column (250 mm × 4.6 mm i.d., 5 µm particle size)maintained at 25ºC.

 

Optimized chromatographic conditions:

Isocratic elution was performed using 0.1% acetic acid and acetonitrile (20:80%, v/v) and the flow rate was 0.8 ml/min. The overall run time was 10 min. The detection was carried at 203nm. 20 µL of sample was injected into the HPLC system and all chromatographic conditions were performed at room temperature (25°C±2°C).

 

Preparation of stock solution:

The stock solution was prepared by transferring accurately 25 mg of APT in to a 25 ml volumetric flask and diluting with mobile phase (1000 μg/ml) and further dilutions were made on daily basis from the stock solution with mobile phase as per the requirement and filtered through 0.45 μm membrane filter prior to injection.

 

Method validation:

Linearity:

A series of solutions (0.5–150 μg/ml) were prepared from the Apremilast stock solution and 20 µL of each solution was injected in to the HPLC system and the peak area of the chromatogram was noted.Calibration curve was plotted by taking the concentration of the solutions on the x-axis and the corresponding peak area values on the y-axis.

 

Precision:

The intra-day precision of the assay method was evaluated by carrying out 9 independent assays for Apremilastat three concentration levels (5, 10 and 20 μg/ml) (n=3) against a qualified reference standard and the %RSD was calculated. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (5, 10 and 20 μg/ml) and the % RSD was calculated.

 

Accuracy:

The accuracy of the assay method was evaluated using standard addition method followed by recovery studies (80, 100 and 120%)

 

Robustness:

The robustness of the assay method was established by introducing very small changes in the optimized HPLC conditions that include detection wavelength, mobile phase composition and flow rate. A 10 μg/mlof Apremilast was used for the Robustness study.

 

Limit of quantification and Limit of detection:

The limit of quantification and limit of detection were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines Q2 (R1) (ICH guidelines, 2005). Sensitivity of the method was established with respect to limit of detection LOD and LOQ for analytes.

 

Forced degradation studies:

Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method (ICH guidelines, 2003). All solutions for this study were prepared at an initial concentration of 50 μg/ml of APT and refluxed for 20 min at 80 ºC and then diluted with mobile phase. 

 

Acidic degradation:

Acidic degradation was performed by treating the drug solution (50 μg/ml) with 0.1 M HCl for 1hour in a thermostat maintained at 80 ºC. The stressed sample was cooled, neutralized with NaOH and then diluted with mobile phase as per the requirement. 20 µL of this solution was injected in to the HPLC system.

 

Alkaline degradation:

Alkaline degradation was performed by treating the drug solution (50 μg/ml) with 0.1 N sodium hydroxide for 1hour in a thermostat maintained at 80ºC. The stressed sample was cooled, neutralized with HCl and then diluted with mobile phase as per the requirement and 20 µL of the solution was injected in to the HPLC system.

 

Oxidation degradation:

Oxidation degradation was performed by treating the drug solution (50 μg/ml) with 30% H₂O₂ for 1 hour in a thermostat maintained at 80ºC. The drug solution mixture was cooled and then diluted with mobile phase as per the requirement and 20 µl of the solution was injected in to the HPLC system.

 

Assay of marketed formulations (Tablets):

Twenty tablets of Apremilast were procured from the medical store, weighed, crushed to a fine powder and powder equivalent to 25 mg of Apremilast was accurately weighed and transferred carefully into a 25 ml volumetric flask and made up to volume with mobile phase. The contents of the volumetric flask were sonicated for 30 min to enable complete dissolution and filtered. The filtrate was diluted with mobile phase as per the requirement. 20 μL of these solutions were injected into the system after filtering through 0.45 μm membrane and the peak area was recorded from the respective chromatogram.

RESULTS AND DISCUSSION:

Method development and optimization:

Initially the stressed samples were analyzed using a mixture of 0.1% acetic acid: acetonitrile (50: 50, %v/v) with a flow rate of 1.0 ml/min in which the peak was obtained at R_t 9.7mins and also the resolution as well as peak symmetry were not satisfactory. Therefore the mobile phase ratio was modified as 40:60, %v/v and the drug sample was analyzed where a sharp peak was eluted at 7.1 mins with tailing. Finally the mobile phase composition was adjusted to 20:80, %v/v and a sharp and symmetrical drug peak was eluted at 5.30 ± 0.02 mins (UV detection 203 nm). The chromatogram of the mobile phase (blank) and the Apremilast standard solution were shown in Fig 2A and Fig 2B respectively.

 

Method Validation:

The method was validated for linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity, system suitability, and robustness (ICH guidelines, 2005).

Linearity:

Apremilast has shown linearity over a concentration range 0.5-150 μg/ml(Table 1) with linear regression equation y = 78597x - 14159 (r² = 0.9999) and the respective chromatogram was shown in Fig 3.

