UHPLC Method development, Validation and Forced degradation study for simultaneous estimation of Vaborbactam and Meropenem in bulk drug

 

B. Ramya Kuber1*, B. Geethika2

1Department of Pharmacognosy, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam, Tirupati (Women’s University) Andhra Pradesh.

2Department of Pharmaceutical analysis, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam, Tirupati (Women’s University) Andhra Pradesh.

*Corresponding Author E-mail: brkuber01@gmail.com

 

ABSTRACT:

The accurate, precise, sensitive and economical spectrophotometric method was developed, validated and force degraded studies for simultaneous estimation of Meropenem and Vaborbactam in bulk drug. The UV method employed was simultaneous equation method. Chromatographic conditions involved in UHPLC C18 column with the mobile phase consisting of methanol and water. The method employs 261nm as λ1 and 273nm as λ2 for formation of equations. Vaborbactam and Meropenem obeys vandeemter equation in the concentration range 20-50µg/mL-1 (0.999) and 20-50µg/mL-1 (0.999). The calibration graph were plotted linear. The mean recovery for vaborbactam and meropenem was found to be 99.6% and 99.4% respectively. The limit of detection and quantification are determined and were found to be 0.025µg/ml and 0.045µg/ml. The developed method were validated according to ICH guidelines and values of accuracy, precision, LOD and LOQ, robustness and ruggedness. were found to be in good accordance with the prescribed values. Upon validation, the developed method effectively detected the drug as a pure compound. The drug was subjected to stress condition of peroxide, photolytic, acid, alkaline and thermal degradation, considerable degradation was found in thermal degradation studies. Thus the proposed methods were successfully applied for simultaneous determination of vaborbactam and meropenem in routine industry work.

 

KEYWORDS: UHPLC, Vaborbactam, Meropenem, System suitability, Precision, and Degradation studies.

 

 


INTRODUCTION:

UHPLC (Ultra High-Performance Liquid Chromatography) is a relatively new technique. UHPLC brings dramatic improvements in sensitivity, resolution and speed of analysis can be calculated[1,2]. It has instrumentation that operates at high pressure but it has no negative impact on column and uses fine particles (<2.5µm) and mobile phases at high linear velocities decreases the length of column, reduces solvent consumption and saves time. The UHPLC is based on the principle on vandeemter equation.

 

The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary gradient. The binary solvent manager is a high-pressure pump that moves solvent through the system. Vaborbactam (VBB) and Meropenem (MPN) standard drugs are used. Vaborbactam is a β-lactamase inhibitor based on a cyclic boronic acid pharmacophore3,4. It is a combination of antibacterial therapy. It is potent inhibition of Klebsiella pneumoniae carbapenemase enzymes and other ambler class A and C enzymes. Meropenem is broad spectrum carbapenem antibiotic. It is active against gram positive and gram-negative bacteria. It was approved for treatment of adult patients with complicated urinary tract infections[3,4]. From the various literature findings it has been seen that vaborbactam in combination with meropenem were analytically validated by different spectroscopic and chromatographic techniques. The major objective of this research is to develop convenient UHPLC for determination of Vaborbactam and Meropenem in bulk drug, to maintain low retention time, better resolution and only two solvents are used in our study when compare to previous other spectroscopic methods.

 

Fig. 1: Chemical structure of Vaborbactam and Meropenem

 

MATERIALS AND METHODS:

Chemicals and reagents:

Meropenem and Vaborbactam were procured from Madras Pharmaceuticals. Ammonium hydrogen phosphate, Acetonitrile, Water, Sodium hydroxide used were of analytical grade (Rankem or Merck chemicals) 0.45µm nylon filter (Madras pharmaceuticals, Chennai, India) was used. All other chemicals and reagents used were analytical grade unless otherwise indicated.

 

Selection of solvents:

On the basis of solubility study methanol was selected as the solvent for dissolving Meropenem and Vaborbactam.

 

Preparation of standard stock solution:

Vaborbactam Stock Solution:

An accurately weighed quantity of vaborbactam (1mg/ml) was taken in 50ml volumetric flask and dissolved in methanol (50ml) with the help of ultrasonication for about 10 min. Then the volume was made up to the mark using methanol to get vaborbactam standard stock solution (1mg/mL).

