INTRODUCTION:

Sulfonation, sulfation and sulfamation reactions involve transfer of SO₃ to the substrate and are carried out using a range of commercially available sulphating reagents e.g. liquid sulfur trioxide, chlorosulfonic acid, sulfuric acid, oleum, sulfamic acid, metal bisulphite and tertiary amine sulfur trioxide complexes viz. pyridine sulfur trioxide, trimethylamine sulfur trioxide, triethylamine sulfur trioxide etc. However tertiary amine sulfur trioxide complexes exhibit variety of advantages over conventional sulfating reagents in terms of reactivity, selectivity, sensitivity and moreover they are easy to handle.

Tertiary amine sulphur trioxide complexes have found multiple uses, for example in the sulfation of dyes, detergents, carbohydrates, sterols and also in the sulfonation of polycyclic compounds such as acid-sensitive heterocycles¹. Denis Barron et al have reported sulfation of flavonoids using sulfur trioxide trimethylamine complex². These complexes have been explored for sulfating a variety of scaffolds containing alcoholic, phenolic, amine, thiol and other functional groups^3-6. In recent years use of pyridine sulfur trioxide complex has been reported in the synthesis of β-lactamase inhibitors like zidebactam, avibactam and relebactam^7-9.

Consistency in the reaction progress and yields depends on the quality of the sulfating reagent in terms of availability of the SO₃ during the reaction. Large variations in the yields in the sulfation processes have been noticed with different batches of amine sulfur trioxide complex. The reason behind is the instability of these complexes, which are extremely hygroscopic and degrade in presence of moisture, the degradation products amine sulfate and H₂SO₄ contaminate the reagent and decrease the potency. Having knowledge of potency of the complex in terms of SO₃content is critical for its synthetic application; it has direct impact on the completion of reaction, product yield and quality. Use of contaminated reagent may lead to the formation of unwanted side products. Therefore it is very important to quantify separately and specifically the SO₃ and H₂SO₄in amine sulfur trioxide complexes.

Though ample number of applications has been reported for this important class of sulfating reagent, hardly ever analytical methodology to ensure quality of these reagents is reported. Only one reference was found in literature for determination of active sulfur trioxide in sulfur trioxide pyridine and sulfur trioxide trimethylamine complexes using Karl Fischer titration¹⁰. The method is based on measuring the amount of water consumed for the hydrolysis of the analyte using wet pyridine. The method was successful because the sulfur trioxide pyridine complex is very labile to hydrolysis. This method did not work for TEA-SO₃;probably because of the relatively good stability of this complex to hydrolysis by water.

Most of the manufactures of tertiary amine sulfur trioxide complexes report the potency based on non-specific analytical methodology e.g. elemental composition either sulfur content or nitrogen content^11-12. However this method does not specifically report the SO₃ content which is an essential moiety for the sulfation process, ultimately fails to justify the stability indicating nature of the analytical method. Hence there was a need to develop and report new specific analytical method to estimate potency of the reagent in terms of SO₃ content as well as its degradation product.

Now a day’s potentiometric titration technique attracts researcher attention due to its wide applicability in the pharmaceutical and chemical industry for various applications like estimation of active drug content, counter ion content, trace level impurity analysis etc^13-16.

Herein we describe potentiometric titrimetry methods for the estimation of SO₃ and H₂SO₄ in the TEA-SO₃ and validations thereof.

EXPERIMENTAL:

MATERIALS AND METHODS:

TEA-SO₃ was synthesised in-house at Wockhardt Research Centre, India. AR grade sodium hydroxide, sulfuric acid and triethylamine were procured from Rankem (India). Primary standard potassium hydrogen phthalate was procured from Merck (India). HPLC grade water was obtained through Milli-Q plus water purification system (Millipore, Milford, MA, USA).

Preparation of TEA-SO₃Complex:

A synthetic process of TEA-SO₃was modified in view of scale up feasibility¹⁷. Modifications were removal of charcolization step and use of reduced volume of solvents. The optimized process was validated in pilot plant. Chlorosulfonic acid (1 eq) was treated with triethylamine (2 eq) in dichloromethane below 10°C. Pure TEA.SO₃ was obtained by recrystallization using dichloromethane and cyclohexane.

