INTRODUCTION:

In Spectrophotometric techniques, derivative spectroscopy gained more significance than any other method [1]. The derivative spectra arises after derivatization of zero-order spectrum may lead to separation of overlapped signals, elimination of background caused by presence of other compounds in a sample [2]. Derivative spectroscopy is important method for qualitative and quantitative data of analytes and derivative spectra is extracted with the help of computer controlled information [3].

In quantitative analysis, derivative spectra increase diversity involving spectra to resolve overlapping bands [4]. The digital algorithm method is known as Savitzky-Golay is the most unusually referred for obtaining derivative spectra [5]. In universal technique involves plotting the rate of change of the absorbance spectrum versus wavelength [6]. The derivative spectra obtained by discriminate zero order spectra by mathematical transformation into first, second or higher order spectra [7]. Zero order derivative is initial step of giving further derivatives i.e., 0^th order spectrum can give n^th order derivative. An increase in order of derivatives increases the sensitivity of determination [8].

Area under curve technique:

The Area Under Curve (AUC) technique is applicable where no sharp or when broad spectra are obtained. It is in the calculation of integrated value of absorbance with respectively to the wavelength between the two selected wavelengths λ1 and λ2 [9]. Area calculation processing atom calculates the area bound by the curve and horizontal axis. The horizontal axis selected by entering the wavelength range over which area has to be calculated. This wavelength range is selected on the basis of repeated observations so as to get the linearity between AUC and concentration the spectrum obtained from zero order and first order derivative was used to calculate AUC [10]. The calibration curve was constructed by plotting AUC and concentration.

Application of Derivative and AUC for Estimation of Apremilast:

Psoriasis is a simple, widespread inflammatory skin disease resulting from the interplay of genetic, environmental and immunological factors [11]. Apremilast is phosphodiesterase 4- inhibitor; Apremilast is a small molecular inhibitor to facilitate by rising cyclic adenosine monophosphate levels, eventually suppressing tumor necrosis alpha production [12]. Apremilast is chemically designated as N-[2-[(1S)-1-(3-ethoxy-4-methoxyphenyl)-2-methylsulfonylethyl]-1,3 dioxoisoindol-4-yl]acetamide [13] shown in Figure 1

Figure 1: Chemical Structure of Apremilast

Literature survey revealed few methods for analysis of Apremilast in bulk, pharmaceutical formulations and biological fluids which includes UPLC–MS/MS [14], High-Performance Liquid-Chromatography method for quantification of impurities of Apremilast [15] and stability-indicating and assay UV- Spectrophotometric method [16, 17]. However, to our attempt no UV-Spectrophotometry method has been studied so for the estimation of Apremilast in bulk material and in-house tablets formulation using derivative spectroscopic techniques. Consequently, our venture is to constitute zero and first order derivative UV-spectrophotometry applying amplitude and also AUC techniques. The current investigate emphasize a simple, sensitive and effective UV-Spectrophotometry methods for estimation of Apremilast in bulk material and in-house tablets formulation. Further, methods were validated as per ICH guidelines.

MATERIALS AND METHODS:

Chemicals:

Pharmaceutical grade Apremilast working standards were obtained as generous gifts from Intas Pharmaceuticals Ltd, Ahmadabad, India. Methanol (HPLC Grade) was purchased from E. Merck Ltd., Mumbai (India).

Instruments:

A double beam UV-VIS Spectrophotometer (UV-2450, Shimadzu, Japan) was used, which was linked to computer installed with spectra manager software UV Probe 2.21with 10 mm quartz cells. The spectra were obtained with following set of instrumental parameters: wavelength range: 400 - 200 nm; scan speed: medium; sampling interval: 10 nm; band width: 1.0 nm; spectral slit width: 1 nm. An electronic balance (Model Shimadzu AUX 120) was used for weighing purpose.

Preparation of Stock Standard Solution:

An accurately weighed sample 10 mg of Apremilast was transferred in 100 mL of volumetric flask and dissolved in methanol to achieve concentration of 100 μg/mL.

