INTRODUCTION:

Tetramethylthionine Chloride is a synthetic basic dye which is also known as Methylene Blue, Methylthioninium Chloride. FDA approved the intravenous form of Tetramethylthionine chloride for the treatment of paediatric and adult patients with acquired methaemoglobinaemia. When administered intravenously in low doses, this agent may convert methemoglobin to haemoglobin by oxidation reduction reaction.

Tetramethylthionine Chloride (INN, or Methylene blue, proposed trade name Rember) is an investigational drug being developed by the University of Aberdeen and TauRx Therapeutics that has been shown in early clinical trials to be Tau protein aggregation inhibitor. The drug is of potential interest for the treatment of patients with Alzheimer’s disease. It is used as a urinary tract antiseptic and for treatment of distributive shock has also been investigated.^1-4

Figure 1: Chemical Structure of Tetramethylthionine Chloride

Cleaning validation is the methodology used to assure that a cleaning process is effective to remove chemical and microbial residues of the active or inactive ingredients of the product manufactured in equipment. The cleaning aids used in the cleaning process and the microbial attributes. In many manufacturing processes, and specially, in the pharmaceutical manufacturing industry, the removal of harmful contaminants from all product contact equipment have vital importance. It ensures the safety, efficacy and quality of all products that are manufactured using the equipment. The objectives of an analytical measurement are to obtain consistent, reliable and accurate data.^5-6

The cleaning procedure utilised in such industries must be validated to verify its effectiveness, based on pre-determined acceptance criteria. Cleaning process validation is thus applied on all product manufacture contact equipment to ensure and verify the removal of all unwanted and harmful contaminants, such as active ingredients, microbes, detergents and impurities, from the equipment prior to its use in another procedure. Cleaning process validation is of the most importance, for example, in situations where pharmaceutical process equipment is used for the manufacture of multiple products in succession, for example, as found on a batch manufacture schedule. In this instance, cleaning process validation ensures that there is no cross contamination between individual batches, different products etc., and thus serves to eliminate the risk of the manufacture of contaminated drug products which is wasteful and expensive.^7-8

Tetramethylthionine Chloride has an appearance of dark green or blue crystal or crystalline powder having a bronze-like luster.⁹ Methylene blue is photodegradable in nature and it can also be obtained from various natural plants.^10-13 Currently, in the pharmaceutical manufacturing industry an equipment cleaning process for methylene blue is typically validated by visual method. Visual method introduces the risk of human error may generate a serious impact on next batch manufacturing by the same pharmaceutical manufacturing equipment. In this method the swabs are subsequently analysed by using UV-Visible spectroscopy.

The main objective of this method was to demonstrate the applicability of UV-Visible method for estimation of the residue of Methylene blue in cleaning control swab sample from manufacturing equipment surfaces. This method may be useful for the verification of the cleaning procedure and detecting the total carryover of Methylene blue and other impurities from previous batches for further batches.

MATERIALS AND METHODS:

Chemicals and reagents:

All the reagents were of analytical grade. Standard Tetramethylthionine chloride and Active Pharmaceutical Ingredient Tetramethylthionine chloride was procured from Sigma-Aldrich Ltd. Swab were obtained from Macsen Drugs, Udaipur, Rajasthan. Double distilled deionized water was used throughout the study.

Equipment and instruments:

UV-Visible spectrophotometer model 1700 (Shimadzu, Japan) was used for this study.

Establishment of cleaning level acceptance criteria for Tetramethylthionine chloride:

The estimation of cleaning limit and acceptance criteria is a crucial element of good cleaning programme. Swab sampling was carried out from equipment used in pharmaceutical manufacturing industries and residues were found in mg/ml. The smallest batch size (BS) subsequent product was selected for calculating the value of the maximal allowable carryover (MAC). Allowable residual limit (ARL) was calculated and compared with different approaches and minimum value of ARL was selected.⁸

Validation of developed method as per ICH guidelines:

The developed method for the quantification of Tetramethylthionine chloride was validated as per ICH guidelines for validation parameters like accuracy, precision, specificity, limit of detection (LOD), limit of detection (LOQ), linearity and robustness and ruggedness.^14-16

Preparation of calibration curve of Tetramethyl thionine chloride:

A primary stock solution of 1mg/ml was prepared in deionised water by dissolving of standard Tetramethylthionine chloride 10mg in 10ml of deionised water. Then dilutions of 1 ppm, 5 ppm and 15 ppm were prepared from the stock solution. Then, the absorbances for these different concentrations were measured at 663nm for obtaining the calibration curve.^14-15

Sampling Technique:

Swab sampling procedure:

Pipette out 5ml of sampling solvent (Deionized water) in the transport container. Transferred the swab in transport container (test tube) containing 5ml of sampling solvent and allowed the swab to soak completely. Took the swab from sampling solvent and squeezed the tip against the inner surface of the test tube to remove the excess solvent in such a manner that excess solvent drips inside the test tube. Using one side of moistened swab wiped the test surface of 5 sq. inch with 10 firm horizontal strokes. Turned the swab over to its other side; wiped the test surface of 5sq. inch with 10 firm vertical strokes. The swab was dropped into the transport container. Plugged the transport container with a stopper.^8,14

Equipment:

Multi Mill:

Multimill which was used in the manufacturing of Tetramethylthionine chloride on Macsen Drugs was sampled through swab sampling procedure to determine the carryover of the API.