Equipments:

Potentiometer (Model: 907 Titrando, Metrohm AG) equipped with Combined pH electrode for aqueous acid/base titrations (Part no. 6.0232.100, Metrohm AG) and magnetic stirrer (Model: 801, Metrohm AG) was used. Tiamo 2.5 software was used for instrument control and data processing.

Volumetric solution preparation:

1N and 0.02N sodium hydroxide solutions were prepared by dissolving appropriate quantity of sodium hydroxide in water. These solutions were standardized by titrating against potassium hydrogen phthalate.

1N sulfuric acid solution was prepared by diluting appropriate quantity of concentrated sulfuric acid with water. The solution was standardized by titrating against 1N sodium hydroxide solution.

Titration procedures:

Sulfuric acid content:

About 0.5g of test substance was dissolved in 40mL of water and titrated against 0.02N sodium hydroxide solution using potentiometer and burette reading at equivalent point (V1) was recorded. A blank titration was performed and burette reading at equivalent point (V2) was recorded. The sulfuric acid content was calculated using following equation.

Each mL of 0.02 N sodium hydroxide is equivalent to 0.9808g of sulfuric acid.

Sulfuric acid content (%, w/w) = (V1 – V2)* N1* 0.9808* 100/W1* 1000 * 0.02

(Wherein, N1 = normality of sodium hydroxide and W1 = weight of test substance in g)

Active sulfur trioxide content:

About 2g of test substance was transferred in a 250mL single neck round bottom flask, 20mL of 1N sulfuric acid solution and 15mL of water were added in to it and mixed. This mixture was refluxed with stirring at 125±5℃ in oil bath for 3 hours with chilled water circulation through the condenser. After reflux, the solution was cooled to room temperature. The mixture was transferred to 200mL glass beaker, the round bottom flask was rinsed twice with 10mL of water and mixed with mixture. This mixture was titrated against 1N sodium hydroxide solution using potentiometer. The burette reading at equivalent point (V3) was recorded. A blank titration was conducted and burette reading at equivalent point (V4) was recorded. Assay of TEA-SO₃ was calculated using following equation.

Each mL of 1N sodium hydroxide is equivalent to 0.18125g of triethylamine sulfur trioxide.

Assay of TEA-SO₃ (%, w/w) = [(V3–V4)* N2* 0.18125 *100/W2] – Sulfuric acid content

(Wherein, N2 = normality of sodium hydroxide and W2 = weight of test substance in g)

Active sulfur trioxide was calculated from the assay by applying molecular weight correction.

Active sulfur trioxide content (%, w/w) = Assay of TEA-SO₃* 80.07/181.25

(Wherein, 80.07 = Mol. Weight of SO₃ and 181.25 = Mol. Weight of TEA-SO₃)

Method validation:

For sulfuric acid content method, specificity was evaluated by analysing blank, test substance and test substance fortified with triethylamine. Method precision was determined by, six replicate analysis of the test sample. Linearity of the method was evaluated by analysing sulfuric acid solutions in the range of 70 to 130% of the specification limit. Accuracy was verified by standard addition method. The test substance was spiked with known amount of sulfuric acid at three levels (about 50%, 100%, and 150% of specification limit) and analysed in triplicate. Recovery was calculated by comparing amount added and amount found. Test solution stability was determined at room temperature for 24 hours by analysing test solution initially and periodically up to 24 hours.

For active sulfur trioxide content, specificity of the method was evaluated by analysing blank, test substance and test substance fortified with triethylamine and sulfuric acid. Method precision was determined by, six replicate analysis of the test sample. Accuracy was determined by varying the weight of test sample in the range of 70 to 130% of the prescribed weight (2g). Test solution stability was determined at room temperature (25±3ºC) for 24 hours. Robustness of the method was evaluated by purposely altering experimental conditions like reflux temperature (±10ºC) and reflux time (±18 min).

RESULTS AND DISCUSSION:

Method development:

It is vital that the analyte remain stable during complete analytical procedure. Our past experience of pyridine sulfur trioxide was essentially reverse. Therefore firstly solid as well as solution state stability of TEA-SO₃was assessed.Stability in solid state was evaluated by performing analytical tests like infra-red spectroscopy and melting point. The TEA-SO₃ was exposed to atmosphere after initial analysis and then periodically analyzed up to 7 days. Stability in aqueous solution was monitored by measuring pH of solution up to 8 h. To our surprise unlike pyridine sulfur trioxide the TEA-SO₃was found very stable in both states as there was no change in IR spectrum, melting point and pH of solution over period of time. Stability of analyte was essential particularly for this compound to estimate true percent of active SO₃ and the degradation product H₂SO₄.

