1. INTRODUCTION:

The manufacturing process is critical in designing the various products and ensuring that cross-contamination is avoided. Meanwhile, many pieces of equipment are present and used to manufacture various products; the cleaning process should be designed in such a way that the residues from the equipment are at an adequate level (Secretariat, P. I. C. O. S. 2007)^1-6.

The pharmacopeial data shown identification and quantification of Rocuronium bromide drug substance official in BP, USP, EP. Analytical Research and Development laboratory developed the Analytical Method (swab analysis) for testing Residual Rocuronium Bromide injection on production equipment after cleaning^7-10. This method was developed in accordance with the Method Development Plan, and it meets the requirements listed below as well as the ideal characteristics specified in USP General Chapter 621 and EP 2.2.46. Rocuronium bromide is a fast-acting, intermediate-acting non-depolarizing neuromuscular blocking agent with all of the typical pharmacological actions of this class in pharmaceuticals (curariform agents). It works by competing with nicotinic choline receptors on the motor end-plate. Acetylcholinesterase inhibitors such as neostigmine, edrophonium, and pyridostigmine inhibit this action¹¹.

Figure 1: Rocuronium bromide

2. MATERIALS AND METHODS:

2.1 Instrumentation:

Shimadzu HPLC-A20, Mettler Toledo pH meter, Heal force milli Q, Diluent system, Mettler semi micro and pan balance, Flycoz air dryer and swab plates SS, PTFE, silicone, and borosil glass.

2.2 Chemicals:

HPLC grade acetonitrile and methanol from Dikma, SCR grade orthophosphoric acid, Aladdin grade tetramethylammonium hydroxide pentahydrate, and Rocuronium Bromide Drug Substance (Aspen).

2.3 Preparation of Solutions:

2.3.1 Specificity:

Standard preparation (12ppm):

Weighed accurately and transferred 30mg of standard into a 25mL volumetric flask, then added diluent and mixed to volume. Pipette out 1 mL and transfer it to 100 mL of diluent.

Stainless Steel Plate:

Add 0.1mL of the 12PPM standard solution to the appropriate plate (SSplate), then use a dryer to dry the plate. Swab pattern-1 procedure: after swabbing, cut off the swab's head and place it in a test tube with 10mL of diluent. Shake by hand for two minutes. Then inject the filtered solution into the HPLC using a 0.45micron Nylon filter.

Silicone Plate:

Add 0.1mL of the 12PPM standard solution to the appropriate plate (a Silicone plate), then use a dryer to dry the plate. Swab pattern-1 procedure: after swabbing, cut off the swab's head and place it in a test tube with 10 mL of diluent. Shake by hand for two minutes. After that, filter the solution using a 0.45-micron Nylon filter before injecting it into HPLC.

PTFE Plate (Polytetrafluoroethylene):

Add 0.1mL of the 12PPM standard solution to the appropriate PTFE plate, and then use a dryer to dry the plate. Swab pattern-1 procedure: after swabbing, cut off the swab's head and place it in a test tube with 10mL of diluent. Shake by hand for two minutes. Next, filter the solution using a 0.45micron Nylon filter before injecting it into the HPLC.

Same as sample blank interference was verified.

2.3.2 Precision:

Standard preparation (12 ppm):

30mg of standard were precisely weighed, transferred, and mixed with diluent to create the desired volume in a 25mL volumetric flask. 1mL should be pipetted into 100mL of water to dilution.

Precision sample:

Add 0.1mL of the 12PPM standard solution to the appropriate plate (SS plate), then use a dryer to dry the plate. Swab Pattern-1 Procedure After swabbing the plate, remove the swab's head and place it in a test tube with 10mL of diluent. Shake by hand for two minutes. After that, filter the solution using a 0.45-micron Nylon filter before injecting it into HPLC.

2.3.3 Intermediate precision (IM):

Standard preparation (12 ppm):

Accurately weigh and transfer 30mg of standard into a 25mL volumetric flask, then add Diluent and mix to volume. Pipette 1mL into a 100mL dilute with water.

IM Precision sample:

Add 0.1mL of 12PPM standard solution to the appropriate plate (SS plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45-micron Nylon filter and injected into HPLC.

2.3.4 Linearity:

The standard solutions for various levels of drug (from LOQ to 200% of the target concentration) were analysed, and the average responses were plotted against the concentration to determine the concentration range with linearity. Six concentration levels were prepared and analysed [LOQ, 25%, 50%, 75%, 100%, 200%, and 300%]. The results are linearly regressed, and the correlation coefficient is computed.

