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ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue VIII August 2025

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QbD Approach to Hplc Method Development and Validation of the

Simultaneous Estimation of Sulbactum and Durlobactum in

Pharmaceutical Dosage Form

Dr.D.Tirumala., Basapuram Meghana., Mothukuru Sainikitha., Jakku Jansi., Md Mamun Reja

Omega college of Pharmacy, India

DOI: https://doi.org/10.51244/IJRSI.2025.120800133

Received: 21 Aug 2025; Accepted: 27 Aug 2025; Published: 13 September 2025

INTRODUCTION

The pharmaceutical industry consistently emphasizes the importance of product quality, safety, and efficacy.

To enhance these aspects, scientific tools such as Quality by Design (QbD) and Process Analytical

Technology (PAT) have been widely implemented. These approaches provide a systematic and science-based

framework for pharmaceutical development and manufacturing, aiming to improve product understanding,

process control, and ultimately, minimize risks while maximizing productivity and quality.

The QbD framework, initially developed for manufacturing processes, has been successfully extended to the

field of analytical method development—a concept now termed Analytical Quality by Design (AQbD).

AQbD applies the principles of QbD to ensure that analytical methods are not only fit for their intended

purpose but also robust and reliable throughout the product lifecycle.

Regulatory bodies, including the U.S. FDA, have recognized the importance of QbD and released guidance

documents specifically for its implementation in immediate-release and extended-release formulations.

Moreover, the International Council for Harmonisation (ICH) encourages the application of QbD principles

through its guidelines Q8 to Q11, supporting its adoption in both formulation and analytical development.

Drug profile:

Sulbactam is a beta-lactamase inhibitor commonly used in combination with other antibiotics (like

ampicillin) to treat various bacterial infections. It works by blocking the enzyme beta-lactamase, which

bacteria use to resist antibiotics. Sulbactam has a molecular formula of C₈H₁₁NO₅S and a molecular weight of

233.24 Da. It's available mainly in injectable form under brand names such as Unasyn and Sulperazon.

Common side effects include skin rash, diarrhea, nausea, and vomiting, and it should be avoided by

individuals allergic to beta-lactam antibiotics.

Durlobactam is an investigational beta-lactamase inhibitor developed to treat serious hospital-acquired

and ventilator-associated bacterial infections, especially those caused by resistant strains like Acinetobacter

baumannii. It has a molecular formula of C₈H₁₁N₃O₆S and a molecular weight of 277.25 g/mol. Durlobactam

is designed to inhibit a broader range of beta-lactamase enzymes (classes A, C, and D) but is not active

against class B metallo-beta-lactamases. It is currently not commercially available, and limited clinical data

exist regarding its side effects and tolerability.

) Tailing Factor Of Sulbactum And Durlobactum:-

ANOVA for Quadratic model Table 1: Response 2: tailing factor

Source

Squares

Square

Value

Prob > F

Model

0.036

3.967E-003

9.01

0.0042

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A-Flow rate

5.119E-003

11.62

0.0113

B-Buffer PH

3.178E-004

0.72

0.4237

C-Organic ration MP

8.137E-005

0.18

0.6802

5.167E-003

11.73

0.0111

9.749E-005

0.22

0.6523

1.481E-004

0.34

0.5802

A^2

8.176E-003

18.56

0.0035

B^2

0.014

31.52

0.0008

C^2

4.654E-003

10.57

0.0140

Residual

3.083E-003

4.404E-004

Lack of Fit

3.083E-003

1.028E-003

Pure Error

0.000

Cor Total

0.039

System Suitability:

Figure 1: Chromatogram for system suitability

Figure 2: Chromatogram for system suitability

Table 2: Results of system suitability parameters

S.No

Name

RT(min)

Area sec)

Height (µ

USP tailing

USP plate count

Sulbactum

2.162

18895

1109

1.0

5797

Durlobactum

3.441

789931

22645

0.85

2357

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Acceptance criteria:

 Resolution between two drugs must be not less than 2.

 Theoretical plates must be not less than 2000.

 Tailing factor must be not more than 2.

 It was found from above data that all the system suitability parameters for developed method were

within the limit.

Validation Parameters:

ASSAY:

Standard

and

sample

solution

injected

described

under

experimental

work.

The corresponding

chromatograms and results are shown below.

