Determination of λ Max of Component:

Aliquots (0.5ml, 1ml, 1.5ml, 2ml, 2.5ml, 3ml) of prepared standard solution were transferred into series of 10ml volumetric flasks and diluted by HPLC grade methanol to give the concentration range of 2-12μg/ml. The above solutions were scanned over the range of 200 nm to 400nm against reagent blank. The absorbance of each solution at 261nm was scanned against HPLC grade methanol as blank.

Overlay Spectra of Ascorbyl palmitate:

The overlain spectrum of drug was recorded (Fig.1) and wavelength 261nm were selected for further study.

Validation of Proposed Method:

The Proposed method was validated as per the ICH guidelines.

Linearity Study for Ascorbyl palmitate:

Fresh aliquots were prepared from standard stock solution-2 ranging from 2-12μg/ml and the absorbance values of each concentration was recorded at 261 nm for this method using HPLC grade Methanol as blank. The drug shows linearity between 2-12μg/ml for this method. Results were shown in Table-1 and figure-2²

Figure 2: Linearity curve of Ascorbyl palmitate

Table 1: Linearity results of Ascorbyl palmitate:

S. No	Concentration (µg/ml)	Absorbance (at 261nm)
1	2	0.06
2	4	0.104
3	6	0.154
4	8	0.196
5	10	0.245
6	12	0.293

Accuracy [Recovery Study]:

Accuracy of the developed method was confirmed by performing recovery studies at three different concentration ranges 50%, 100%, 150% each one in triplicate. From the recovery studies it was clear that the method is very accurate for quantitative estimation of tablet as the statistical results were within the acceptance range. Results were shown in Table-2.

Amount Found =

test absorbance 20 10 2 potency

––––––––––––––––× ––– × –––× –– × ––––––– × 1000

std absorbance(100%absorbance) 100 50 10 100

Amount found

% Recovery = –––––––––––––––×100

Amount added

The results are reported in (Table 2).

Table 2: Recovery Study.

S. No	Spike Level	µg/ml added	µg/ml found	% recovery	Mean % recovery
1	50%	6	6.22	103.6	103.60%
2	50%	6	6.13	102.1
3	50%	6	6.3	105
1	100%	8	7.87	98.3	100.12%
2	100%	8	8.04	100.5
3	100%	8	8.12	101.5
1	150%	10	10.36	103.6	103.60%
2	150%	10	10.2	102
3	150%	10	10.53	105.3

Precision:

In intraday study, concentration of replicates of drug was calculated on the same day for two times. In inter-day study the concentration of drug were calculated on two successive days which expresses the laboratory variation in different days. In both intra and inter day precision study for the methods %RSD was calculated and results are shown in Table 3 and 4.

Precision Studies:³

Table 3: Intra-day precision (Repeatability) data of proposed method

S. No	Concentration (µg/ml)	Absorbance (at 261nm)
S. No	Concentration (µg/ml)	Morning	Evening
1	12	0.293	0.302
2	12	0.298	0.305
3	12	0.298	0.299
4	12	0.296	0.298
5	12	0.292	0.298
6	12	0.304	0.307
Average		0.297	0.301
S.D		0.004	0.003
%R.S.D		1.451	1.272

Table 4: Inter-day precision (Reproducibility) data of proposed method

S. No	Concentration (µg/ml)	Absorbance (at 261 nm)
S. No	Concentration (µg/ml)	Day 1	Day 2	Day 3
1	12	0.293	0.222	0.301
2	12	0.298	0.227	0.306
3	12	0.298	0.226	0.304
4	12	0.296	0.226	0.298
5	12	0.292	0.224	0.297
6	12	0.304	0.229	0.294
Average		0.297	0.225	0.300
S.D		0.004	0.002	0.004
%R.S.D		1.451	1.073	1.33

Robustness:

Robustness of the method was determined by carrying out the analysis at three different wavelengths (±2nm). The respective absorbance was noted and the result was indicated by % RSD and results were shown in Table-5⁴