Preparation of standard:

Accurately weigh and transfer 30mg of standard into 25 mL volumetric flask add Diluent and mix it made up to volume. Pipette out 1mL transfer it into 100mL dilutes with water.

Preparation of LOQ 25%, 50% sample:

Weigh and transfer 30mg of standard into a 25mL volumetric flask, then add Diluent and mix to volume. Pipette out 1mL and dilute with water in 100mL. This ranges from 4mL to 100mL (LOQ), 3mL to 10mL (25%), and 5mL to 10mL (50%).

Preparation of 100%, 200%, 300% linearity sample:

Transfer 30mg of the standard into a 25mL volumetric flask, add Diluent, and mix it up to volume. Linearity samples ranged from 1mL to 100mL (100%), 2mL to 100mL (200%), and 3mL to 100mL (300%).

2.3.5 Accuracy and Recovery:

Stainless steel plate:

SS Plate LOQ level sample:

Add 0.005mL of 12PPM standard solution to the appropriate plate (SS plate) and dry the plate with a dryer. Swab pattern-1 procedure: After swabbing the plate, remove the swab head and place it in a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter before being injected into the HPLC. Three samples were prepared and injected in triplicate.

SS Plate 50% level sample:

Add 0.05mL of 12PPM standard solution to the appropriate plate (SS plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Samples were prepared and injected in triplicate.

SS Plate 100% level sample:

SS Plate 300% level sample:

Add 0.3 mL of 12 PPM standard solution to each plate (SS plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10 mL diluent test tube. Manually shake for 2 minutes. The solution is then filtered with a 0.45-micron Nylon filter before being injected into the HPLC. Samples were prepared and injected in triplicate.

Silicone Plate LOQ level sample:

Add 0.005 mL of 12 PPM standard solution to the appropriate plate (Silicone plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter before being injected into the HPLC. Three samples were prepared and injected in triplicate.

Silicone Plate 50% level sample:

Add 0.05mL of 12 PPM standard solution to each plate (Silicone plate) and dry plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2 minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Samples were prepared and injected in triplicate.

Silicone Plate 100% level sample:

Add 0.1mL of 12 PPM standard solution to the appropriate plate (Silicone plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Samples were prepared and injected in triplicate.

Silicone Plate 300% level sample:

Add 0.3mL of 12PPM standard solution to a separate plate (Silicone plate) and dry plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Three samples were prepared and injected in triplicate.

PTFE Plate LOQ% level sample:

Add 0.005mL of 12PPM standard solution to the appropriate plate (PTFE plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Interference was verified in the same way that sample blank interference was.

PTFE Plate 50% level sample:

Add 0.5mL of 12PPM standard solution to each plate (PTFE plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Interference was verified in the same way that sample blank interference was.

PTFE Plate 100% level sample:

Add 0.1mL of 12 PPM standard solution to the appropriate plate (PTFE plate) and dry the plate with a dryer. After swabbing the plate keep, cut off the head of the swab and transfer it into a 10mL diluent test tube. Manually shake for 2minutes. The solution is then filtered with a 0.45micron Nylon filter and injected into HPLC. Interference was verified in the same way that sample blank interference was.

PTFE Plate 300% level sample:

Fill each plate (PTFE plate) with 0.3mL of 12PPM standard solution and dry the plate with a dryer. After swabbing the plate keep, remove the swab's head and place it in a 10mL diluent test tube. Shake for 2 minutes by hand. Before being injected into HPLC, the solution is filtered with a 0.45micron Nylon filter. Interference was tested in the same manner as sample blank interference.

2.3.6 Robustness:

The standard solution system suitability was investigated using deliberate variation of the actual method, flow rate (10% variation), pH buffer (10% variation), wavelength (+ or - 3nm), and column temperature (10% variation).

3. RESULTS AND DISCUSSION:

3.1 Method development^12-16

3.1.1 Selection of detector wavelength:

The UV spectrums of Rocuronium Bromide and a 10 mg/mL standard are injected into a PDA system and recorded. The obtained spectrum, shown in figure, was chosen based on data at 210nm. The experimental results are consistent with the Pharma Europe monograph.

Figure 2: Spectrum of Rocuronium Bromide

3.1.2 Chromatographic conditions optimization:

The assay method and monograph conditions are used as a guide; the assay method run time is 15 minutes, and the diluent is Acetonitrile; the main goal is to shorten the runtime and change the diluent proportion with Diluent or Diluent solvent mixture.

Figure 2: Chromatogram of Standard

The retention of the analyte at 3.177 and the chromatography were found to be satisfactory, meeting the system suitability criteria of reducing runtime. And no blank interference was found in the blank and residue plate blank interference.

