INTRODUCTION:

Analytical chemistry may be defined as the science and art of determining the composition of materials in term of the element and compounds on them. It is concerned with the chemical characterization of matter both quantitative and qualitative¹. Analytical method development validation¹² plays important role in the discovery and development of pharmaceuticals². The official test methods obtained from these processes are used by quality control laboratories^3,4 to ensure identity, purity, potency and performance of drug^5,6.

Evening primrose oil:

Evening primrose oil (EPO) contains an omega-6 essential fatty acid, called gamma-linolenic acid (GLA), which is believed to be EPO's active ingredient. Essential fatty acids are required by the body for growth and development, and must be obtained from the diet⁷.

Chemical Structure Evening primrose oil⁸:

In view of its pharmaceutical applications it is necessary to develop a suitable analytical method for the estimation of evening primrose oil in pharmaceutical preparations. As the literature survey shows that no UV- spectroscopic method has been developed for evening primrose oil, the present work involves development of a new UV spectroscopic analytical method which is sensitive, precise and accurate for evening primrose oil, validate it and apply for evening primrose oil formulation⁹.

MATERIALS AND METHODS:^10,11

Reagents and chemicals:

Analytical standards such as evening primrose oil were obtained from Sigma-Aldrich The tablets (efem) were purchased from local pharmacy. All the solvents used were of HPLC grade are purchased from Merck, Mumbai, India. All the chemicals used for developing method were of AR grade and purchased from sigma Aldrich.

Instrument and analytical conditions:

UV-Visible double beam spectrophotometer (UV-1800, SHIMADZU Limited, Japan) with 1cm matched quartz cells wavelength accuracy of ±0.5nm (with automatic wavelength correction) was used. The drug analyses data were acquired and processed using UV Probe software (Version 2.0, Shimadzu, Japan). For scanning, the wavelength range selected was from 200nm to 400nm.

Preparation of Standard Stock Solutions:

Accurately weighed 100mg of evening primrose oil in 100ml volumetric flask and dissolved in cyclohexane and the volume were made up to the mark with the same solvent. This gave the concentration of 1000µg mL^-1 of evening primrose oil, from this five dilutions of evening primrose oil were prepared in between 100-500µg mL^-1 with cyclohexane and used in UV spectrophotometric estimation.

Preparation of Working Standard:

Solution Suitable aliquots of 1000mg mL-1 solution were diluted up to the mark with cyclohexane to get the concentration range of 100, 200, 300, 400 and 500mg mL-1 of evening primrose oil. The absorbance was measured at 233nm.

Sample Solution Preparation:

Efem capsule containing Evening primrose oil taken were weighed accurately and the average weight was calculated and respective concentrations were prepared.

Selection of Wavelength:

The absorbance of the solutions containing Evening primrose oil at 100µg/ml was determined in the UV range 200-400nm using an appropriate blank. The λ _max was found to be 233nm which is shown in fig no. 1b

Amount of evening primrose oil in each capsule was calculated by using following formula

Sample Absorbance × Standard Dilution × Average Weight (1)

Standard Absorbance Sample Dilution

% Content = Amount Present / Label Claim × 100 (2)

Preparation of efem capsule and Assay Procedure:

Efem capsules containing 100mg of evening primrose oil were weighed and transferred to 100ml volumetric flask containing 25ml of cyclohexane and sonicated for 10 min. The above solution was carefully filtered through Whatmann filter paper (No.41) and the residue was washed with 3 portions of 5ml of solvent. The volume was made up to the mark with cyclohexane. From this solution, required dilutions for UV spectroscopic method were prepared by using cyclohexane as a solvent.