Due to the aqueous solution stability of the analyte, H₂SO₄ present in the test substance could be easily estimated by simple potentiometric titration against sodium hydroxide solution using pH glass electrode. Here potentiometric end point detection scores over visual color change using indicator due to detection sensitivity and reproducibility in detecting small changes. The sample quantity and titrant strength was optimized in such a way that at least 0.02% of H₂SO₄should be accurately and precisely quantified in TEA-SO₃. Based on the several experiments, 0.5g of sample quantity and 0.02 N sodium hydroxide solution were found optimum to achieve the desired sensitivity.

For SO₃determination, the TEA-SO₃need to be hydrolysed to H₂SO₄ and later could be titrated against alkali. In first attempt the TEA-SO₃was dissolvedin water and warmed at 60°C for 1h, there was very little hydrolysis, the condition was stressed to 100°C (reflux) for 3 h but no improvement. The experiment was repeated with addition of catalytic amount of alkali, but in vain. In second attempt the TEA-SO₃was refluxed with excess sodium hydroxide solution and excess alkali was titrated against standardized H₂SO₄, however the TEA-SO₃did not hydrolyse completely with 1N sodium hydroxide even after refluxing for 3 h. In the third attempt H₂SO₄ was used as hydrolysing agent, reflux for 3 h at drastic condition like temperature of 125°C was required for complete hydrolysis. The excess of H₂SO₄ and H₂SO₄ generated due to hydrolysis of TEA-SO₃was titrated against sodium hydroxide solution. The reaction stoichiometry is presented in Fig. 1. Hydrolysis of the TEA-SO₃withexcessH₂SO₄ yields triethylamine sulphate. Triethylamine sulphate is titrated against sodium hydroxide to produce sodium salt of triethylamine sulphate. The reaction stoichiometry is confirmed on the basis of consumption of sodium hydroxide for neutralization. Optimized potentiometer parameters of the both methods are provided in Table 1. Representative titration curves are presented in Fig. 2.

Figure 1. Reaction stoichiometry in the analysis of TEA-SO₃.

A B

C D

Figure 2. Representative titration curves of A: Blank of sulfuric acid content; B: Test solution sulfuric acid content; C: Blank of active sulfur trioxide content; D: Test solution of active sulfur trioxide content.

Table 1 Optimized potentiometer titration parameters

Parameter	H₂SO₄ content	SO₃ content
Stirring rate	5	5
Start volume (mL)	0.0	25
Dosing rate (mL/min)	Maximum	5
Pause time (second)	10	10
Sensor	pH electrode	pH electrode
Signal drift (mV/min)	50	50
Volume increment (mL)	0.1	0.05
Temperature (ºC)	25	25
EP Criteria (mV)	20	20
EP Recognition	Greatest	Greatest

Method validation:

Method validation exercise imparts the confidence on the developed method^18-23. The developed methods were validated in order to prove their suitability for its intended reason and capability to produce legitimate analytical results consistently²⁴.

For H₂SO₄ content method, specificity was confirmed as the presence of triethylamine did not impact the performance of the analysis.

Table 2 Method validation summary of H₂SO₄content

Parameter	Result
Method precision-Analyst-1 (%RSD)	0.75
Intermediate precision-Analyst-2 (%RSD)	0.73
Overall precision (%RSD)	1.22
Linearity Range (%)	70-130
Linearity (r²)	1.000
Mean recovery (%)	102.80±1.08

Method was reproducible with RSD for six independent determination of H₂SO₄ in TEA-SO₃was 0.75%. Intermediate precision was determined by performing experiment by different analyst on different day. The overall RSD for total 12 analyses by two different analysts was 1.22%. The linear correlation was found between H₂SO₄ concentration and volume of titrant with the regression equation y=0.1067+0.0519 and correlation coefficient 1.000. The mean recovery of H₂SO₄was 102.80±1.08%. The test solution was found stable for 24 hours at room temperature with maximum absolute deviation of 0.01%. The validation data is presented in Table 2.

