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Application of Chloramine-T, Methylene Blue in the Assay of Mesna in Tablets, Injections and Wastewater Samples

Nief Rahman Ahmed1*, Lujein Mohammad Kasim2 and Muna Sobhi Abdullah3

1Department of Environmental Technology, College of Environment, University of Mosul, Iraq

2Manawa Health Department, Primary Health Care Sector, Al-Hamdaniyah, Iraq

3Department of Chemistry, College of Science, University of Mosul, Iraq

*Corresponding Author:
Nief Rahman Ahmed
Department of Environmental Technology
College of Environment
University of Mosul, Iraq
Tel: 91+09886658520
E-mail: [email protected]

Received Date: 18/04/2019; Accepted Date: 11/05/2019; Published Date: 01/06/2019

Visit for more related articles at Research & Reviews: Journal of Pharmaceutical Analysis


A new, accurate, sensitive and simple indirect spectrophotometric method for the estimation of sodium 2-mercaptoethanesulfonate (mesna) has been developed. This procedure base on the drug oxidation with excess Chloramine-T in acidic solution, followed by the reaction of the excess oxidants with methylene blue, and read the absorbance at 664 nm .The absorbance results increased with increasing concentration of mesna .The calibration curve was linear at the range 0.1-2.0 ppm. Apparent molar absorbance, Sandell’s sensitivity values were 7.832×104 Lmol-1cm-1. And 47 ngcm-2, respectively. The (RSD) of this method was less than 1.8 and average recovery (accuracy) is 100 ± 0.95. The method applied successfully for estimation of mesna in waste water sample and in (tablets and injections pharmaceutical formulations).


Mesna, Indirect spectrophotometric, Pharmaceutical preparations, Waste water


Mesna is an important thiol compound. The chemical name is sodium 2-mercapto ethane sulfonate Figure 1. Mesna used as antioxidant for prevention of urothelial toxicity in patients treated by antineoplastic, cyclophosphamide or ifosfamide. In the kidney, by neutralizing the highly reactive urotoxic metabolites of oxazaphosphorines locally in the urine [1-3], mesna recently used as antioxidant against acetaminophen toxicity [4]. Mesna oxidized to disulfide, and stabilized using EDTA, sodium hydroxide and inert gas atmosphere in pharmaceutical formulation [5]. The reducing character of mesna should be considered in the design of any analytical methods [5].


Figure 1: Chemical Structure of Mesna.

The literature revealed that mesna has been determined by means of a few analytical methods. These include these include: HPLC [5-8]. Chemiluminescence-flow injection [9], spectrophotometric methods [10-12], Spectrofluorimetric [13]. BP and USP described a tedious titrimetric estimations [14,15]. The present method work is accurate, simple, sensitive spectrophotometric method for estimation of mesna in pure, pharmaceutical dosage forms and waste water sample (Industrial waste water sample provided from the state company for drug industries and medical appliances Mosul-Iraq (NDI). Utilize Chloramine-T and methylene blue as a new reagents.



Optima SP 3000 plus UV-Visible spectrophotometer with 1.0 cm quartz cells was used (OPTIMA USA, INC).


The chemical reagents used were of pharmaceutical and analytical purity grade, distilled water, ethanol were used throughout.

A standard Chloramine T (CAT) solution (Merck) (0.1%): Prepared by dissolving 0.1 g in 100 mL of distilled water. This solution stored in a dark bottle [15].

Methylene blue (Merck) 0.01 %: Prepared by dissolving 0.01 g by distilled water and diluting it to 100 mL in calibrated flask.

Sulfuric acid solution (2M)

Standard solution of mesna (100 ppm): Prepared by dissolving 0.1 g of mesna in 1 L distilled water. It was later diluted to get concentration of 10 ppm by distilled water.

Recommended Procedure

Different amount of standard solution of mesna equivalent 2.5-50 μg were transferred into a series of 25 mL calibrated flasks, 2 mL of 2M H2SO4, and 3 mL of CAT solution were added. Mixed well, let stand for 5 min with occasional shaking, and then added 5 mL of 0.01% methylene blue solution, the volume was diluted to the mark with distilled water. The absorbance was measured at 664 nm against a reagent blank.

Procedures for Pharmaceutical Dosage Forms

Tablets: To minimize a possible variation in the composition of the tablets (were provided from the company for pharmaceutical industries (NDI) Mosul-Iraq). The mixed content of 10 tablets (Mesnan tablets 400 mg of Mesna/tablet), supplied from (NDI) Mosul- Iraq were weighed and grounded, then powder equivalent to 100 mg of mesna was dissolved well in 1 L distilled water, filtered by filter paper and 10 mL of this solution was diluted to 100 mL by distilled water and 3 mL of this solution was treated as under recommended procedure.