3.1.3 Extraction/Recovery study/Diluent selection:

Rocuronium Bromide is a water-soluble compound. Based on the drug's solubility and the swabbing solvent's impact (to destroy the equipment material) on Material Water, Methanol, and IPA as used in the initial swabbing studies.

3.1.4 Recovery simple preparation:

Prepared standard solution of 8PPM solution and add 0.1mL of 8PPM solution on a respective plate (SS plate) and dried plate with the help of dryer.

3.1.5 Swabbing solution adding patterns:

Swab solution added in all four types, best recovery observed in pattern-4, for further experiments pattern-4 selected.

Pattern-1 Pattern-2

Pattern-3 Pattern-4

Figure 3: Swabbing pattern

Maintain a swab in the test tube. After drying the solution on the plate, add 10mL of swabbing solvent. Squeeze the swab wall against the test tube wall. Swab the plate's surface with a swab, following the plate swabbing procedure.

3.1.6 Way of Plate Swabbing:

Swab- Side- 1 Swab- Side- 2

Swab- Side- 3 Swab- Side- 4

Figure 4: Plate Swabbing pattern

After swabbing the plate keep, cut off the swab's head and place it in a diluent test tube. Manually shake for 2 minutes. The solution is then filtered with the appropriate filter and injected into the HPLC.

Table 2: Recovery study

S. No	Plate material	Diluent/Swab solvent	Area	Recovery%
1	SS-1	Diluent	71632	88.1
2	SS-2	Diluent	66632	82.0
3	Silicone-1	Diluent	81244	100.0
4	Silicone-2	Diluent	83207	102.4
5	PTFE-1	Diluent	79816	98.2
6	PTFE-2	Diluent	82206	101.1

Percent recovery should find more than 80% with Diluent on the SS, silicone, PTFE. Diluent selected as swabbing solvent.

3.1.7 Rinse samples preparation:

Rinse samples prepared with the swabbing solvent mentioned above. Prepared a standard solution of 8PPM solution and added 0.1mL of it to a respective plate (SS plate) and dried it with a dryer. Prepared a standard solution of 12PPM solution and added 0.1mL of the solution to each plate (Silicone and PTFE plate) and dried the plate with a dryer. After drying the plate, slowly pour 10mL of swab solvent and collect the solvent, then filter solution and inject into HPLC. Nylon 0.45micron filter is given good data after initial rinse and swab samples to perform different filter study.

Table 3: Rinse recovery study

S. No	Plate material	Diluent/Swab solvent	Area	Recovery %
1	SS-1	Diluent	101925	104.9
2	SS-2	Diluent	101950	104.9
3	Silicone-1	Diluent	125456	129.1
4	Silicone-2	Diluent	125651	129.3
5	PTFE-1	Diluent	106102	109.2
6	PTFE-2	Diluent	106005	109.1

4. METHOD VALIDATION^17,18

4.1 Specificity and system suitability:

The analyte is specific to quantify, no interference was observed on SS, PTFE, and Silicone materials during analyte retention, and the peak purity of rocuronium passed. Assay SS plate (101.2%), PTFE plate (94.2%), and Silicone plate (97.2%) content was discovered. The SS plate was chosen for precision testing. Figures 5 and 6 show SS and PTFE, Silicone materials interference, standard, sample chromatograms, and table no. 4 shows system suitability.

Figure 5: Shows the standard chromatogram

Figure 6: Shows the sample chromatogram

4.2 Precision and intermediate precision:

The obtained data demonstrated that the method was precise despite instrument and analyst variation. The obtained assay was found to be in the 50 to 130% range, as shown in table no. 5.

4.3 Linearity:

Rocuronium's correlation coefficient was calculated. The quantification limit at 300% is 0.55PPM to 36.40 PPM. The obtained result was deemed satisfactory; the correlation coefficient was found to be 0.998.

Figure 7: Linearity graph of Rocuronium Br.

4.4 Recovery:

The analyte recovery was estimated using SS, PTFE, and Silicone, with a 50 to 130% recovery found. Table no.6 summarizes the findings.

Table 6: Accuracy and recovery

Stain less steal plate
LOQ Level (Avg 3 prp)	50% (Avg 3 prp)	100% (Avg 3 prp)	300% (Avg 3 prp)	Limit	Overall limit
97.7 %	98.1 %	102.5 %	103.4 %	50 to 130%	<20%
PTFE plate
LOQ Level (Avg 3 prp)	50% (Avg 3 prp)	100% (Avg 3 prp)	300% (Avg 3 prp)	Limit	Overall limit
91.5 %	100.2 %	102.3 %	103.8 %	50 to 130%	<20%
Silicone plate
LOQ Level (Avg 3 prp)	50% (Avg 3 prp)	100% (Avg 3 prp)	300% (Avg 3 prp)	Limit	Overall limit
96.4 %	99.3 %	100.6 %	103.9 %	50 to 130%	<20%

4.5 Robustness:

Small deliberate chances in the actual analytical method result in no impact on analyte retention and chromatography. The results are shown in table 7.

