The ICH guidelines in practices: Stress degradation studies on botulinum toxin and validation of developed stability-indicating HPLC method

Sonal Bhale

Introduction

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Botox is a botulinum neurotoxin, a neurotoxin made by a bacterium called Clostridium botulinum. This is the same type of bacteria that causes botulism. Botulism is a progressive, potentially fatal infection that can cause symptoms such as muscle paralysis, slurred speech, and drooping eyelids. But when this neurotoxin is administered by injection, its effects are concentrated and not dangerous.1

Figure 1

Structural formula of botulinum toxin

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Materials and Methods

Chemical and reagent

Sodium phosphate buffer and acetonitrile were ordered by Merck Private Ltd, Mumbai, India. API of Botulinum toxin as bulk drug were purchased from Teva API India Ltd (2927057), Bijnor, Uttar Pradesh India.

Instrumentation

HPLC model SHIMATZU with pump SPD-20AD (LC-20A) having variable wavelength, UV-Visible detector and Rheodyne injector (20 µl). Column- The analytical column was Phenomenex 100-5C-18, 5 µm 100 A º, 250 mm × 4.6 mm.

Stock solution and standard solution preparation

Standard solutions and its preparations

Weighed accurately 10 mg of Botulinum toxin and transferred to volumetric flask(100 ml). Then add 70 ml HPLC grade water and dissolve it, sonication was done for arround15 min and made the final volume with HPLC grade water. Take 10 ml of stock solution and dissolve in 100ml volumetric flask with HPLC grade water. Filter the stock solution with suction pump assembly.

Preparation of sample solution

Add 1 ml of WFI (water for injection), and shake well. Take out 0.1 ml of sample solution and dissolve in 100 ml HPLC grade water.

Validation of method

HPLC assay method was validated following ICH guidelines Q2R1for linearity and range, accuracy, detection limit, quantitation limit, stability study, etc.2

Results

Development of method and its optimization

The development of RP-HPLC analytical method for Botulinum toxin was done using C8 column after optimization of chromatographic conditions for specificity, retention time and resolution. The suitable mobile phase was sodium phosphate buffer and acetonitrile in the ration of 30:70 at 214 nm. 2.1 min was the retention time at the flow rate of 2ml/min. When the flow was reduced, the resultant chromatogram showed increase in retention time with tailing effect.

Figure 2

Chromatogram of botulinum toxin at 214 nm.

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Table 1

HPLC method development parameters for botulinum toxin

Sr. No.	Parameter	HPLC method
1	Column	C8
2	Flow rate	2ml/min
3	λ max	214 nm
4	Run time	20 min
5	Injection volume	6 µl
6	Mobile phase	Sodium phosphate buffer: Acetonitrile

Validation method

The validation of HPLC assay were carried out following ICH guidelines (Q2 R1).3

System suitability studies

The six replication of standard solution was inserted into the HPLC and observed that parameters for system suitability are within the limit

Table 2

System suitability parameters

Sr. No.	Parameters	Result
1	Peak Area	27572
2	Theoretical plates	5232
3	Tailing factor	1.8
4	Resolution	0.09

Linearity and range

The standard solutions were analysed for concentration against peak area in different concentrations in the range of 2, 4, 6, 8, 10 µg/ml. The regretion equation was found to be y=28351x+10591 and correlation coefficient was 0.9947.

Figure 3

Calibration curve of Botulinum toxin

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Table 3

Result of linearity and range

S. No	Con. (µg/ml)	Peak Area
I	2	70604
II	4	120025
III	6	175072
IV	8	247324
V	10	290467

Accuracy

Recovery study was carried out to find out accuracy was with 3 different concentration levels (80%, 100%, 120%). Botulinum toxin standard of concentration 8µg, 10 µg, 12µg were prepared and recovery study was done. The values of recovery study were observed in the specific limit of 98-102%.

Table 4

Result of accuracy

Sr. No.	% Level	% Recovery	Average
1	80 %	100.4	100.1
2		100.1
3		99.8
4	100 %	100.2	100
5		100.1
6		99.7
7	120%	100.0	99.6
8		99.6
9		99.3

Precision

Precision of the developed method was carried out by repeatability, intermediate precision.

Repeatability-6 replicates of the standard stock were analysed for SD and RSD

Table 5

Result of repeatability

Analyte	Concentration	% RSD
Botulinum toxin	5 µg/ml	0.43

Intermediate precision-6 replicates of the standard stock was analysed on interday, intraday, different analyst

Table 6

Resultof intermediate precision

Analyte	Concentration	% RSD
Botulinum toxin	5 µg/ml	0.32

Detection Limit and Quantification Limit

The detection limit concentration of Botulinum toxin was 1µg/ml and quantitation limit concentration was 3.3 µg/ml.4

Stability

Degradation effect

Botulinum toxin sample was subjected to various degradation studies. Stress degradation parameters were carried out to find the developed HPLC assay is liable for decomposed materials. This study provides results about the conditions wherein the drug is unstable.5

Table 7

Resultsof degradation study

Degradation parameters	% degradation
Alkali Degradation	64.2%
Acid Degradation	57.3%
Unstressed Degradation	100 %
Photolytic Degradation	58.2%
Neutral Degradation	90.3%
Peroxide Degradation	87.6%
Thermal Degradation	37%

Discussion

Research studies have used reversed-phase HPLC for the elution of botulinum toxin. We chose his C18 column with a flow rate of 2 mL/min. The mobile phase chosen consisted of sodium phosphate buffer (0.05 M) at pH 2.8 and acetonitrile in a ratio of 30:70 respectively at 214 nm. It is within ICH and FDA limits. In addition, analysis of a commercial formulation of botulinum toxin showed that the drug elutes without interfering peaks generated by excipients in the commercial product.6, 7, 8, 9, 10, 11 Therefore, we found the method results to be stable for different parameters.

Conclusion

In the current study, a fast, simple, accurate, well-defined, and linear HPLC method for demonstrating botulinum toxin stability was developed and validated and can be rented for routine quality control analysis. The analytical technique and mobile phase solvent conditions provided good separation of botulinum toxin. In addition, the main features of the developed method are short run time and retention time of about 3.1 min. The method has been validated according to ICH guidelines. The method is robust enough to reproduce accurate and precise results under a variety of chromatographic conditions.

List of Abbreviation

Table 0

Sr. No	Abbreviation	Name of Abbreviation
1	BTX	Botulinum Toxin
2	RPHPLC	Reversed Phase High Performance Liquid Chromatography
3	ICH	International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use
4	SD	Standard Deviation
5	RSD	Relative Standard Deviation

Source of Funding

None.

Conflict of Interest