Injection: Ampoule of 100 mg of mesna (provided from (NDI) Mosul- Iraq). The content of 5 ampoules was mixed well in 500 mL beaker. An aliquots equivalent to 100 mg of mesna was transferred into 1 L volumetric flask and diluted up to the mark with distilled water, 10 mL of this solution was diluted to 100 mL with distilled water and 3 mL of this solution was treated as recommended procedure.

Procedure for Real Waste Water Sample

Industrial waste water sample provided from the state company for drug industries and medical appliances Mosul-Iraq (NDI) were analyzed, by added the amount 5-50 μg/25 mL of mesna and this solution was treated as recommended procedures.

Results and Discussion

The proposed method was based on the oxidation of mesna to dimesna by a constant excess of CAT in sulfuric acid medium, and then estimation of residual CAT by reacting with a constant amount of methylene blue and measuring at 664 nm. The first step in the assay procedure is the estimation of the upper limit of methylene blue that can be measured at 664 nm, this found 5 mL of 0.01%.This was completely destroyed to a colorless product by 3 mL of 0.1% of CAT. Hence, different amounts of drug was reacted with 3 mL of 0.1% of CAT and the un-reacted oxidant was determined as described under recommended procedure. The formation of the results might be written as [16].

2HS-CH2-CH2-SO3Na+CAT (Known excess) → NaO3-CH2-CH2-S-CH2-CH2-SO3Na + Un-reacted CAT

Di mesna

Excess CAT + Methylene blue→ Bleached methylene blue (measured at 664 nm).

Mesna reacted with CAT and the determination of CAT by reacting with methylene blue. H2SO4 medium (2 mL of 2 M) was found to be good result. The reaction time of 5 minute up to 6 hours was good. A linear graph was found between absorbance and concentration Figure 2.


Figure 2: The calibration graph of mesna.

The optical parameters values were given in Table 1.

Parameter Proposed method
ג max (nm) 664
Beer’s law limit (µg/mL) 0.1-2.0
Molar absortivity (Lmol-1cm-1) 7.832x104
Sandell,s Sensitivity (ngcm-2) 47
Intercept 0.0064
Slope 0.477
Correlation coefficient, R 0.998
Recovery % 100 ± 0.95
Relative standard deviation (%) Less than 1.8

Table 1. Analytical parameters of the present method.

Precision, Accuracy of the Present Method

The precision and accuracy of the present method was analyzed at four different concentrations, each estimation repeated six times. The accuracy and (RSD) values were shown in Table 2. From Table 2, it was clear that relative error of ±0.95% was as accurate Moreover ,the method was found to be sensitive and accurate with (RSD) values <1.8%. The standard deviation at concentration 0 (blank) was calculated and this value was used for the calculation of the limit of detection and limit of quantification. The Limits of Detection (LOD) and Quantification (LOQ) were calculated using the following equation: LOD=(3.3 σ/s) and LOQ=(10 σ/s) where σ is the standard deviation of the response and s is the slope of the regression line [17]. Limit of Detection (LOD) and Limit of Quantification (LOQ) were found 0.01 μg/mL and 0.03 μg/mL respectively. The results indicate that the method was sensitive enough to detect a concentration of 0.01 μg/mL and able to quantify concentrations above 0.03 μg/mL.

Mesna taken (µg) Mesna exists (µg) Recovery (%)a RSD (%)
5 5.04 0.8 1.3
10 10.093 0.93 1.7
30 30.285 0.95 1.6
50 50.44 0.88 1.2

Table 2. Precision, accuracy and RSD of the method.

Analytical Application

Tables 3 and 4 gives the results of the estimation of mesna which reveals there was almost close agreement between the label claim for pharmaceutical preparations and present method and the accuracy results of real waste water sample was more than 98%, indicating that very good applicability of this method.

Pharmaceutical formulations Label claim
Exists* (mg) % Recovery (n=6) %RSD
Mesna tablet (NDI) 400 403 100.75 0.89
Mesna ampoule (NDI) 100 99.8 99.8 1.2

Table 3. Estimation of mesna in pharmaceutical dosage forms.

Industrial waste water samples Mesna (µg/25mL)★ % Recovery (n=10) %RSD
  Taken Found    
1 5 5.05 101 1.3
2 10 9.95 99.5 0.75
3 20 19.8 99.0 0.83
4 30 30.3 101 1.2

Table 4. Estimation of mesna in real waste water sample.


The proposed method developed was simple, selective and offers the advantages of high sensitivity and a wide range of determinations without the need for heating or solvent extraction .The method un affected by slight variations in acidity and other reagents .This method do not take more than 10 minutes and can be used for routine quality control analysis of mesna in pure form and pharmaceutical formulations .and used to the estimation of mesna in pharmaceutical formulations (tablets and ampoules) .and environmental industrial waste water sample.


The first author (Dr. Nief Raman Ahmad.) wishes to express gratitude to his former company [the state company of drug industries and medical appliance (NDI) Mosul, Iraq for providing gift samples of mesna standard materials and pharmaceutical preparations (tablets and vials).