Department of Pharmaceutical Chemistry, Rani Chennamma College of Pharmacy, Rajiv Gandhi University of Health Science, Belagavi 590001, Karnatak, India.
To develop and optimize UV- Spectrophotometric method for Berberine Hydrochloride API. To validate newly developed UV- Spectrophotometric method using various parameter according to ICH guidelines. Materials and method: UV-Spectrophotometric method was developed using Phosphate Buffer pH 7.4 the developed method was standardized in terms of validation parameter such as simple, sensitive, precise, linear, accurate, robust, reproducible as per ICH Q2 (R1) guidelines for estimation of Berberine Hydrochloride in marketed formulation this newly developed method was successfully applied. Results: Berberine Hydrochloride exhibits ? max at 275nm and in the concentration range 50 to 250 ?g/ml beers law was obeyed. The Limit of Detection was found to be 42.83 ?g/ml and Limit of quantification was found to be 142.79 ?g/ml. Recovery of Berbeshine in Marketed formulation was obtained in range of 102-115%, All the precision and repeatability results were observed within the acceptance range i.e. less than 2%. Assay of Berbeshine was found to be in range of 95-97%. Conclusion: The method was found to be simple accurate and precise reproducible and marketed formulation of Berberine Hydrochloride was estimated.
1.1 Berberine Hydrochloride:1,2,3
Berberine Hydrochloride is an Isoquinoline alkaloid found in a variety of medicinal plants, mainly in the Berberis genus and the family is Berberidaceae [Berberis vulgaris]. It is Bright yellow in colour and is widely used in traditional medicines. Charka Samhita prescribed that the extract of the plant be taken internally for the treatment of haemorrhage, piles, pruritus and alopecia. Sushruta Samhita described it is being useful internally in indigestion deficiency of breast milk and in uterine and vaginal disorders. It also possesses activity against diabetes, High cholesterol, Hypertension, Anti-microbial.
Drug Profile:4
Table no.1 Drug profile of Berberine Hydrochloride
Drug Name |
Berberine Hydrochloride |
Structures: 2D Str |
3D Str
|
Iupac Name |
5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benz0dioxolo[5,6a]-quinolizinium |
Solubility |
Phosphate Buffer pH 7.4 |
Mechanism Of Action |
Berberine Hydrochloride inhibits pseudomonas aeruginosa biofilms growth by down- regulating pslA and pelA. It inhibits inflammation in mice. |
Molecular Formula |
C20H18C1NO4 |
Molecular Weight |
371.81gm/ml |
Category |
Diabeties, High cholesterol, Hypertension, Anti microbial |
Colour |
Bright yellow colour |
Odour And Taste |
Charecteristics odour and taste |
Melting Point |
204-206 |
State |
Solid |
Storage |
Store in dark place |
Part Used |
Root, Rizhome and Bark |
|
|
1.2 UV-Spectroscopy
Basic Principles of spectroscopy5
Spectroscopy deals with the production, measurements and interpretation of spectra arising from the interaction of electromagnetic radiation with matter. There are many different spectroscopic methods available for solving a wide range of analytical problems. The methods differ with respect to species to be analysed (e.g. molecular or atomic spectroscopy), the type of radiation matter interaction to be monitored (e.g. absorption, emission or diffraction) and the region of the electromagnetic spectrum used in the analysis. Spectroscopic methods are very informative and widely used for the both quantitative and qualitative analyses. Spectroscopic method based on the absorption or emission of radiation in the ultraviolet (UV), visible(vis), infrared (IR) and radio (nuclear magnetic resonance NMR) frequency ranges are most commonly encountered in traditional food analysis laboratories. Each of these methods is distinct in that it monitors different types of molecular or aromatic transitions.
Spectroscopy Method:6,7
It is the branch of science dealing with the study of interaction between Electromagnetic radiation and matter. It is a most powerful tool available for the study of atomic and molecular structure/s and is used in the analysis of wide range of samples o [tical spectroscopy includes the region on electromagnetic spectrum between 100 A0 and 400 μ m. The region of electromagnetic spectrum.