Calculation:

% assay of Evening primrose oil =

AT WS DT P Avg. Wt

----- × ----- × ----- ×---- × ----------------- × 100

AS DS WT 100 Label Claim

Where:

AT = Absorbance of Evening primrose oil obtained with test

preparation

AS = Absorbance of Evening primrose oil obtained with standard

preparation

WS = Weight of working standard taken in mg; WT = Weight of

sample taken in mg

DS = Dilution of Standard solution; DT = Dilution of sample solution

P = Percentage purity of working standard

Table no. 01Assay of Evening Primrose oil

Brand name	Standard absorbance	Sample absorbance	% Assay
Brand I	0.83	0.83	100.58

METHOD VALIDATION:

1. Precision: a. Repeatability (Intra-day precision):

Intra-day precision of the test method was determined by using six samples prepared by spiking Evening primrose oil raw material with the equivalent amount of placebo or on the tablets in the same day over a short interval of time. The precision of test procedure was evaluated for Evening primrose oil by performing the assay as per the test method. The results were summarized in table no. 2

Table no.2: Intra-day precision

Sample Name	Sample Absorbance	% Assay
Sample-1	0.61	99.9
Sample-2	0.6	98.26
Sample-3	0.61	99.9
Sample-4	0.62	101.54
Sample-5	0.6	98.26
Sample-6	0.61	99.9
Average	0.60	99.62
%RSD	1.12	1.12

a. Intermediate Precision (Inter-day precision):

Inter-day precision of the test method was determined by using six samples prepared by spiking Evening primrose oil raw material with the equivalent amount of placebo or on the tablets between days (in next three days). The precision of test procedure was evaluated for Evening primrose oil by performing the assay as per the test method. The results were summarized in table no.3

Table no.3: Inter-day precision

Sample Name	Sample Absorbance	% Assay
Sample-1	0.64	99.99
Sample-2	0.65	101.54
Sample-3	0.64	99.99
Sample-4	0.66	101.94
Sample-5	0.65	101.54
Sample-6	0.64	99.99
Average	0.64	99.99
%RSD	1.26	1.26

Acceptance Criteria:

The % Relative Standard Deviation of % Assay of EPO from the six sample preparations should be not more than 2.0%. The individual % Assay of EPO should not be more than 98.0% and not less than 102.0%.

2. Accuracy:

Recovery:

To determine the accuracy of the test method samples were prepared by spiking Evening primrose oil raw material with the equivalent amount of placebo at 50%, 100% and 150% of the target concentration 3 samples were prepared at highest and lowest concentration levels and three samples were prepared at 100% concentration level. The average % recovery of Evening primrose oil was found to be within the limits. The results were summarized in Table No: 4

Acceptance Criteria:

The Correlation Coefficient should be not less than 0.999.

Report:

All the absorbance values are proportional to the concentration. So that our method is linear.

4. Range:

The range of an analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of accuracy, precision and linearity. Data from linearity, precision, accuracy sections was considered to establish range of the method. The results were summarized in table no.06

Table. no 06: Range

Parameter	Result
Linearity	0.999
Precision	1.12-1.26
Accuracy	99.66-100.58

Acceptance Criteria:

For linearity, correlation coefficient shall be ≥ 0.999.

For precision, %RSD of assay of 6 replicate sample preparations shall be NMT 2.0%.

For accuracy, individual recovery at each spike level 98.0% to 102.0%

5. Specificity and selectivity:

The analyte should have no interference from other extraneous components and be well resolved from them. The specificity of the method was evaluated with regard to interference due to presence of any other excipients.

6. Limit of detection (LOD) and Limit of quantitation (LOQ):

The limits of detection and quantitation, LOD and LOQ, were calculated by use of the equations LOD = 3.3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the blank and S is the slope of the calibration plot.

7. Ruggedness: Analyst to analyst/ System to system variability:

Ruggedness of assay method was conducted on Evening primrose oil tablets using two different systems by different analysts and analyzed under similar conditions as per the test method. The results were summarized in table no.07 and 08

Analyst to analyst variability:

Table no.07 Analyst to analyst variability

No.

System suitability

Observed value

Analyst-1

Analyst-2

%RSD for Evening primrose oil in standard solution

1.12

1.26

System to system variability:

Table no.08 System to system variability

No.

System suitability

Observed value

System-1

System-2

%RSD for Evening primrose oil in standard solution

0.98

0.16

Acceptance Criteria:

The % Relative Standard Deviation for the absorbance’s of evening primrose oil from the six standard replicates results should be not more than 2.0%.

