Determination Of Trometamol Content In Gadobutrol Solution For Intravenous Administration, 1 mmol / mL Using RP-LC With Refractive Index Detector

Background: Extremely responsive technique designed for the willpower of trometamol for instance tris(hydroxymethyl)aminomethane in gadobutrol by RP-LC technique. Quantication of trometamol content in gadobutrol samples by HPLC with refractive index detector (RID). Trometamol was UV inactive compound. Methods: Trometamol was determined by RP-LC method using Inertsil NH 2 (250x4.6mm, 5µm) column as motionless segment. Column temperature maintained 30°C, inoculation quantity 10µL, ow velocity was 0.3 ml/min, sample cooler temperature 25°C. The mixture of phosphate buffer and cyanomethane in the ratio of 990:10 (volume/volume) was utilized as movable segment. The retention time of trometamol determined 10.95 minutes respectively. The acceptance limit of the trometamol content is 0.9%-1.5%. eciency gadobutrol injection

Gadobutrol solution is a sterile, clear, colorless to pale yellow solution containing 604.72 mg gadobutrol per mL (equivalent to 1 mmol/mL) as the active ingredient and the excipients calcobutrol sodium, trometamol, hydrochloric acid (for pH adjustment) and water for injection. Gadavist contains no preservatives.
Multifunctional characteristics of Tris amino in pharmaceutical products: Buffering agent in both small and large volume parenterals Neutralization of carbomer polymers in topical gels or creams In literature, no analytical method was reported for the determination of Trometamol in Gadobutrol solution for intravenous administration. Hence the author was aimed towards the development of rapid, speci c and robust methods for the determination of Trometamol in Gadobutrol solution for intravenous administration.

Chemicals and reagents
Trometamol purchased from Merck India., Mumbai, India. Potassium dihydrogen ortho phosphate, milli-Q water and acetonitrile were procured from Merck, India. Chromatographic conditions RP-LC analysis was carried out on Waters 2690 separation module with Refractive index detector model:2414. Inertsil NH 2 (250x4.6mm, 5µm) column was used as stationary phase. The mixture of phosphate buffer and Acetonitrile in the ratio of 990:10 (v/v) was used as mobile phase. The ow rate of the mobile phase was kept at 0.3 mL/min. The injection volume was set as 10µL. Column oven temperature and auto sampler temperature were set as 30°C and 25°C, respectively. Refractive index detector temperature 30°C and sensitivity 1028.

Method development
A Spiked solution containing Trometamol and Gadobutrol solution was run in 0.5 mL/min ow rate.
Trometamol peak closely eluted to placebo peak and hence the ow rate of the mobile phase was decreased from 0.5 mL/min to 0.3 mL/ min. In this condition Trometamol peak eluted at an optimum retention time, placebo peak and Trometamol peaks are well separated. Hence, the elution order was observed from the chromatogram (Figures.3.0 to 7.0).

Method validation [6]
3.2.1 Speci city Blank interference: Blank was prepared and injected as per test method. It was observed that no blank peaks were interfering with analytical peaks.

System Precision:
Perform the analysis of Diluted standard six times and determine the percentage relative standard deviation of peak area, Asymmetry and Theoretical plates of replicate injections of Trometamol. System precision results is tabulated in Table: 1.0. The % relative standard deviation for peak area of Trometamol peak from six replicate injections of standard solution 0.4%. The asymmetry for Trometamol peak from standard solution is 1.12 The theoretical plates for the Trometamol peak from standard solution is 18443.

Method Precision
Precision was determined by injecting six sample solutions spiked Trometamol at speci cation level. The samples were prepared as per the method and the result for precision study is tabulated in Table: 2.0. The method precession was performed with six replicate solutions prepared and the results found within the acceptance criteria

Linearity and Range
The linearity is determined by injecting the solutions in duplicate Trometamol ranging from 25% to 150% of the speci ed limit. Perform the regression analysis and determine the correlation coe cient and residual sum of squares. Linearity results is tabulated in Table:3.0. Figure shown in Figure: 11.0.

Accuracy
Recovery of Trometamol was performed. The sample was taken and varying amounts of Trometamol representing 50% to 150 % of speci cation level were added to the asks. The spiked samples were prepared as per the method and the results are tabulated in Table 4.0.

Solution stability of analytical solutions:
Trometamol standard and sample solutions were kept for about 48 hrs at room temperature in transparent bottles in auto sampler and in refrigerator 2-8°C. The stability of standard and sample solutions was determined by comparison of "old" prepared standard solutions with freshly prepared standard solutions. Solution stability results is tabulated in Table: 5.0 to 8.0.  The standard and sample solutions are stable up to 48 Hrs on bench top and when stored in refrigerator (2-8°C) condition.

Robustness
To validate the method robustness the chromatographic performance at changed conditions was evaluated compared to nominal conditions of the method. Standard solution was injected at each of the following changed conditions: Robustness results is tabulated in Table:9.0. The developed method was applied for the determination of Trometamol content in Gadobutrol in injection formulations obtainable in the local market. The test injection sample was prepared at a concentration of 60.0mg/mL and analyzed by the proposed method three times. The percent recovery and %RSD of Trometamol was calculated. The mean percent recovery and relative standard deviation of Gadobutrol was found to be 95.24% and 0.50% (Table 10). In this article, the authors have performed no experiments with any of the animal and human subjects.

Consent for publication:
Not applicable Availability of data and materials: All data and material are available upon request.

Not applicable
Funding: No funding was received Authors' contributions: We have assured that "all authors have read and approved the manuscript." All the authors have equal contribution and participation in this research work. SB has analyzed all samples on HPLC instrument and completed the experimental work and was a major contributor in writing the manuscript. He had completed his work under the supervision of HB a who help him to elaborate the methodology as well as theoretical approach. linearity of detector response for Trometamol