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Standard Operating Procedure
TITLE PAGE NO. :
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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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QC/Val/096/135 USP,ICH

FPREPARED BY : DATE : CHECKED BY : DATE: APPROVED BY : DATE:

Senior Q.C. Officer Asst. Q.C. Manager Sr. Q.C. Manager

CONTENTS

1. PURPOSE
2. SCOPE
3. DISCRIPTION
4. INTRODUCTION
5. EXPERIMENTAL
6. DEVELOPMENT OF THE HPLC METHOD
7. ASSAY METHOD
8. INSTRUMENT PRECISION
9. OUTLINE FOR THE VALIDATION PROTOCOL
10. STATISTICAL DATA & ACCEPTANCE CRITERIA
11. DISCUSSION

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Standard Operating Procedure
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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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1. Purpose:
To describe the procedure for validation or verification of analytical test methods and build confidence in measurements taken on
samples of incoming materials and products.

2. Scope
This SOP is applicable for the validation of HPLC analytical method for Anthocyanidin in Syrup formulation.

3. Description
Product Name: Anthocyanidin
Analyte: Anthocyanidin

4. Introduction
Morus nigra, called black mulberry or blackberry (not to be confused with the blackberries which are various species of Rubus), is
a species of flowering plant in the family Moraceae, native to southwestern Asia.
Morus nigra is a deciduous tree growing to 12 m (39 ft) tall by 15 m (49 ft) broad. The leaves are 10–20 cm (4–8 in) long by 6–
10 cm (2–4 in) broad - up to 23 cm (9 in) long on vigorous shoots, downy on the underside, the upper surface rough with very short,
stiff hairs.
The edible fruit is dark purple, almost black, when ripe, 2–3 centimetres (0.8–1.2 in) long, a compound cluster of several
small drupes; it is richly flavoured, similar to the red mulberry (Morus rubra) but unlike the more insipid fruit of the white mulberry
Anthocyanins are a type of flavonoid, a class of compounds with antioxidant effects. Found naturally in a number of foods,
anthocyanins are the pigments that give red, purple, and blue plants their rich coloring. In addition to acting as antioxidants and
fighting free radicals, anthocyanins may offer anti-inflammatory, anti-viral, and anti-cancer benefits.

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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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In herbal medicine, anthocyanin-rich substances have long been used to treat a number of conditions (including high blood
pressure, colds, and urinary tract infections). Recent research suggests that anthocyanins may also help fend off major health
problems, including heart disease and cancer.
Research on Anthocyanins
Here's a look at several key findings on anthocyanins and their health effects.
1) Heart Disease
Anthocyanins may enhance heart health, according to a 2010 report published in Nutrition Reviews. The report's authors note that
anthocyanins appear to improve cholesterol levels and blood sugar metabolism, as well as fight oxidative stress (a process known
to play a role in heart disease).
Dietary intake of anthocyanins may also help prevent high blood pressure (a major risk factor for heart disease), according to a 2011
study from the American Journal of Clinical Nutrition.
2) Obesity
Preliminary research suggests that anthocyanins may protect against obesity. In a 2008 study from the Journal of Agricultural and
Food Chemistry, scientists found that mice fed an anthocyanin-enriched high-fat diet for eight weeks gained less weight than mice
fed a high-fat diet without anthocyanins.
3) Cancer
Anthocyanins may aid in the prevention of breast cancer, according to a laboratory study published in Phytotherapy Research in
2010. In test-tube experiments, scientists showed that anthocyanins extracted from blueberries helped inhibit the growth of breast
cancer cells.
Using Anthocyanins for Health
Getting your fill of anthocyanin-rich fruits and vegetables may help boost your overall health by offering up an array of nutrients.
However, scientists have yet to determine whether taking high concentrations of anthocyanins in supplement form can help treat or
prevent any specific health condition.
Keep in mind that supplements haven't been tested for safety and due to the fact that dietary supplements are largely unregulated,
the content of some products may differ from what is specified on the product label. Also keep in mind that the safety of supplements
in pregnant women, nursing mothers, children, and those with medical conditions or who are taking medications has not been
established. You can get tips on using supplements, but if you're considering the use of anthocyanins, talk with your primary care
provider first. Self-treating a condition and avoiding or delaying standard care may have serious consequences.

5. Experimental
5.1 SAMPLES
The reference substance Morus Nigra (as-signed purity Anthocyanidin 16.36%) and Morus Nigra Active raw material was brought
from Shaanxi Joryherb Biotechnology Co., Ltd China.
The reference substance, as well as raw material, was always kept at room temperature.
5.2. Reagents and Solvents
All other chemicals were of analytical grade. The water used was freshly distilled.