Selection of Appropriate wavelength for Determination of Apremilast:

An appropriate volume of 1 mL was transferred into 10 mL volumetric flask and the volume was adjusted to the mark with same solvent to obtain concentration of 10 μg/mL. The resulting solution was scanned in the UV range 400 - 200 nm. In Method A (Zero order Spectrophotometry) absorbance was recorded at 230 nm shown in Figure 2. While in Method B (Zero order Spectrophotometry- AUC) was selected in the wavelength range of 226.20 - 233.20 nm shown in Figure 2.

The calibration curves were constructed by plotting concentration versus absorbance and AUC of zero order spectrum in Method A and B, respectively shown in Figure 3. For the Method C (First order Spectrophotometry) Method D (First order Spectrophotometry- AUC), the spectra of above solutions were derivatized into first order using software UV- Probe 2.21with delta lambda 2 and scaling factor 10. In Method C, amplitude was recorded at 233.50 nm shown in Figure 2 while in Method D; AUC was selected in the wavelength range of 233 - 234.60 nm shown in Figure 2. The calibration curves were constructed by plotting concentration versus amplitude and AUC of first order spectrum in Method C and D, respectively, is shown in Figure 3.

Preparation of Sample Solution:

Since the pharmaceutical formulation of Apremilast is not obtainable in the Indian market; therefore, in-house tablets were prepared with 30 mg of Apremilast and common excipients. The sample solution was prepared from in-house formulated Apremilast tablets.

Twenty in-house tablets were taken, and powder was collected and weighed. An accurately weighed portion of the powder equivalent to 30 mg of Apremilast was taken in 100 mL of volumetric flask. About 50 mL of methanol was added to this flask and sonicated in an ultrasonic bath for 15 min and volume was makeup to the mark with same solvent. The resulting solution was filtered through a 0.45 μm filter (Millifilter, Milford, MA, USA). From the filtrate, measured volume was taken and diluted with methanol to achieve concentration 6 μg/mL for all methods. The resulting solutions were scanned using UV-Spectrophotometer in the range of 400 - 200 nm. Results was shown in Table 1.

Table 1: Analysis of in-house Tablets

Methods	(Mean ± SD)	% RSD
I	98.28 ± 0.61	0.62
II	99.17 ± 0.50	0.51
III	98.98 ± 0.83	0.84
IV	99.11 ± 0.30	0.30

Validation of Method:

The proposed method was validated as per the ICH guidelines.

Linearity Study:

From the stock standard solution, aliquots in the range of 0.2–1.2 mL were transferred into a series of 10 mL volumetric flasks and diluted up to mark using the same solvent to obtain a concentration in the range of 2–12 µg/mL. The resulting solution was scanned in the UV range 400 - 200 nm. The overlain zero order spectrum of Apremilast was shown in Figure 4.

Figure 4: The overlain zero order spectrum of Apremilast

Accuracy and Precision:

The accuracy of the anticipated methods were assessed as, recovery studies performed at three different levels i.e. 80, 100 and 120%. To the pre - analyzed sample solution a known quantity of standard drug solution was added at three different levels.

Precision of the method was studied as repeatability, intra-day and inter-day precision. Repeatability was determined by analyzing Apremilast (4 μg/mL) for six times. Intra-day precision was determined by analyzing the 4, 8 and 10 μg/mL of Apremilast for three times in the same day. Inter-day precision was determined by analyzing the same concentration of the solutions daily for three days.

Sensitivity:

Sensitivity of the proposed method was estimated in terms of Detection Limit (DL) and Quantification Limit (QL). The DL and QL were calculated by the use of the equation DL = 3.3 Х ASD/S and QL = 10 Х ASD/S; where, ‘ASD’ is Average standard deviation of the peak height and areas of the drug (n = 3), taken as a measure of noise, and ‘S’ is the slope of the corresponding calibration curve.

Ruggedness:

Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slot by two analysts using same operational and an environmental condition was performed by using concentration 4 μg/mL.

RESULTS AND DISCUSSION:

Developed methods were validated as for accuracy, precision, ruggedness and sensitivity as per the ICH guidelines.