 

 

Table.1. Linearity of Apremilast

Conc. (mg/ml)	*Mean peak area ± SD	RSD (%)
0.5	39425 ± 72.80	0.18
1	78015 ± 89.74	0.11
5	386574 ± 3830.9	0.99
10	776979 ± 2190.55	0.28
20	1557458 ± 2165.66	0.13
40	3125478 ± 19549.81	0.62
50	3881245 ± 10677.53	0.27
100	77612457 ± 122506.2	0.15
120	79169915 ± 124789.8	0.16
150	11642572 ± 154578.1	0.89

*Mean of three replicates

 

 

Fig 2. Typical chromatograms of blank [A], Apremilast standard (10 μg/ml) [B], OTEZLA (10 μg/ml) [C]

 

Fig 3.Calibration curve of Apremilast

Table.2. Precision and accuracy studies of Apremilast

*Mean of three replicates

 

Table.3. Robustness study of Apremilast

*Mean of three replicates

 

Accuracy:

The method accuracy was proved by the recovery test at three different concentrations (80, 100 and 120 %). A known amount of Apremilast standard (5 μg/ml) were added to aliquots of sample solutions and then diluted to yield the total concentrations of 9, 10 and 11 μg/ml as described in Table 2. The % RSD was found to be 0.30- 0.48 (<2.0 %) with a recovery of 98.88 - 99.52 %.

 

Precision:

The intra-day precision of the method was determined by assaying three samples of each at three different concentration levels (5, 10 and 20 µg/ml) on the same day. The inter-day precision was calculated by assaying three samples of each at three different concentration levels (5, 10 and 20 µg/ml) on three different days. The % RSD for intra-day precision was found to be 0.20-0.31whereas the inter-day precision was found to be 0.72-0.79 (Table 2).

 

Robustness:

Slight changes in flow rate, detection wavelength, mobile phase composition etc. affects the chromatographic response such as retention time, tailing factor and theoretical plates etc and the results were given in Table 3.  The % RSD obtained was 0.11-0.28 for APT (< 2.0%) indicating that the proposed method is robust.

 

The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability for routine analysis. The robustness of the method was evaluated by assaying the same sample under different analytical conditions deliberately changing from the original condition. The detection wavelength was set at 201 and 205 nm (± 2 nm), the ratio of percentage of 0.1% acetic acid: acetonitrile in the mobile phase was applied as 18:78 and 22:82 (± 2, %v/v), the flow rate was set at 0.7 and 0.9 ml/min (± 0.1 ml/min). The results obtained (Table 3) from assay of the test solutions were not affected by varying the conditions and were in accordance with the results for original conditions. The % RSD value of assay determined for the same sample under original conditions and robustness conditions was less than 2.0% (0.11-0.28) indicating that the method is robust.

 

Analysis of commercial formulations:

The proposed method was applied for the determination of Apremilast in marketed formulations available and the % recovery was found to be 99.12 (Table 4). The resultant chromatogram obtained for the marketed formulation was shown in Fig 2C.

 

_{Table.4. Analysisof}Apremilast _{in commercialformulation(Tablet)}

Formulation	Labelled claim (mg)	Amount found* (mg)	Recovery* (%)
Brand I	10	9.91	99.12

* Mean of three replicates

 

Stress degradation studies:

The stability indicating capability of the method was established from the separation of Apremilast peak from the degraded samples. The degradation of Apremilast was found to be very similar for both the marketed formulation and standard. Typical chromatograms obtained following the assay of stressed samples are shown in Fig 4(A-G). A slight decomposition (< 20 %) was observed when APT drug was exposed to acidic (7.51 %), alkaline (11.72 %) thermal (0.03 %) and oxidative degradation (8.82 %) (Table 5).

The system suitability parameters for all the degradation studies were shown in Table 5. The number of theoretical plates (N) is used to determine the performance and effectiveness of the column. The efficiency of a column can be measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical plates, indicating good column and system performance. Columns with theoretical plates ranging from 4,000 to 100,000 plates / meter are ideal for a good system. The theoretical plates were found to be more than 4000 and the tailing factor was less than <1.5–2 or <2 indicating that the method is more selective and specific.

 

Fig 4.Typical chromatograms of blank [A], Apremilast standard (10 μg/ml) [B], acidic [C], alkaline [D], oxidative [E], and thermal [F] degradations

 

Table.5. Stress degradation studies of Apremilast

*Mean of three replicates

 

CONCLUSION:

The proposed stability-indicating HPLC method was validated as per ICH guidelines and applied for the determination of Apremilast in pharmaceutical dosage forms and can be successfully applied to perform long-term and accelerated stability studies of Apremilast formulations. It was observed that Apremilast is more sensitive towards the alkaline environment during the forced degradation studies.

 

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM University, Visakhapatnam for providing the research facilities. The authors have no conflict of interest.

REFERENCES:

1.        Gulen H, Melike M, Recep T, Cengiz K, Ozturk T, Hasan Y and Yusuf Y, Apremilast for Behçet's Syndrome — A Phase 2, Placebo-Controlled Study. New England Journal of Medicine, 372, 2015,1510-1518.

2.        Syedakulsum, Vidyasagar G, Afreenbutul, Saba Fatima, Samiuddin MD, Method development and validation of forced degradation studies of Aapremilast by using UV spectrophotometric method”, world journal of pharmacy and pharmaceutical sciences, 2016, 5(6), 1595-1601.

3.        KaiheXiong, Xingling Ma, Na Cao, Lei Liu, Lili Sun, Qiaogen Zou, Ping Weia, Identification, characterization and HPLC quantification of impurities in Apremilast. Royal society of chemistry, 2016, 8, 1889-1897.

4.        ICH stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, 2003.

5.         ICH validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization, 2005.

 

 

 

Accepted on 30.04.2017           © RJPT All right reserved