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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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Formic Acid (Scharlau Chemie S A); Methanol (Merck Pvt Ltd); Acetonitrile (Merck Pvt Ltd).

5.3 Instrumentation and Conditions:-

All analytical works were performed on HPLC Shimadzu LC-20AT Autosampler equipped with Quaternary pump with UV-Visible
detector SPD-20A. All weights were taken on (Sartorius) electronic balance.

6.0 Development of the HPLC Method

In order to evaluate Anthocyanidin from excipients,
Mobile Phase Preparation:
MOBILE PHASE

SOLUTION A: 10% FORMIC ACID

SOLUTION B: 55% SOLUTION A +22.5% ACN + 22.5% METHANOL

Preparation of standard solutions

Take 20mg of Anthocyanidin in 200ml of volumetric flask Add 1% phosphoric acid solution to dissolve and make up the volume
with same solvent.
From the spectra, wavelength 520nm was selected. Maximum solubility was observed in 1%. The linearity was observed in the
range of 80-120mcg mL -1 for Anthocyanidin and Precision was also achieved in this range.

7.0 Assay method

Mobile Phase Preparation:
MOBILE PHASE

SOLUTION A: 10% FORMIC ACID

SOLUTION B: 55% SOLUTION A +22.5% ACN + 22.5% METHANOL

Diluent: 1% Phosphoric acid solution Preparation of standard solutions

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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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Take 20mg of Anthocyanidin in 200ml of volumetric flask Add 1% phosphoric acid solution to dissolve and make up the volume
with same solvent.

Sample preparation
Take 5 ml of sample containing 20mg of Anthocyanidin in 100ml of volumetric flask and diluted to 200 ml with1% phosphoric
acid solution. sonicate for 10 mins and make up the volume upto 100ml with the same solvent.
Detector: 520 nm
Column: 4.6-mm × 15-cm; packing L1
Flow rate: 0.5 mL/min
Injection size: 20 µL

Calculations: As x Cst x 100

Ast x Cs

8.0 Instrument Precision

Mobile Phase Preparation:
MOBILE PHASE

SOLUTION A: 10% FORMIC ACID

SOLUTION B: 55% SOLUTION A +22.5% ACN + 22.5% METHANOL

Diluent: 1% Phosphoric acid solution

Preparation of standard solutions

Take 20mg of Anthocyanidin in 200ml of volumetric flask Add 1% phosphoric acid solution to dissolve and make up the volume
with same solvent.
Take Chromatograms for five times. %age of RSD obtained with replicate Areas of Standard Preparation should NMT 2.0%.
Results are listed as follows:-

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Anthocyanidin in Syrup Formulation by HPLC
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Absorbance Area of Standards

Sr.No: Statistical Calculation
(Anthocyanidin )
1.
1163354 Mean= 1168586
2.
1160651 Std Deviation= 7223.52
3.
1167516 RSD= 0.62%
4.
1172886
5.
1178523
Hence, the system precision is achieved.

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9.0 Out Line for the Validation Protocol:

The Detailed describe in below as follows:-

VALIDATION:
Establishing documented evidence that a system does what it purports to do. Analytical validation essentially means the examination
of five basic attributes: linearity and range, precision, accuracy, sensitivity and Specificity.

9.1. Linearity
The linear relationship between the concentration and the instrument response as areas at 520nm was confirmed by plotting the
graph between the concentrations and areas in the range of 80, 90, 100, 110 and 120 % of the target value (100 mcg mL -1).
The calibration graph is shown below, indicates that linear relationship observed between the Concentration and area of by shaking
mechanically the solution.
The correlation coefficient of calibration data was calculated to 0.9991.This indicates that the test procedure obeys the Beer’s law.
Standard Preparation for Linearity:-
Mobile Phase Preparation:
MOBILE PHASE

SOLUTION A: 10% FORMIC ACID

SOLUTION B: 55% SOLUTION A +22.5% ACN + 22.5% METHANOL

Diluent: 1% Phosphoric acid solution

Preparation of standard solutions

Take 200mg of Anthocyanidin in 200ml of volumetric flask Add 1% phosphoric acid solution to dissolve and make up the volume
with same solvent.
Transfer 4.0 mL, 4.5mL, 5.0mL, 5.5mL, 6.0mL of this solution to a 50 mL volumetric flasks, dilute with Diluent to volume, and
mix to obtain 80%, 90%, 100%, 110%, 120% concentration. Pass through a filter having a 0.45-µm or finer porosity. Inject replicate
injections of each dilution for 80%, 90%, 100%, 110% and 120 %.
Preparation as follows:-