Fig No.1 UV- Spectrophotometer
Instrumentation of UV- Spectrophotometry8
UV-Visible spectrophotometry is one of the most frequently employed technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measures the ratio, or function of ratio, of the intensity of two beams of light in the UV-Visible region are called Ultraviolet-Visible spectrophotometers. In qualitative analysis, organic compounds can be identified by use of spectrophotometer, if any recorded data is available, and quantitative spectrophotometric analysis is used to ascertain the quantity of molecular species absorbing the radiation. Spectrophotometric technique is simple, rapid, moderately specific and applicable to small quantities of compounds. The fundamental law that governs the quantitative spectrophotometric analysis is the Beer -Lambert law. Beer’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases exponentially with the number of absorbing molecules. In other words, absorbance is proportional to the concentration.
Lambert’s law: It states that the intensity of a beam of parallel monochromatic radiation decreases exponentially as it passes through a medium of homogeneous thickness. combination of these two laws yields the Beer-Lambert law.
Beer-Lambert law: When beam of light is passed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity of light may occur.
Mathematically, Beer Lambert law is expressed as
A=a b c
Where,
Both b and a are constants so a is directly proportional to the concentration c when c in gm/100, then the constant is called A (1%, 1cm)
A=A1%/1cm’bc
Quantification of medicinal substance using spectrophotometer may carried out by preparing solution in transparent solvent and measuring its absorbance at suitable wavelength. The wavelength normally selected is wavelength of maximum absorption (λ max), where small error in setting the wavelength scale has little effect on measured absorbance. Ideally, concentration should be adjusted to give an absorbance of approximately 0.9 around which the accuracy and precision of the measurements are optimal. The assay of single component sample, which contains other absorbing substances, is then calculated from the measured absorbance by using one of three principal procedures.
Fig No.2 Schematic instrumentation of UV spectrophotometer
The materials required to carry out the work includes reagents, chemicals and analytical instruments as given below:
Chemicals and Apparatus:
Chemicals: Drug sample Berberine Hydrochloride API was procured from Zyrex Ayurveda India for method development of Berberine Hydrochloride. Methanol, Ethanol, Phosphate Buffer6,8,7,7.4 and Distilled water. For Method Validation: Phosphate Buffer 7.4, Berberine Hydrochloride extract, Distilled water, tablet marketed formulation. Apparatus: 100ml volumetric flask, 10ml of volumetric flask, beakers, funnels, micro pipette, measuring cylinders, cuvette, soft tissue papers, aluminium foil. Instruments: Shimadzu UV 1800 Spectrophotometer.
Gravimetric Method10: Take 10 ml of each solvent and add 100mg of drug to it. Stir continuously for 24hours.filter the solution and evaporate the solvent using hot air oven at 400 c. After drying weigh the residue. Calculate the solubility by using the formula.
Table No.2 Solubility
Solvents |
W1 |
W2 |
W3=W2-W1 |
Ethanol |
1.08 |
1.108 |
0.028 |
Methanol |
1.08 |
1.103 |
0.023 |
PH 7 |
1.08 |
1.141 |
0.061 |
PH 7.4 |
1.08 |
1.108 |
0.028 |
PH 6.8 |
1.08 |
1.136 |
0.056 |
Distilled water |
1.08 |
1.12 |
0.043 |
Formula: W3= W2- W1
By performing above gravimetric analysis we have come to the conclusion that we should use Phosphate Buffer pH 7.4 should be used.
Calculation:
Note:
Methodology for UV-spectroscopy:
First select a suitable solvent then selection of maximum absorbance range and wavelength and determination of wavelength then determination of UV- Spectrophotometric method.
Determination of Maximum Wavelength (λ max):
Take 0.1gm of Berberine Hydrochloride powder API is dissolved in 100ml of Phosphate Buffer pH 7.4 transfer 1ml of above solution into 10ml of vol flask containing 10ml of Phosphate Buffer pH 7.4 then the solution was scanned against black for the entire UV visible wavelength 200-400nm. Based on the spectrum λ max of 275nm was selected for further analysis.
0.1gm of Berberine Hydrochloride API was weighed accurately then dissolve in 100ml 0f Phosphate Buffer pH7.4 transfer 0.5, 1, 1.5, 2, 2.5 ml of above solution into 10ml vol. flask make up the mark with phosphate Buffer pH 7.4 to make 50, 100, 150, 200, 250 μ g/ml solutions then take absorbance of these standard measured at λ max of 275nm. The standard curve was drawn by plotting concentration v/s absorbance.