Table 7: Robustness

Parameter	% RSD of RT	% RSD of Area	% RSD of Tailing	% RSD of NTP
Parameter	% RSD of RT	% RSD of Area	% RSD of Tailing	% RSD of NTP
Optimized Method	0.53	0.54	0.21	0.31
Flow rate 1.8mL/min	0.52	0.52	0.58	0.56
Flow rate 2.2mL/min	0.5	0.48	0.52	0.54
Temperature 25°C	0.48	0.46	0.51	0.52
Temperature 35°C	0.46	0.44	0.47	0.48
Buffer pH at 7.20	0.44	0.42	0.45	0.44
Buffer pH at 7.60	0.42	0.41	0.43	0.43
Organic phase +10%	0.41	0.38	0.41	0.42
Organic phase -10%	0.38	0.34	0.38	0.41
Wavelength at 207 nm	0.36	0.32	0.37	0.39
Wavelength at 213 nm	0.32	0.31	0.33	0.36

5. CONCLUSION:

The method for analysing residue swabs was finalised and validated. The Rocuronium injection residue method was accurate and precise, with no interference of subsidiary peaks at the analyte peak. The Rocuronium analyte peak was retained for about 3.5 minutes, and the system suitability was found to be satisfactory; the sample run time was 8 minutes. The linearity test was carried out from the LOQ level to the 300% level, and a linear correlation coefficient of R² 0.998 was identified. The estimated recovery of SS, PTFE, and Silicone was found to be between 50 and 130%. The precision and intermediate precision assays average 92.4% and 101.1%, respectively. As a result, the analytical residue method was suitable for routine and daily laboratory analysis. The swab recovery studies are appropriate for producing reproducible and precise results. The cleaning validation revealed that the cross-contamination is well within the acceptable range and the cleaning procedure used can keep drug residues to an acceptable level.

6. CONFLICT OF INTEREST:

None.

7. REFERENCES:

1. Błażewicz, A., Fijałek, Z., Warowna-Grześkiewicz, M., and Boruta, M. Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection. Journal of Chromatography A. 2007; 1149(1): 66-72. doi: 10.1016/j.chroma.2006.12.041

2. Narendra Chotai, Vishnu Patel, Harsha Patel, Uren Patel, Rajendra Kotadiya. Cleaning Validation Study of Amoxycillin Trihydrate. Research J. Pharm. and Tech. 2009; 2(1): 147-150. Available on: https://rjptonline.org/AbstractView.aspx. PID=2009-2-1-76

3. Raj Pal, G., Arya, R., Joshi, T., and Bisht, D. A Review on Cleaning Validation In Pharmaceutical Industry. Journal of Drug Delivery and Therapeutics, 2018; 8(3): 138-146. https://doi.org/10.22270/jddt.v8i3.1695

4. El Houssini OM, Abd El-Rahman MK, Fahem DK, Zaazaa HE. Application of ICH Guidelines for Studying the Degradation Behavior of Rocuronium Bromide Coupled with Stability-Indicating RP-LC Method. J Chromatographic Science. 2022: 60(3): 217-223. doi: 10.1093/chromsci/bmab065.

5. Kishan Malviya, Monika Maheshwari, Mahendra Singh Rathore. Spectroscopic method for the quantification of residue of Tetramethylthionine chloride on swab from manufacturing equipment in support of cleaning validation. Research Journal of Pharmacy and Technology. 2022; 15(4): 1499-4. doi: 10.52711/0974-360X.2022.00249

6. Kamal Hossain, Kamrun Nahar, Ehsanul Hoque Mazumder, Tony Gestier, Tanvir Ahmed Khan, Kaiser Hamid. Development of a Cleaning Validation Protocol for an Odd Case Scenario and Determination of Methoprene residues in a Pharmaceutical Manufacturing Equipment Surfaces by using a Validated UFLC Method. Research J. Pharm. and Tech. 2017; 10(11): 3789-3794. doi: 10.5958/0974-360X.2017.00687.4

7. Swetha Sri. R, Dr. Bhavya Sri. K, Mounika. Ch. A Review on Comparative study of HPLC and UPLC. Research J. Pharm. and Tech. 2020; 13(3): 1570-1574. doi: 10.5958/0974-360X.2020.00284.X

8. Patel Payal, K., Patel, N. M., and Patel, P. M. An overview on cleaning validation. International Journal of pharmaceutical and Biological Archives, 2011; 2(5): 1332-1336.