For SO₃ content method, specificity was established as presence of externally added triethylamine and H₂SO₄in test sample did not hamper the analysis. Method was precise with RSD of 0.25% for six independent analysis of single lot of TEA-SO₃. Similarly intermediate precision for twelve determinations determined by two different analysts was 0.29%. The method was accurate with mean recovery of 99.92±0.78%. Two major sample preparation parameters viz. reflux temperature and reflux time was varied as 125±10ºC and 180±18 min respectively. At each varied condition, no significant change in the method performance was observed. The test solution was found stable for 24 hours at room temperature with absolute deviation in SO₃ estimation of 0.44%. The validation data is presented in Table 3.

Table 3 Method validation summary of SO₃ content

Parameter	Results
Method precision-Analyst-1 (%RSD)	0.25
Intermediate precision-Analyst-2 (%RSD)	0.23
Overall precision (%RSD)	0.29
Mean recovery (%)	99.92±0.78
Robustness (SO₃ content, %, w/w)
a. Reflux temperature (-10°C)	43.75
b. Reflux temperature (+10°C)	43.87
c. Reflux time (-18 min)	43.94
d. Reflux time (+18 min)	43.97

Application of the method for batch analysis:

Several batches of TEA-SO₃ were analyzed using validated methods. In these batches, SO₃ content and H₂SO₄content were in the range of acceptance limits. An excellent mass balance was achieved by summing up water content, H₂SO₄ content and assay of TEA-SO₃substantiating overall accuracy of the analysis. Data of batches analysis is shown in Table 4.

Table 4 TEA-SO₃batches analysis data

Batch Code	TEA-SO₃ (%, w/w)	H₂SO₄ (%, w/w)	Water (%, w/w)	Mass balance (%, w/w)
A	99.28	0.13	0.17	99.58
B	98.81	0.43	0.48	99.72
C	98.63	0.44	0.43	99.50

CONCLUSION:

Specific analytical methods have been developed toquantify SO₃ and H₂SO₄ in TEA-SO₃. The developed methods score over the elemental analysis method used to judge quality by the reputed manufactures of TEA-SO₃. The advantage of the procedure is that it does not need any highly sophisticated instrument, which makes it cost effective. The developed methods were reproduced using phenolphthalein as an indicator for wider industrial application. The crux of the developed method is optimization of hydrolysis condition for TEA-SO_3.During analytical method development it is also learnt that the TEA-SO₃is chemically and physically stable reagent as compared to other amine sulfur trioxide complexes e.g. pyridine sulfur trioxide complex. This property makes the reagent user friendly in view of packaging, transportation and longer shelf life. The developed methods were validated and found specific, sensitive, precise, accurate and robust. The validated method was successfully applied for the quality control of in-house manufactured TEA-SO₃.

ACKNOWLEDGEMENT:

Authors thank the management of Wockhardt Limited for the facility and encouragement to support this research.

CONFLICT OF INTEREST:

The presented work is original research work of Wockhardt Limited and all the authors are employees of Wockhardt Limited.

FUNDING:

The research work presented in this manuscript is a part of drug discovery research program of Wockhardt Limited.

REFERENCES:

1. Gilbert EE. The reactions of sulfur trioxide, and its adducts, with organic compounds. Chemical Reviews. 1962; 62(6): 549–589.

2. Barron D, Ibrahim RK. Synthesis of flavonoid sulfates. III. synthesis of 3',4'-ortho disulfates using sulfur trioxide-trimethylamine complex, and of 3'-suIfates using aryl sulfatase. Zeitschrift für Naturforschung A. 1988; 43(9): 631–635.

3. Dusza JP, Joseph JP, Bernstein S. The preparation of estradiol-17β sulfates with triethylamine-sulfur trioxide. Steroids. 1985; 45(3–4): 303–315.

4. Kelly D. Sulfur trioxide-amine adducts, Spotlight 76, SYNLETT. 2003; 14: 2263–2264.

5. Kanetani F, Yamaguchi H. Studies of the reaction of amine with sulfur trioxide IV. One step synthesis of N-substituted Imidobissulfates from amines. Bulletin of the Chemical Society of Japan. 1974; 47(11): 2713–2716.

6. Tidwell TT. Sulfur trioxide pyridine. Encyclopedia of reagents for organic synthesis, University of Toronto, Toronto, Ontario, Canada. 2017.