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Acceptance criteria:-
Correlation Coefficient (r²) = -1 to +1

Sr.# Volume of the %age Conc. Of Target Concentration Absorbance Areas of

Solution Value (mcg mL -1) the Test Solution
1. 4.0 mL 80% 80 mcg mL -1
1005014
2. 4.5 mL 90% 90 mcg mL -1
1110297
3. 5. 0 mL 100% 100 mcg mL -1
1205168
4. 5.5 mL 110% 110 mcg mL -1
1321987
5. 6.0 mL 120% 120 mcg mL -1
1427109

Linearity (Anthocyanidin)
1600000
1400000
1200000
1000000 y = 10559x + 158035
Area

800000 R² = 0.9991
600000
400000
200000
0
50 70 90 110 130
Concentration

Conclusion:
The Calibration chart has shown the linear relationship between the absorbance and the concentration of analyte. Thereby indicating
that the procedure follows Beer’s Law.

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9.2 Range
Acceptance Criteria:
The range of analytical procedure is the interval between the upper and lower concentrations of analytes for which the procedure
has a suitable level of precision, accuracy and linearity.
 %age RSD of triplicate assay values obtained at each level (80%, 100%, 120%). NMT 2.0%.
 Average of triplicate assay values obtained at each level (80%, 100%, and 120%).
 Between 99-101%.
Procedure:
The range 80% to 120% is when the accuracy precision and linearity 80% and 120% are established , %age RSD of triplicate assay
at each level (80%,100%,120%) is less than 2.0% and the average of triplicate assay values obtained at each level (80%,100%,120%)
are between 99.0-101.0%. Hence, the range from 80% to 120% is established.

9.3 Specificity
The specificity is defined as the ability to disunite the analyte in the presence of other components like matrix components.
Specificity shows that the procedure is unaffected by the presence of impurities or excipients. Specificity is performed by running
a standard solution (as identification test) comparing with a placebo run.
I prepared a placebo without addition of active (Anthocyanidin) and make a test solution and run against the standard. The retention
time of standard peak is 3.370min and no peak response is shown by placebo at this time.

Mobile Phase Preparation:

MOBILE PHASE

SOLUTION A: 10% FORMIC ACID

SOLUTION B: 55% SOLUTION A +22.5% ACN + 22.5% METHANOL

Diluent: 1% Phosphoric acid solution

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Analytical Method Development & Validation of
Anthocyanidin in Syrup Formulation by HPLC
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Preparation of standard solutions

Take 200mg of Anthocyanidin in 200ml of volumetric flask Add 1% phosphoric acid solution to dissolve and make up the volume
with same solvent.

Placebo Preparation:
Sample solution: Nominally 0.1 mg mL -1of syrup without active in diluent prepared as follows. Transfer a suitable portion to a
suitable volumetric flask from NLT 20 finally powdered Tablets. Add diluent equivalent to 80% of the flask volume. Dissolve and
fill with diluent to volume. Pass the solution through a suitable filter of 0.22-um pore size.

CHROMATOGRAM
SAMPLE
(Peak Area / Spectrum)
Standard
1249666.5/Positive
(Anthocyanidin)
Placebo Negative

9.4 Method precision

Precision of an analytical procedure is the degree of agreement among individual test results when the procedure is applied
repeatedly to multiple samplings of a homogeneous sample.
Precision is usually expressed as Relative Standard Deviation (Coefficient of variation). Precision is performed through;
a-Repeatability
b- Reproducibility.
-1
Sample solution: Nominally 0.1 mgmL of Anthocyanidin in diluent prepared as follows. Transfer a suitable portion of
Anthocyanidin to a suitable volumetric flask from NLT 20 finally powdered Tablets. Add diluent equivalent to 80% of the flask
volume. Dissolve and fill with diluent to volume. Pass the solution through a suitable filter of 0.22-um pore size.
9.4.1. REPEATABILITY:
Assay is performed on three separate samples in 2 replicate sets.
Concentration of Reference: 100 mcg mL -1
Wavelength: 520nm
Avg. absorbance Area of the Reference: 1227246