Figure 3: Chromatogram for Standard

Figure 4: Chromatogram for Sample

Table:3 Retention Times Of Sulbactum,Durlobactum

S.No

Name( STD)

RT(min)

Area(µV sec)

Height (µV)

USP tailing

USP plate count

Sulbactum

2.162

18895

1109

1.0

5797

Durlobactum

3.441

789931

22645

0.85

2357

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Table:4 Retention Times Of Sulbactum,Durlobactum

S.No

Name (Sample)

RT(min)

Area(µV sec)

Height (µV)

USP tailing

USP plate count

Sulbactum

2.170

18899

1129

1.2

5799

Durlobactum

3.437

789938

22695

0.89

2351

Table 5: Results of Assay for Sulbactum and Durlobactum

Label Claim (mg)

% Assay

Sulbactum and Durlobactum

0.5 g + 1.0 g

101.2

Linearity:

The linearity range was found to lie from 10µg/ml to 50µg/ml of Sulbactum and Durlobactum and

chromatograms are shown below

Figure 5: Chromatogram for linearity-1

Figure 6 : Chromatogram for linearity-2

Figure 7: Chromatogram for linearity-3

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Figure 8: Chromatogram for linearity-4

Figure 9: Chromatogram for linearity-5

Table 6: Area of different concentration of Sulbactum and Durlobactum

Concentration (µg/ml) Sulbactum and Durlobactum

Areas Durlobactum

Areas of Sulbactum

6299

273312

12599

526625

18899

789938

25198

1053250

32498

1316563

Figure 10: Calibration graph for Sulbactum

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Figure 11: Calibration graph for Durlobactum

Table 7: Analytical performance parameters of Sulbactum and Durlobactum

Parameters

Sulbactum

Durlobactum

Slope (m)

649.97

26131

Intercept (c)

400.5

7999.5

Correlation coefficient (R

)

0.999

Acceptance criteria:

Correlation coefficient (R

) should not be less than 0.999

The correlation coefficient obtained was 0.999 which is in the acceptance limit.

Precision:

Precision of the method was carried out for both sample solutions as described under experimental

work. The corresponding chromatograms and results are shown below.

Figure 12: Chromatogram for Precision -1

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Figure 13 : Chromatogram for Precision -2

Figure 14: Chromatogram for Precision -3

Figure 15: Chromatogram for Precision -4

Figure 16: Chromatogram for Precision -5

Figure 17: Chromatogram for Precision -6

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Table 8: Results of Precision for Sulbactum and Durlobactum

Injection

Area

Injection-1

7970152

16726

Injection-2

8065041

16157

Injection-3

7899251

16878

Injection-4

7842995

16504

Injection-5

7926488

16948

Injection-6

7951230

16631

Average

7942526

16640.67

Standard Deviation

74679.64

286.6243

%RSD

0.94

1.72

Acceptance criteria:

 %RSD for sample should be NMT 2

 The %RSD for the standard solution is below 1, which is within the limits hence method is precise.

Intermediate Precision (ruggedness)

There was no significant change in assay content and system suitability parameters at different conditions

of ruggedness like day to day and system to system variation.

Figure 18: Chromatogram for ID Precision -1

Figure 19: Chromatogram for ID Precision -2

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Figure 20: Chromatogram for ID Precision -3

Figure 21: Chromatogram for ID Precision -4

Figure 22: Chromatogram for ID Precision -5

Figure 23: Chromatogram for ID Precision -6

Table 9: Results of Intermediate precision for Sulbactum Durlobactum

Injection

Area

Injection-1

16726

7970152

Injection-2

16557

8065041

Injection-3

16678

7899251

Injection-4

16514

7842995

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Injection-5

16928

7926488

Injection-6

16631

7951230

Average

16672.33333

7942526

Standard Deviation

147.2123183

74679.64

%RSD

0.8

0.9

Acceptance criteria:

 %RSD of five different sample solutions should not more than 2

 The %RSD obtained is within the limit, hence the method is rugged.

ACCURACY:

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was

calculated.