Analysis of in-house Tablets:

The percentage amounts of Apremilast estimated from in-house tablets using all methods shown in Table 1. The % amount estimated from tablet formulation indicates that there is no interference from excipients present in it. Analysis of in-house tablets studies performed at 6 μg/mL.

Accuracy study:

The solutions were re-analyzed by proposed methods; results of recovery studies was reported in Table 2.

Precision Study:

The precision of the developed method was expressed in terms of % relative standard deviation (% RSD). These results show reproducibility of the assay. The % RSD values found to be less than 2 indicate that the methods were precise for the determination of drugs in formulation Table 3.

Table 2: Accuracy Studies

Methods	Excess of drug added to the analyte (%)	% Recovery (Mean ± SD)	% RSD
I	80	99.60 ± 0.39	0.39
	100	99.06 ± 0 .93	0.94
	120	99.30 ± 0.79	0.80
II	80	99.53 ± 1.15	1.15
	100	99.38 ± 1.41	1.42
	120	99.53 ± 0.77	0.77
III	80	99.53 ± 1.14	1.16
	100	99.38 ± 1.40	1.42
	120	99.53 ± 0.65	0.77
IV	80	99.60 ± 0.25	0.39
	100	99.06 ± 0.76	0.94
	120	99.03 ± 0.74	0.80

Table 3: Precision Studies [Intra-day and Inter-day]

Methods	Concentrations (μg/mL)	% Amount found (Mean ± SD)	% RSD
Intra-day Studies
I	4	98.54 ± 0.35	0.50
	8	99.75 ± 0.43	0.65
	10	99.35 ± 0.46	0.72
II	4	98.96 ± 0.63	0.52
	8	99.65 ± 0.58	0.69
	10	99.38 ± 0.63	0.79
III	4	100.48 ± 0.15	0.18
	8	99.89 ± 0.19	0.25
	10	98.87 ± 0.25	0.71
IV	4	99.88 ± 0.32	0.22
	8	99.16 ± 0.36	0.35
	10	101.33 ± 0.45	0.47
Inter-day studies
I	4	98.86 ± 0.69	0.60
	8	99.78 ± 0.45	0.75
	10	99.41 ± 0.56	0.85
II	4	99.20 ± 0.46	0.63
	8	99.68 ± 0.45	0.83
	10	99.31 ± 0.56	1.12
III	4	100 ± 0.45	0.56
	8	99.75 ± 0.62	0.59
	10	98.93 ± 0.49	0.69
IV	4	100 ± 0.45	0.25
	8	99.33 ± 0.95	0.78
	10	100 ± 0.25	0.54

Sensitivity:

The DL and QL for Apremilast were found to be 0.078 μg and 0.220 μg (Method A), 0.011 μg and 0.034 μg (Method B), 0.020 μg and 0.63 μg (Method C), and 0.020 μg and 0.60 μg (Method D), respectively.

Ruggedness Study:

The peak area was measured for the same concentration solutions, six times for all methods. The results were in the acceptable range for the drug. The results showed that the % RSD was less than 2% in Table 4.

Table 4: Ruggedness studies

Methods	Analysts-I % Amount found (Mean ± SD)	% RSD	Analysts- II % Amount found (Mean ± SD)	% RSD
I	99.80 ± 0.30	0.30	99.66 ± 0.96	0.34
II	100.46 ± 0.33	0.96	100.30 ± 0.95	0.95
III	99.43 ± 0.19	0.25	99.62 ± 0.28	0.14
IV	99.95 ± 0.15	0.28	99.75 ± 0.40	0.40

CONCLUSION:

Overall four methods were established for quantitative analysis of Apremilast in bulk and in-house tablets using zero order, first order and AUC technique of UV-Spectrophotometry. The results and statistical limits show that the developed UV-Spectrophotometric methods are simple, accurate and precise and specific. Therefore, these methods can be routinely used for estimation of Apremilast in bulk and pharmaceutical formulations.