The Equipment Multimill has the two major contact surface areas – Sieve plate and discharging hopper.

Figure 2 Multi Mill

Surface Area of Sieve Plate – 78.5sq.cm

Surface Area of Discharging Hopper – 3673 sq.cm

After the completion of cleaning activity, the swab sample was collected, one from the sieve plate and another from the discharging hopper.

RESULTS:

Calculation of total carryover limits based on therapeutic dosage:

Considering the pharmacological potency of Tetramethylthionine chloride, lowest allowable residue value is calculated according to the following formula.

Where, ARL is the acceptance residual limit, STD is the API smallest therapeutic dose of previous product A (mg/unit dose), SBS is the smallest batch size of any subsequent product (mg) to be manufactured in the small equipment train, SF is the safety factor i.e. 1/1000 or 0.001, MDD is the maximum daily dose of the product to be manufactured in the same equipment train, M is the surface area/swab (25cm²), SSA is the shared equipment surface area.

Calculation of Actual Determination of Carryover Residue of Tetramethylthionine Chloride from the Equipment:

The determination of carryover residue from the equipment through the UV Method Technique is calculated according to the following formulas

AT × CS × Dillution Factor × SSA

Limit of Carryover = --------------------------------------------------

Residue (mg) AS × M

Where, ATis the absorbance of the Sample solution, ASis the absorbance of the Standard Solution, CSis the concentration of the Standard Solution, Dilution Factor is the volume (mL) of solvent used in dilution of sample, and SSA is the shared equipment surface area. M is the surface area/swab (25cm²).

Method Validation:

Accuracy:

Accuracy of this method is estimated by conducting recovery studies with the appropriate matrix spiked with three concentrations (50%, 100% and 150%) of the analyte covering the range of method. For each concentration, three sets were prepared and absorbances were noted. The achieved spike recovery for 50.0%, 100.0% and 150.0% were found 99.13, 97.76 and 101.18 respectively. % RSD was found below 2 %which indicate that the proposed method was accurate.

Table 1: Accuracy (Recovery)

Con.	Weight of Sample	Sample Conc. (mg/mL)	Quantified Value	% Recovered
50 %	5.1 mg	0.00255 mg/mL	49.56 %	99.13 %
100 %	10.3 mg	0.00515 mg/mL	97.76 %	97.76 %
150 %	15.0 mg	0.0075 mg/mL	151.77 %	101.18 %

Precision:

Intra-day Precision (Repeatability):

The repeatability of the analytical procedure is assessed by measuring the concentrations of six independently prepared replicates of the test concentration. In intra-day precision, all the replicates were prepared on the same day and statistical validation was carried out. The results was represented as relative standard deviation (% RSD). Low value of %RSD was found which indicates the good method precision. The % RSD of Method Precision was achieved 0.04% which is demonstrating good repeatability and reproducibility of the method.

Table 2: Method Intraday Precision

Preparation No.	Standard Weight	Standard Con. (mg/mL)	Abs.
1.	10.1 mg	0.00505 mg/mL	1.0843
2.	10.0 mg	0.00500 mg/mL	1.0842
3.	10.1 mg	0.00505 mg/mL	1.0840
4.	10.2 mg	0.00510 mg/mL	1.0849
5.	10.2 mg	0.00510 mg/mL	1.0845
6.	10.0 mg	0.00500 mg/mL	1.0853
Average			1.08453
Std Deviation			0.00048
%RSD			0.04 %
Acceptance Criteria:			%RSD Not more than 1.0%

Inter-day Precision:

Day to day precision was performed by analysis of 6 concentrations with three replicates. In inter-day precision, all concentration was prepared and absorbances were recorded. Low value of %RSD indicates the good method precision. Sample solutions analyzed on different days, the %RSD were 0.04% and 0.07% which was within the acceptance limit.

Robustness and ruggedness:

Robustness and ruggedness were performed to evaluate the influence of deliberate variation in experimental conditions for the quantification of tetramethyl thionine chloride. Ruggedness of the method was determined by changing the analysts. Two Analysts analysed on the same day, the %RSD between two analysts analysis were 0.04% and 0.07% which was within the acceptance limit.

Specificity:

In UV-Vis measurements, specificity is ensured by the use of a standard wherever possible and is demonstrated by the lack of interference from other components present in the matrix. The specificity of this method investigated by analyzing the sample to demonstrate the absence of interference with the elution of analyte. Three replicates of Standard solution and three replicates of the sample solution were analysed. The interference of blank or any other analyte was absent in the parameter of Specificity.

Limit of Detection:

The Limit of detection (LOD) of the method was estimated by calculating the following formula as per ICH guidelines on six replicate measurements of a blank solution.