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Repeatability Results
Samples Concentration of analyte Variation from
Absorbance Area %age Results
(mcg mL -1) Theoretical Results
I 1 100 1202912 98.02 1.98%
2 100 1217183 99.18 0.82%
II 3 100 1215585 99.05 0.95%
4 100 1228314 100.09 0.09%
III 5 100 1233282 100.49 0.49%
6 100 1228370 100.09 0.09%
Average of 6 Results: 99.49%
Standard Deviation: 0.9144
RSD of 6 Results: 0.919%
9.4.2. Reproducibility
Reproducibility refers to the use of the analytical procedure by different analyst within the same laboratory. Assay is performed on
three separate samples in each case.
Concentration of Reference: 100 mcg mL -1
Wavelength: 520nm
Avg absorbance Area of the Reference: 1229400

Sa Repeatability Results
mp Concentration of Analyte Variation from
les Absorbance Area %age Results
(mcg mL -1) Theoretical Results
1 100 1239662 100.83 0.83%
2 100 1249322 101.62 1.62%
3 100 1248820 101.58 1.58%
Average of 3 Results: 101.34%
Standard Deviation: 0.4423
RSD of 3 Results: 0.436%
9.5 Accuracy
A measure of exactness of the analytical method. The method is said to be accurate if on the average the method provides the true
answer. Accuracy implies there is no inherent systematic error or bias - (bias is the deviation from true value). Through the use of
reference standards the accuracy of a method can be measured and extent of bias that may exist can also be determined.

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Actually Accuracy of an analytical procedure is the closeness of test results obtained by that procedure to the true value. It is
determined by the addition of known amounts of analyte to the sample with different quantities (3X3 replicates) and is calculated
as the percentage of recovery by the assay of the known added amount of analyte in the Placebo.
Concentration of Reference Standard: 100 mcg mL -1
Wavelength of the Reference Standard: 520nm
Avg. Absorbance Area of the Reference Standard 80%: 1009052
Avg. Absorbance Area of the Reference Standard 100%: 1243728
Avg. Absorbance Area of the Reference Standard 120%: 1503481
Content of Active Added in
80% 100% 120%
Placebo
80 mcg mL -1 100 mcg mL -1 120 mcg mL -1
(% of label claim)

Abs. area of the test solution 1011190 1009054 1242367 1241867 1498335 1497709

%age of label claim 100.21% 100.00% 99.89% 99.85% 99.66% 99.62%

Variation from theoretical
0.21% 0.00% 0.11% 0.15% 0.34% 0.38%
Results or difference
Average or %age Recovery 100.10% 99.87% 99.64%

9.6 Limit of Detection and Limit of Quantitation

9.6.1 Limit of Detection
The limit of detection is the smallest amount of analyte that can be detected, and not necessarily determined, in quantitative fashion.
LOD is calculated by the formula:
LOD= 3×SER*/Slope
*Standard Error of Regression
The Limit of Detection of Anthocyanidin is 1.61mcg/mL.
9.6.2 Limit of Quantitation

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The limit of detection is the lowest concentration of an analyte in a sample that may be determined with acceptable accuracy and
precision. LOQ is calculated by the formula:
LOQ= 3.3×LOD
The limit of quantitation of Anthocyanidin is 5.30mcg/mL.

10.0 Statistical data & Acceptance criteria

10.1 Performance Characteristics Acceptance Limits and Results

Parameter Acceptance Limit Result Remarks

Linearity Correlation Coefficient -1…+1 0.993 OK
Instrument Precision Relative Standard deviation
NMT 1.5% 0.62% OK
Precision Repeatability:
Relative Standard Deviation NMT 2.0% 0.919%
Reproducibility:
Results should reproducible by different OK
analyst with Relative Standard Deviation 0.436%
NMT 2%

Accuracy For Assay + 2% of Theoretical Complies OK

concentration
Robustness Results Should be reproducible at slight Complies OK
variation in Temperature
Specificity The Analyte must be Quantities in the Complies OK
presence of impurities (Excipients)

Limit of Quantitation Acceptable precision and accuracy 1.61mcg/mL OK

mcg/mL

Limit of Detection No impurity detected 5.30mcg/mL OK

mcg/mL

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11.0 Discussion
The proposed methods are simple, accurate, precise, reproducible, economic and rapid for analysis of Anthocyanidin in Raw
material and in Syrup formulation.
Accuracy of the method was evaluated by carrying out recovery studies. Low values of %RSD are indicative of high precision of
the methods.
Based on the validation study data, it can be concluded that the proposed method is accurate and precise for the analysis of drug.

No interference was found from excipients used in the syrup formulation and hence the method is suitable for Anthocyanidin in

Raw material and in Syrup formulation.

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