Table 5: Robustness study

S. No	Concentration ( µg / ml)	Absorbance
S. No	Concentration ( µg / ml)	260 nm	261 nm	262 nm
1	10	0.089	0.088	0.087
2	10	0.089	0.088	0.088
3	10	0.089	0.088	0.087
4	10	0.087	0.086	0.085
5	10	0.088	0.086	0.085
6	10	0.089	0.087	0.085
Average		0.088	0.087	0.086
S.D		0.0008	0.0009	0.001
%R.S.D		0.945	1.127	1.542

Ruggedness:

Ruggedness of the method was determined by carrying out the analysis by two different analysts and the respective absorbance was noted. The result was indicated by % RSD and results were shown in Table-6.

Table 6: Ruggedness Study.

S. No	Concentration (µg/ml)	Absorbance (at 261 nm)
S. No	Concentration (µg/ml)	Analyst 1	Analyst 2
1	10	0.249	0.246
2	10	0.252	0.248
3	10	0.247	0.244
4	10	0.25	0.25
5	10	0.247	0.248
6	10	0.246	0.247
Average		0.248	0.247
S.D		0.002	0.002
%R.S.D		0.908	0.825

Limit of Detection and Limit of Quantification:

The limit of detection and limit of quantification of Ascorbyl palmitate by proposed method were determined using calibration graphs.⁵

LOQ and LOD were calculated as,

LOD = 3.3 X S.D/S

3.3 × 0.00083666

= –––––––––––––––

0.023

= 0.1200μg/ml

LOQ = 10 X S.D/S

10 × 0.00083666

= –––––––––––––––

0.023

= 0.363μg/ml

Where S is the slope of the calibration curve and SD is the standard deviation of response of least concentration of calibration curve in three replicates.

test absorbance

Assay: ––––––––––––––––––––– × 100

standard absorbance

0.149

= ––––––––– × 100

0.154

= 96.75

The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (98.94% to 100.80%) of the drug were obtained at each added concentration, which indicates that the method was accurate. The LOD and LOQ were found to be in sub-microgram level, which indicates the sensitivity of the method. The method was also found to be robust and rugged as indicated by the %RSD values which are less than 2%. The results of assay show that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery (%).

Table 7: Assay results of in Tablets

Formulation	Brand name	Label Claim	Amount Found	% Assay
Ascorbyl palmitate	Ascorbyl palmitate	500mg	483mg	96.75%

ACKNOWLEDGEMENT:

We wish to thank Management and Dr. M. Chinna Eswaraiah, Principal of Anurag Pharmacy College, Kodad for their moral support and blessings and also express our heartful thanks to them for motivating us with strength and spirit.

REFERENCES:

1. W.M. Cort; Antioxidant Activity of Tocopherols, Ascorbyl Palmitate and Ascorbic Acid and Their Mode of Action, Journal of The American Oil Chemists Society, 1974, Vol-51, Issue-7.

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3. V.P Devamurari; Simultaneous Spectrophotometric Determination of Lamivudine and Abacavir in the Mixture, Asian Journal of Research in Chemistry, 2016, Vol-I, Issue –7, 707-709.

4. Indrani Chandra, Ripunjoy Bardoloi, Debarupa D. Chakraborty, Prithviraj Chakraborty, Smita Rekha Chanda Das; Development & Validation of UV-Spectroscopic Method for Estimation of Niacin in Bulk & Pharmaceutical Dosage Form, Journal of Applied Pharmaceutical Sciences, Vol-7, Issue-09, 81-84.

5. K. Anandakumar, K. Anusha, V. Kokila Vani, V.P, Sangeetha, R. Saranya, M. Jambu, Lingam, J Ramesh; Method Development and Validation of UV Spectroscopic Method for the Determination of Lamivudine as an active Pharmaceutical Ingredient and In Tablet Dosage Form, International Journal of Pharmaceuticals & Health Care Research, 2017, Vol-5, Issue -04. 129-137.

Received on 17.07.2020 Modified on 16.09.2021

Research J. Pharm. and Tech. 2022; 15(6):2737-2740.

DOI: 10.52711/0974-360X.2022.00458