Where σ is the mean standard deviation of y-intercept of the regression line, S is the slope of the standard curve.

The Limit of Detection of Instrument was determined at the absorbance of 0.0001 and the analyte’s lower limit of Detection was achieved at 0.01 ppm of Tetramethylthionine chloride as the absorbance of 0.0019 thus indicating high sensitivity of the method.

Limit of Quantification:

The LOQ (LOQ) can be estimated by calculating the following formula as per ICH guidelines on six replicate measurements of a blank solution.

Where σ is the mean standard deviation of y-intercept of the regression line, S is the slope of the standard curve.

The Limit of Quantification of Instrument was determined at the absorbance of 0.0001 and the analyte’s lower limit of Quantification was achieved at 0.1 ppm of Tetramethylthionine chloride on the absorbance of 0.0105 thus indicating high sensitivity of the method.

Table 3: Limit of Detection (LOD) and Limit of Quantification (LOQ)

Sr. No.	Product Name	LOD Value	Level	LOQ Value	Level
1.	Tetramethylthionine Chloride	0.0019	0.1%	0.0105	1.0%

Acceptance Criteria: LOD – Absorbance Not less than 0.001 and LOQ – RSD not more than 1.0%

Linearity and Range:

Linear relationship between the analyte concentration and UV-Vis response is demonstrated by the preparation of five standard solutions at concentrations of 50%, 70%, 100%, 130% and 150% encompassing the anticipated concentration of the test solution. The operational range of an analytical instrument and the analytical procedure is the interval between the upper and lower concentrations of analyte in the sample for which it has been demonstrated that the instrumental response function has a suitable level of precision, accuracy, and linearity. The Correlation coefficient of the linearity of Tetramethylthionine Chloride was found to be 0.999 thus indicating that the variance of response is homogeneous.

Figure 3: UV Spectra showing maximum absorbance at 663 nm (λ_max)

Figure 4: UV spectra for tetramethylthionine chloride for different Concentrations

Table 4: Quantitative Parameters of UV-Visible Spectrophotometric Method

Parameters	Results
λ_max	663.0 nm
Beer’s law limits (ppm)	0.1 ppm – 15 ppm
Slope	0.081
Intercept	0.022
Correlation coefficient (R²)	0.999
Instrument LOD	0.0001
Instrument LOQ	0.0001
LOD (Analyte)	0.01 ppm
LOQ (Analyte)	0.1 ppm

Table 5: Linearity

Concentration (ppm)	Concentration as % of analyte target	Mean Abs.	Standard Deviation	RSD (%)
2.5 ppm	50%	0.5499	0.0006	0.06%
3.5 ppm	70%	0.7513	0.0012	0.08%
5.0 ppm	100%	1.0844	0.0062	0.30%
6.5 ppm	130%	1.4196	0.0059	0.22%
7.5 ppm	150%	1.6257	0.0094	0.17%
Correlation Coefficient (r2) =		*0.999*

Acceptance Criteria: Correlation Coefficient (r2) should be greater than 0.995

Figure 5: Linearity graph for tetramethylthionine chloride

DISSCUSSION:

The Analytical Method for estimation and quantification of Tetramethyl Thionine Chloride by UV-Visible Spectrometry was developed and validated according to the ICH guideline of analytical method validation.¹⁵ The accuracy or recovery of the method at three different concentrations was achieved within the acceptable limit. The Specificity of this method by blank bracketing was determined by no interference of blank to the analyte. The LOD and LOQ of the instrument and Analyte were determined. UV-Visible Spectrometer has lower It is determined in the validation parameter of LOD and LOQ. The Linearity of Tetramethylthionine Chloride is found to be in the range of 2.5ppm – 7.5 ppm with linear correlation coefficient of 0.999. Various researchers have performed quantification and method validation by using UV spectrophotometer.^17-21 This method is very efficient, reliable, accurate and precise for quality control purpose to evaluate the cleaning. The results from this method validation can be used to judge the quality, reliability and consistency of analytical results, which are an integral part of any good analytical practice.

CONCLUSION:

Tetramethylthionine chloride is used in the treatment of acquired methemoglobinemia. The cleaning validation of Tetramethylthionine chloride has a considerable impact on the pharmaceutical industries. Several pharmaceutical companies are searching for efficient cleaning validation method for quantification of Tetramethylthionine chloride. But nowadays, no efficient method is available for the cleaning validation method for the Tetramethylthionine chloride. Only visual analysis of cleaning is performed in pharmaceutical manufacturing industries which may be harmful for the safety of pharmaceutical products. Hence in the future, This analytical method can be used to judge the quality, reliability and consistency for the validation of cleaning method.

Acknowledgements:

Authors are also thankful to the management of Macsen Drugs. and Geetanjali Institute of Pharmacy, Udaipur for providing necessary

Conflict of Interest:

The authors declare that they have no conflict of interest.

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Received on 26.12.2020 Modified on 11.05.2021

Research J. Pharm.and Tech 2022; 15(4):1499-1504.

DOI: 10.52711/0974-360X.2022.00249