9. Secretariat, P. I. C. O. S. Recommendations on validation master plan, installation and operational qualification, non-sterile process validation, cleaning validation. Documents for inspectors. PI, 2007, 006-3.

10. SONG, G., JIANG, J., ZHOU, L., and YAN, K. Determination of Related Substances in Rocuronium Bromide and Its Injection by HPLC [J]. Chinese Journal of Pharmaceuticals, 2010, 6.

11. Komal P. Shinde, Akash D. Rajmane. A Review UV Method Development and Validation. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 122-0. doi: 10.52711/2231-5675.2023.00021

12. Manojkumar K. Munde, Nilesh S. Kulkarni, Nikita B. Rukhe, Dhanya B. Sen. A Comprehensive Review on Analytical Method Development and Validation for SGLT-2 Inhibitors by HPLC in Its API and Dosage Form. Research J. Pharm. and Tech. 2020; 13(7): 3472-3479. doi: 10.5958/0974-360X.2020.00616.2

13. Saish Naik, Celina Nazareth, Sanelly Pereira. A Novel HPLC cleaning Validation and Assay method for the simultaneous estimation of Perindopril and Amlodipine. Research J. Pharm. and Tech. 2020; 13(12): 5919-5923. doi: 10.5958/0974-360X.2020.01033.1

14. Namratha, S., and V. A. “Method development and validation of lopinavir in tablet dosage form using reversed-phase high-performance liquid chromatography”. Asian Journal of Pharmaceutical and Clinical Research. 2018; 11(16): 125-8. doi: https://doi.org/10.22159/ajpcr.2018.v11s4.31715

15. Sunkara Namratha et al. Analytical method development and validation of valacyclovir in dosage forms by uplc techique. Journal of Global Trends Pharm Sciences. 2020; 11 (2): 7590 – 7594.

16. Akash D. Rajmane, Komal P. Shinde. A Review of HPLC Method Development and Validation as per ICH Guidelines. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 143-1. doi: 10.52711/2231-5675.2023.00024

17. Rahul D. Khaire, P. Y. Pawar. A Review on Development of Stability Indicating Analytical Methods for drugs in Bulk and Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2022; 12(4): 261-3. doi: 10.52711/2231-5675.2022.00043

18. Saish Naik, Celina Nazareth, Sanelly Pereira. A Novel HPLC cleaning Validation and Assay method for the simultaneous estimation of Perindopril and Amlodipine. Research J. Pharm. and Tech. 2020; 13(12): 5919-5923. doi: 10.5958/0974-360X.2020.01033.1

Received on 29.08.2023 Revised on 17.10.2024

Accepted on 12.02.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4241-4247.

DOI: 10.52711/0974-360X.2025.00609

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	HPLC conditions
01	Mobile Phase	Buffer solution: Transfer 4.53 g of tetramethylammonium hydroxide pentahydrate to a 1000-mL volumetric flask, dissolve in water, and mix well. Adjusted the pH: 7.4 with phosphoric acid solution and mixed well. filter through 0.2 µm membrane filter. Mobile phase: Mix pH 7.4 buffer: Acetonitrile in the ratio of 40:60 v/v.
02	Diluent	Stock in Diluent and further diluted with 70% Acetonitrile
03	Column Details	Hypersil silica (250x4.6) mm, 5µm
04	a) Detector Wavelength	210 nm
	b) Flow rate	2.0 mL/min
	c) Column Temperature	30°C
	d) Injection Volume	5 µL
	e) Run time	8 min
05	Blank details	Water and Acetonitrile mixture
06	Test sample/ Standard Concentration	0.04 mg/mL and 0.08 mg/mL

Area	Retention time	Tailing	Plate count	Interference
120368	3.268	1.444	6686	No

Instrument-1 and Analyst-1 precision assay limit (50 to 130 %)
Test-1	Test-2	Test-3	Test-4	Test-5	Test-6	Avg	%RSD	Limit
94.4	98.8	89.0	90.0	95.1	85.7	92.4	5.2	>20
Instrument-2 and Analyst-2 precision assay limit (50 to 150 %)
Test-1	Test-2	Test-3	Test-4	Test-5	Test-6	Avg	%RSD	Limit
98.2	102.1	99.5	101.0	99.9	100.2	100.1	1.3	>20