Table No:3 Analytical Instruments
Instruments |
UV- Spectroscopy |
Model |
UV1800 |
Software |
UV probe |
Company |
Shimadzu |
Solvent |
Phosphate Buffer pH 7.4 |
Scanning wavelength range |
400-200nm |
Maximum absorbance of Berberine Hydrochloride |
275nm |
Intraday precision: For intraday precision prepare triplicates of 50 μ g/ml, 150 μ g/ml, 250 μ g/ml from stock solution in 10ml vol. flask make up the volume with phosphate buffer pH 7.4 up to the mark then scanned at 275nmand % RSD was found to be less than 2%. The results are shown in Table No. 5
Interday Precision: For interday precision prepare triplicates of 50 μ g/ml, 150 μ g/ml, 250 μ g/ml from stock solution in 10ml vol. flask make up the volume with phosphate buffer pH 7.4 up to the mark and scanned at 275nm and %RSD was found to be less than 2%. The results are shown in Table. No 6
Where,
σ – Standard deviation of Y intercept of calibration curve
S - Slope of regression equation, the results are shown Table No. 9
Where,
σ – Standard deviation of Y intercept of calibration curve
S - Slope of regression equation, the results are shown Table No.10
Assay: 10 tablets of Berbeshine were weighed that contains 500mg of Berberine Hydrochloride. Average weight of tablet is calculated then calculate the theoretical drug content. Prepare stock solution of Berberine Hydrochloride and stock solution of Berbeshine and filter the solution through Whatman filter paper then 100 μ g/ml concentration sample solution and standard solution scanned at 275nm then calculate the practical yield.
Formula:
%Purity=Practical drug content Theoretical drug content× 100
Average weight of 10 tablets = 842mg
Lable claim = 500mg Berberine Hydrochloride
(Limit for % purity of drug is 90-120%)
The results are shown in Table No. 10
For 50%: From the above standard stock solution pipette out 0.5ml standard solution and 0.25ml of sample solution transferred into 10 vol. flask and the volume is made up to the mark using phosphate buffer pH 7.4. solvent three replicates were prepared to performed the study and calculate % recovery.
For 100%: From the above standard stock solution pipette out 0.5ml standard solution and 0.5ml of sample solution transferred into 10 vol. flask and the volume is made up to the mark using phosphate buffer pH 7.4. solvent three replicates were prepared to performed the study and calculate % recovery.
For 150%: From the above standard stock solution pipette out 0.5ml standard solution and 0.75ml of sample solution transferred into 10 vol. flask and the volume is made up to the mark using phosphate buffer pH 7.4. solvent three replicates were prepared to performed the study and calculate % recovery.
(Limit for % recovery of drug is 90-120%)
The results were shown in Table No. 11
Discussion: The UV- Spectroscopic method was developed for estimation of Berberine Hydrochloride using phosphate buffer pH 7.4 at 275nm. Validation was performed as per ICH guidelines.
UV-spectroscopic method development and validation: UV method was developed by using Shimadzu UV 1800 spectrophotometer instrument using a solvent system phosphate buffer pH 7.4 at 275nm.
Fig No. 3: UV spectra of Berberine Hydrochloride
Linearity: The linearity range of Berberine Hydrochloride was in the range of 50-250 μ g/ml correlation coefficient :0.999
Table No. 4
Sr No |
Concentration |
Absorbance |
1 |
0 |
0 |
2 |
50 |
0.184 |
3 |
100 |
0.371 |
4 |
150 |
0.522 |
5 |
200 |
0.761 |
6 |
250 |
0.898 |
|
r2 |
0.999 |
SLOPE |
0.0036 |
|
LOD |
42.83 |
|
LOQ |
142.79 |
Report: The results of UV spectroscopic method were within the acceptance limit. The calibration curve of the drug gives correlation coefficient value 0.999
Specificity and selectivity: Solvent used in method developed was interfered with the drug hence the method was found to be specific.