Figure 24: Chromatogram for Accuracy 50%-1

Figure 25: Chromatogram for Accuracy 50%-2

Figure 26: Chromatogram for Accuracy 50%-3

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Figure 27: Chromatogram for Accuracy 100%-1

Figure 28: Chromatogram for Accuracy 100%-2

Figure 29: Chromatogram for Accuracy 100%-3

Figure 30: Chromatogram for Accuracy 150%-1

Figure 31: Chromatogram for Accuracy 150%-2

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Figure 32: Chromatogram for Accuracy 150%-3

Table 10: Accuracy (recovery) data for Sulbactum and Durlobactum

%Concentration (at

specification Level)

Area*

Sulbactum

Amount Added

(mg)

Amount

Found (mg)

Recovery

Mean

Recovery

50%

9449.5

12.5

12.2

97.6

98.1

100%

18899

24.5

150%

28348.5

37.5

37.1

98.9

%Concentration (at

specification Level)

Area*

Durlobactum

Amount

Added (mg)

Amount

Found (mg)

% Recovery

Mean

Recovery

50%

394969

12.5

12.2

97.6

98.1

100%

789938

24.5

150%

1184934

37.5

37.1

98.9

*Average of three determinations

Acceptance Criteria:

The percentage recovery was found to be within the limit (98-102%).

The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence method is accurate.

Limit Of Detection For Sulbactum And Durlobactum

The lowest concentration of the sample was prepared with respect to the base line noise and measured the

signal to noise ratio.

Figure 33: Chromatogram of Sulbactum and Durlobactum showing LOD

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Table 11: Results of LOD

Drug name

Baseline noise(µV)

Signal obtained (µV)

S/N ratio

Conc. In ppm

Sulbactum

243

2.96

0.38

Durlobactum

223

2.97

0.08

 Signal to noise ratio shall be 3 for LOD solution

 The result obtained is within the limit.

Limit Of Quantification For Sulbactum And Durlobactum

The lowest concentration of the sample was prepared with respect to the base line noise and measured the

signal to noise ratio.

Figure 34: Chromatogram of Sulbactum and Durlobactum showing LOQ

Table 12: Results of LOQ

Drug name

Baseline noise(µV)

Signal obtained (µV)

S/N ratio

Conc. In ppm

Sulbactum

818

9.97

1.2

Durlobactum

Sulbactum Durlobactum

745

9.93

0.2

 Signal to noise ratio shall be 10 for LOQ solution

 The result obtained is within the limit.

Robustness:

The standard and samples of Sulbactum and Durlobactumwere injected by changing the conditions of

chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric

factor, and plate count.

Variation in flow

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Figure 35: Chromatogram showing less flow

Figure 36: Chromatogram showing more flow

Variation of mobile phase organic composition:

Figure 37: Chromatogram showing less organic composition

Figure 38: Chromatogram showing more organic composition

Table 13: Results for variation in flow for Sulbactum and Durlobactum

S. No

Flow

(ml/min)

System Suitability Results of Sulbactum

System Suitability Results of Durlobactum

USP Plate Count

USP Tailing

USP Plate Count

USP Tailing

0.8

5721

1.1

2349

1.01

5799

1.2

2351

0.89

1.2

5793

0.98

2345

0.96

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* Results for actual flow (1 ml/min) have been considered from Assay standard.

Table 14: Results for variation in mobile phase composition for Sulbactum and Durlobactum

Change in Organic

Composition in the

Mobile Phase

System Suitability Results of Sulbactum

System Suitability Results of

Durlobactum

USP Plate Count

USP Tailing

USP Plate Count

USP Tailing

10% less

5721

1.1

2349

1.01

*Actual

5799

1.2

2351

0.89

10% more

5793

0.98

2345

0.96

* Results for actual Mobile phase composition have been considered from Accuracy standard.

Acceptance criteria:

The Retention time, USP plate count, USP tailing factor obtained for change of flow rate, variation in mobile

phase was found to be within the acceptance criteria. Hence the method is robust.

CONCLUSION

The application of QbD principles in the development and validation of the HPLC method for simultaneous

estimation of sulbactam and durlobactam proves beneficial in enhancing method reliability and efficiency. By

systematically identifying and controlling key variables during method development, the method's robustness

and suitability for pharmaceutical analysis are significantly improved.

Furthermore, the establishment of a thorough method validation protocol ensures that the method meets

regulatory requirements for accuracy, precision, linearity, specificity, and robustness. The validated method

provides a reliable tool for routine analysis of sulbactam and durlobactam in pharmaceutical dosage forms,

contributing to the overall quality control and assurance in pharmaceutical manufacturing processes.

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