7. Joshi SN, Wankhede KS, Jadhav SB, Pawar SS, Ahirrao VK, Bhawsar SB, Deshpande PK, Yeole RD, Patel MV. A process for preparation of (2S,5R)-7-Oxo-6-sulphooxy-2-[N'-((3R)-piperidin-3-carbonyl)-hydrazinocarbonyl]-1,6-diaza-bicyclo[3.2.1]octane. US Patent US9657021B2.

8. Aszodi J, Lampilas M, Musicki B, Rowlands A, Colette P. New heterocyclic compounds and their preparations and their use as medicaments, in particular as anti-bacterial agents. 2013, US Patent US2007/0299108 A1.

9. Mangion IK, Ruck RT, Rivera N, Huffman MA, Shevlin M. A concise synthesis of a β-lactamase inhibitor. Organic Letters. 2011; 13(20): 5480–5483.

10. Sieh DH, Dunham JM. Determination of active sulfur trioxide in sulfur trioxide pyridine and sulfur trioxide-trimethylamine complexes. Analytical Chemistry. 1982; 54: 1216–1217.

11. Specification sheet of triethylamine sulfur trioxide. https://www.sigmaaldrich.com/catalog/substance/ sulfurtrioxidetriethylaminecomplex 1812576101311? lang=en&region=I.

12. Specification sheet of triethylamine sulfur trioxide. https://www.tcichemicals.com/JP/en/p/T2136.

13. Rele RV, Gurav PJ, Sawant SA. A validated non-aqueous potentiometric titration method for the quantitative determination of lafutadine from pharmaceutical preparation. Asian Journal of Research in Chemistry. 2012; 5(6): 722-724.

14. Thakur SV, Ware RL, Farooqui M, Naikwade SD. Equilibrium studies on Imipramine Hydrochloride-metal ion in 20%ethanol-water mixture. Asian Journal of Research in Chemistry. 2012; 5(12):1464-1465.

15. Rele RV, Tiwatane PP. A validated non-aqueous potentiometric titration method for quantitative determination of alprazolam from pharmaceutical preparation. Asian Journal of Research in Chemistry. 2016; 9(11): 597-600.

16. Rele RV, Rane DG. Validated non-aqueous potentiometric titration method for quantitative determination of clopidogrel bisulphate from pharmaceutical preparation. Asian Journal of Research in Chemistry. 2017; 10(1): 26-28.

17. Nair V, Bernstein S. A convenient procedure for the preparation of triethylamine-sulfur trioxide. Organic Preparations Procedure. International. 1987; 19(6): 466–467.

18. Landge KR, Siddheshwar S, Ghogare R. Analytical method development and validation of ramipril and hydrochlorothiazide- A review. Research Journal of Science and Technology. 2020; 12(3):211-221.

19. Deokar AU, Siddheshwar S, Kakad SB. Analytical method development and validation of rivaroxaban- A review. Research Journal of Science and Technology. 2020; 12(1): 36-46.

20. Nageswara Rao T, Patrudu TB, Sunil kumar A, Rao KN, Karri A. A new analytical method validation and quantification of residual solvents in telmisartan bulk drug product by headspace chromatographic method. Research Journal of Science and Technology. 2018; 10(2).

21. Nagamallika J, Aruna M. Development and validation of spectrophotometric method for the estimation of aripiprazole in tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2011; 1(3): 46-49.

22. Dahiya J, Singh A, Gupta SK, Kumar B. Spectrophotometric estimation of dextromethorphan in bulk drug using hydrotropic solubilization technique. Asian Journal of Pharmaceutical Analysis. 2013; 3(3): 90-93.

23. Ganji S, Satyavati D. Development and validation of RP-HPLC method for the analysis of Cobicistat and related impurities in bulk and pharmaceutical dosage forms. Asian Journal of Pharmaceutical Analysis. 2015; 5(1): 1-8.

24. International conference on harmonization (ICH Q2). (2005). Validation of Analytical Procedures: Text and Methodology. https://www.ich.org/page/quality-guidelines.

Received on 18.08.2021 Modified on 08.12.2021

Asian J. Pharm. Ana. 2022; 12(1):17-22.

DOI: 10.52711/2231-5675.2022.00004