Precision: Precision was implemented intraday and interday Precision. The % RSD was found within acceptance criteria
Table No. 5: Intraday precision of Berberine Hydrochloride
Sample no |
Conc μ g/ml |
Absorbance |
%RSD |
Average %RSD |
||
|
|
Morning |
Afternoon |
Evening |
|
0.49% |
1 |
50 |
0.252 |
0.269 |
0.263 |
0.75% |
|
2 |
150 |
0.514 |
0.521 |
0.523 |
0.48% |
|
3 |
250 |
0.749 |
0.752 |
0.753 |
0.26% |
Table No. 6: Interday precision of Berberine Hydrochloride
Sample no |
Conc μ g/ml |
Absorbance |
%RSD |
Average %RSD |
||
|
|
Day 1 |
Day 2 |
Day 3 |
|
0.4% |
1 |
50 |
0.253 |
0.266 |
0.268 |
0.8% |
|
2 |
150 |
0.515 |
0.518 |
0.523 |
0.4% |
|
3 |
250 |
0.750 |
0.755 |
0.755 |
0.2% |
Ruggedness: : By change in analyst ruggedness was performed. %RSD was found within acceptance criteria.
Table No.7: Ruggedness Study
Analysts |
Sample no. |
Conc μ g/ml |
Absorbance |
SD |
%RSD |
Analyst 1 |
1 |
50 |
0.189 |
0.0031 |
1.65% |
Analyst 2 |
2 |
150 |
0.487 |
0.001 |
0.21% |
Analyst 3 |
3 |
250 |
0.774 |
0.0015 |
0.20% |
Robustness: : By change in wavelength the robustness was performed. The %RSD was found within acceptance criteria
Table No.8
Sample no |
Conc μ g/ml |
Wavelength |
%RSD |
Average %RSD |
||
|
|
273nm |
275nm |
277nm |
|
0.66% |
1 |
50 |
0.148 |
0.149 |
0.147 |
1.34 |
|
2 |
150 |
0.469 |
0.474 |
0.472 |
0.44 |
|
3 |
250 |
0.725 |
0.733 |
0.731 |
0.21 |
Sensitivity: LOD and LOQ data of Berberine Hydrochloride:
Table No.9
Drug |
LOD μ g/ml |
LOQ μ g/ml |
Berberine Hydrochloride |
42.83 |
142.79 |
Accuracy: By taking the absorbance of Standard solution and Marketed solution Accuracy was performed. %Recovery was within acceptance criteria.
Table No. 10 Accuracy of Berberine Hydrochloride
Total conc μ g/ml, |
Std conc of BH |
Sample conc of BH |
Absorbance 275nm |
Conc Y=mx+c |
Sample conc difference μ |
% Recovery |
|
std |
sample |
||||||
50% |
25 |
25 |
0.184 |
0.186 |
50.83 |
25.83 |
103.3% |
150% |
25 |
75 |
0.371 |
0.373 |
102.7 |
77.7 |
103.65 |
250% |
25 |
125 |
0.522 |
0.540 |
149.1 |
124.1 |
99.25% |
Assay:
Table No:11Assay of Berberine Hydrochloride
Sr no |
Sample |
Absorbance |
%Assay |
1 |
Standard solution |
0.512 |
105.5% |
2 |
Marketed solution |
0.229 |
CONCLUSION: It was revealed that the newly developed UV-Spectrophotometer method for determining the Berberine Hydrochloride in its pharmaceutical dosage form was simple, specific and cost effective. Using the ICH criteria all validation parameters were found to be acceptable ranges.
Summary: Method was developed by using Phosphate buffer pH 7.4 as solvent at 275nm.method was validated by using phosphate buffer pH 7.4 as solvent and accuracy was done by using market formulation (Berbeshine). By using ICH guidelines, the validation was performed
ACKOWLEDGMENT: The completion of our project work has been an intense learning period for us, not only in scientific arena, but also a personal level. In this acknowledgement we take privilege to express sincere gratitude towards the fine individuals for their inspiration guidance and support that we received. First and foremost, we thank the almighty who blessed us with this opportunity. We would like to express our gratitude to our guide Kiran patil Lecturer of Dept of pharmaceutical chemistry, Rani chennamma college of pharmacy. Her valuable guidance encouragement timely suggestions throughout our work will always remembered. We wish to express our thanks to principal of Rani chennamma college of pharmacy, Belagavi. We express our sincere gratitude to Udaykumar Bolmal sir and all teachers for their valuable Feedback and profound co-operation offered during the time of research work. Thanks to one all……
REFERENCES
Kiran Patil*, Abishek Shedabal, Kalpana Gulappagol, MD Umar Gokak, Pushpa Kadalagi, Vidya Bellivari, UV-Spectrophotometric Method Development and Validation of Berberine Hydrochloride, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 2825-2836. https://doi.org/10.5281/zenodo.15704448