ACKNOWLEDGMENT:

Authors are thankful to Principal of R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist: Dhule (MS) for providing necessary laboratory facility and Intas Pharmaceuticals Ltd. for providing gorgeous gift sample of Apremilast for project work.

Disclosure of interest:

The authors declare that they have no competing interest.

REFERENCE:

1. Rojas FS, Ojeda CB. Recent development in derivative ultraviolet/visible absorption spectrophotometry: 2004–2008: A review. Analytica Chimica Acta. 2009; 635: 22 -44.

2. Kowalczuk D, Hopkała H. Application of derivative UV spectrophotometry for the determination of cinoxacin and oxolinic acid in pure and dosage forms. Chemia Analityczna 2003; 48: 97 -106.

3. Karpińska J. Derivative spectrophotometry- recent applications and directions of developments. Talanta 2004; 64: 801 -822.

4. Erk N. Application of first derivative UV-spectrophotometry and ratio derivative spectrophotometry for the simultaneous determination of candesartan cilexetil and hydrochlorothiazide. Die Pharmazie-An International Journal of Pharmaceutical Sciences 2003; 58: 796 -800.

5. Jeffery GH, Bassett J, Mendham J, Denney RC. Textbook of quantitative chemical analysis. New York, Longman. 1989.

6. Willard HH, Merritt Jr, LL, Dean JA, Settle Jr, FA. Instrumental methods of analysis. New Delhi, CBS Publishers. 1988.

7. Mendham L, Denney R, Barnes J, Thomas M, Shivasankar B. Vogel’s Textbook of Quantitative Chemical Analaysis. New York. John Wiley and Sons Inc.

8. Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. New Delhi, CBS Publications and Distributors. 1997.

9. Patil AS, Shirkhedkar AA. Development and Validation of Five Simple UV-Spectrophotometry Methods for Estimation of Anagliptin in Bulk and in-house Tablets. Pharmaceutical Methods. 2016; 7: 127.

10. Dhangar KR, Shirkhedkar AA. Estimation of Delafloxacin Using Derivative Spectrophotometry and Area Under Curve in Bulk Material and in Laboratory Mixture. Journal of Pharmaceutical Technology and Research Management 2016; 4: 81-87.

11. Di Meglio P, Nestle FO. Immunopathogenesis of Psoriasis. In Clinical and Basic Immunodermatology. Springer International Publishing; 2017.

12. Schafer P. Apremilast mechanism of action and application to psoriasis and psoriatic arthritis. Biochemical Pharmacology 2012; 83: 1583 -1590.

13. Ruchelman AL, Connolly TJ. Enantioselective synthesis of the Apremilast aminosulfone using catalytic asymmetric hydrogenation . Tetrahedron Asymmetry 2015; 26: 553 -559.

14. Iqbal M, Ezzeldin E, Al-Rashood ST, Imam F, Al-Rashood KA. Determination of Apremilast in rat plasma by UPLC–MS/MS in ESI-negative mode to avoid adduct ions formation. Bioanalysis. 2016; 8(14):1499-508.

15. Xiong K, Ma X, Cao N, Liu L, Sun L, Zou Q, et al. Identification, characterization and HPLC quantification of impurities in Apremilast. Analytical Methods. 2016; 8(8):1889-97.

16. Panchumarthy R, Sulthana MS, Babu PS. Development and validation of stability-indicating UV spectrophotometric method for determination of Apremilast in bulk and pharmaceutical dosage form. Ind. J. Res. Pharm. and Biotech. 2017;5(1):147.

17. Chakravarthy AV, Sailaja BBV, Kumar PA. Method development and validation of ultraviolet-visible spectroscopic method for the estimation of assay of sugammadex sodium, Apremilast, riociguat, and vorapaxar sulfate drugs in active pharmaceutical ingredient form. Asian Journal of Pharmaceutical and Clinical Research. 2017; 10: 241 -250.

Received on 06.11.2017 Accepted on 10.01.2018

Asian J. Pharm. Res. 2018; 8(1):11-16.

DOI: 10.5958/2231-5691.2018.00003.5