Table-9: Optical characteristics

S. No.	Parameter	Result
1	Absorption Maximum (nm)	233nm
2	Linearity	2-10µg/ml
3	Standard Regression Equation	y= 0.0021x+0.0005
4	Slope	0.0021
5	Intercept	0.0005
6	Correlation Coefficient (r)	0.999
7	Molar extinction coefficient (L.mol^-1cm^-1)	9.733 × 10³
8	Sandell’s sensitivity (µg/cm²- 0.001absorbance units)	0.07
9	Accuracy (% Recovery)	99.66-100.58
10	Precision (Intra-day) %RSD (Inter-day) %RSD	1.12 1.26
11	LOD (µg/ml)	7.5
12	LOQ (µg/ml)	23
13	Standard error	0.00483

ASSAY

Table No.1: Assay

Brand name	Standard absorbance	Sample absorbance	% Assay
Brand I	0.83	0.83	100.58

DISCUSSION:

The objective of the proposed work was to develop some new and sensitive analytical methods for the determination of Evening primrose oil and to validate the methods according to ICH guidelines and applying the same for its estimation in pharmaceutical formulations.

In the start of the method development for this drug, different solvents were tested such as cyclohexane, toluene, alcohol. Due to greater solubility and reproducible readings of maximum absorbance 233nm, cyclohexane was taken under consideration for further work. The UV spectrum of Evening primrose oil was obtained by using cyclohexane as a solvent and then validated. The λ_max of drug in cyclohexane was determined using UV-Spectrophotometer. The λ _max was determined by scanning 100μg/mL solution of drug in the cyclohexane in the range of 200-400nm. The wavelength 233nm was selected because it showed maximum absorbance by the solvent cyclohexane.

Calibration curve of drug were prepared in cyclohexane at λ_max 233nm. The absorbance values with their standard deviations at different concentration in the range of 100-500μg/mL are given in table no.6 and calibration curve was prepared by plotting graph between absorbance and concentration (µg/ml).

The accuracy of the method was determined by recovery studies by adding known amount of the pure drug to the formulation. Thus, for accuracy, recovery studies were carried out and the percentage recovery was found to be in the range of 98.76% - 99.73%, which was within the recommended limits, indicating that the method has required accuracy. The results are shown in table no. 4.

The precision of an analytical method or a test procedure is referred to as the degree of closeness of the result obtained by the analytical method or the test procedure to the true value. For evaluation of the precision, the %RSD was determined and the % assay was calculated. For intra-day precision, the %RSD was found to be 0.29 and % assay was found to be 99.13. For inter-day precision, the %RSD was found to be 0.47 and % assay was found to be 99.04. The results are given in Table 6 and 7. It is suggested that the analytical method may be considered validated in terms of precision if the precision around the mean value does not exceed 2% RSD. The results obtained in intra-day precision and inter-day precision expresses the precision of the method.

Linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. Data from the regression line is helpful to provide mathematical estimates of the degree of linearity. Linearity data was prepared in cyclohexane. The extract was found to obey Beer’s law in the concentration range of 100-500μg/ml with correlation coefficient (r) values 0.999. The regression equations were calculated as y=0.0021x+0.0005 for cyclohexane. The results were shown in Figure 1 and table No.9. The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity, slope (b), intercept (C), correlation coefficient (r) obtained from different concentrations, and percent relative standard deviation values were presented in table 9.

Limit of detection (LOD) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under a stated experimental condition and the limit of quantitation (LOQ) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. These two parameters are required for assay validation as per ICH Q2A guidelines. Limit of detection and limit of quantitation of calibration curve were calculated which was based on the standard deviation of y-intercept of regression line (SD) and the slope (S) of the calibration curve at levels approximating the LOD and LOQ, LOD = 3.3 (SD/S) and LOQ =10 (SD/S). LOD and LOQ of calibration curve of drug prepared in Cyclohexane were found to be 1.6 and 4.9µg/ml, respectively. Ruggedness of the proposed method was determined by analysis of aliquots from homogeneous slots by using two different systems by different analysts and analyzed under similar operational and environmental conditions. The % RSD reported was found to be less than 2. The results were shown in the table 7 and 8, indicating that the cyclohexane is rugged.

SUMMARY AND CONCLUSION:

An efficient UV Spectrophotometric method was developed and validated for estimation of Evening primrose oil in capsule dosage forms. The method was developed using cyclohexane as solvent and the λmax was found to be 233nm. The method was validated with respect to linearity, precision, accuracy, sensitivity and ruggedness. The calibration plot for the method was constructed. The method was established according to ICH guideline and definition. Accuracy was investigated by analyzing marketed formulations and percentage recovery was found to be within the limits. Therefore, it can be said that the method was highly accurate. The interday and intraday relative standard deviation (RSD) values with low percentage RSD values were obtained. This indicated that the precision of the method was found to be good. The proposed method based on UV spectrophotometer is precise, accurate, simple to perform and economy in practice. It does not require expensive or sophisticated and chemicals in contrast with chromatographic method.

ACKNOWLEDGMENT:

The authors are thankful to the management of Vishnu Institute of Pharmaceutical Education and Research, Narsapur-Medak T.S., for providing necessary chemicals and analytical facilities and to Sigma Aldrich Pvt. Ltd. India, for providing pharmaceutical grade Evening Primrose oil as gift sample.

REFERENCES:

1. Sharma BK. Instrumental Methods of Chemical Analysis. 7th ed. Meerut: Goel Publishing House; 2000. p. 1-2.

2. Beckett H., et al., Practical Pharmaceutical Chemistry. 4th edition. New Delhi (Ind): C.B.S Publications; 2002. Page.1.

3. Willard HH., et al., Instrumental Methods of Analysis, 7^th edition, CBS Publishers and Distributors: New Delhi, 1986; page.1-5

4. Bassett J., et al., Vogel’s Text book of quantitative inorganic analysis. 4th ed. England: Longman group; 1986.

5. Hohat H. Willard., et al. Instrumental Methods of Analysis, 7^th edition New Delhi: CBS Publishers; 1999.

6. Gurdeep R. Chatwal, et al., Instrumental Methods of Chemical Analysis, 5^th edition New Delhi: Himalaya Publishing House; 2008.

7. Elżbieta Skrzydlewska., et al., Evening Primrose (Oenothera biennis) Biological Activity Dependent on Chemical Composition, Antioxidants (Basel) 2018 Aug; 7(8): 108.

8. Laura (Shih-Shiuan) Lin, Evening primrose oil- chemical structure www.naturaldatabase.com on April 11, 2013.

9. Mohaddese Mahboubi et al., Evening Primrose (Oenothera biennis) Oil in Management of Female Ailments, J Menopausal Med. 2019 Aug; 25(2): 74–82.

10. Syed Ansar Ali et al., Analytical method development and validation for vetiver oil in microsphere formulation by uv spectroscopic method, World Journal of Pharmacy and Pharmaceutical Sciences, 6(10), 1232-1241; 25/9/2017.

11. William W. Christie et al ., The analysis of evening primrose oil, Elsevier Science Industrial Crops and Products 10 (2), 73-83, September 1999,

12. David CL, et al., Webb. Pharmaceutical Analysis. London: Black well publishing; 1994. p. 2-4.

Received on 24.01.2020 Revised on 09.06.2020

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):133-138.

DOI: 10.52711/2231-5675.2021.00023

S. No.	Concentration (µg/mL)	Absorbance
1	100	0.231
2	200	0.438
3	300	0.638
4	400	0.852
5	500	1.034
Regression coefficient (r²)		0.9999
Correlation coefficient (r)		0.9999

Sample No.	Recovery/ Spike level at about (in %)	Amount of EPO added (mg)	Amount of EPO found (mg)	% Recovery	Mean % Recovery
1	50	50	50.29	100.58	100.58
2	50	50	50.29	100.58
3	50	50	50.49	100.62
1	100	100	99.99	99.99	99.66
2	100	100	99.00	99.00
3	100	100	99.99	99.99
1	150	150	150.97	100.65	100.53
2	150	150	148.5	99
3	150	150	150.87	101.96