 

Meropenem Stock Solution:

An accurately weighed quantity of Meropenem (1mg/ml) was taken in 50ml volumetric flask and dissolved in methanol (50ml) with the help of ultrasonication for about 10min. Then the volume was made up to the mark using methanol to Meropenem.

 

Validation Method:

Vaborbactam and Meropenem were validated by various parameters like system suitability, precision, linearity, accuracy, limit of detection, quantification, robustness and ruggedness[5].

 

Forced degradation study was carried out in alkaline, peroxide, acid and thermal methods according to ICH guidelines[5].

METHOD DEVELOPMENT:

Determination of λ max of individual component:

Appropriate necessary dilutions are made from standard stock solutions to get the concentration range of 10µg/ml of vaborbactam and 10µg/ml of meropenem. Drug solutions were scanned separately between 200-400nm. Vaborbactam shows at 261nm while meropenem shows at 273nm respectively.

 

Overlay spectra of Vaborbactam and Meropenem:

The overlain spectrum of both drugs was recorded (Fig:2) and two wavelengths 261nm(λ max of vaborbactam) and 273nm(λ max of meropenem) were selected for further study.

 

 

Fig. 2: Isobestic point of Vaborbactam and Meropenem

 

OPTIMIZATION METHOD:

Preparation of mobile phase solution:

Mix 700ml of methanol and add 300ml of water and it is filtered through 0.45µm membrane filter. The solution was sonicated up to 10min and again the solution was filtered through 0.22µm membrane filter and again sonicated for degassing and proper mixing purpose. The column temperature has to be maintained at 50°C with gradient composition pressure.

 

Chromatographic condition:

By acquity UHPLC (Zodiac, C18(150x4.6x5µm) with isocratic elution with flow rate 1ml/min. The column temperature was maintained at 40°C. The prepared standard solutions automatically injected through column.

 

 

Fig. 3: Optimized method of Vaborbactam and Meropenem

Observation:

In this trail, observed that, good resolution and good peak shape. So, this trail is considered as optimized method.

 

RESULTS AND DISCUSSION:

System suitability:

System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as a whole[1,2]. System suitability is the checking of a system to ensure system performance before or drying the analysis unknowns. To verify that the analytical system is working properly and can give accurate and precise results were evaluated by 100µg/ml of MPN and 100µg/ml of VBB were injected six times and the chromatograms were recorded for the same. Various parameters such as theoretical plates tailing factor, retention time and resolution factor were reported as per the ICH and USP guidelines[5,6]. The relative standard deviation and tailing factor not more than 2.0 within the acceptance criteria.

 

 

Fig. 4:System suitability chromatogram

 

Precision:

Precision can be defined as “the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample”. A more comprehensive definition proposed by the International Conference on Harmonization (ICH) divides precision into three types[2,3,4]:

·       Repeatability

·       Intermediate precision and

·       Reproducibility

 

Repeatability is the precision of a method under the same operating conditions over a short period of time. Intermediate Precision is the agreement of complete measurements (including standards) when the same method is applied many times within the same laboratory. Reproducibility examines the Precision between laboratories and is often determined in collaborative studies or method transfer experiments[7,8,9].

 

Procedure:

Method precision was determined by injecting six different solutions of sample solutions MPN (100μg/mL) and VBB (100μg/mL) for six times are prepared separately. The relative standard deviation and tailing are 2.0 within the limits. The results of precision were shown in table 1.

 

Table 1: Precision readings of Vaborbactam and Meropenem

VABORBACTAM

MEROPENEM

Area

%Assay

Area

%Assay

1

535328

100.0

2877968

100.4

2

561133

100.5

271334

99.8

3

565523

99.9

262343

100.4

4

544261

99.0

273760

98.6

5

571227

99.0

295910

99.8

6

622800

98.5

295173

99.5

Average

566712

99.5

279414.7

99.7

SD

27951.72

0.8

12323.61

0.6

%RSD

0.049322

0.8

0.441036

0.6

 

Linearity:

The Linearity of a method is a measure of how well a Calibration plot of response against concentration approximates a straight line. Linearity can be assessed by performing single measurements at several analyte concentrations. The data are then processed using a linear least-squares regression. In the resulting plot slope, intercept and Correlation coefficient provide the desired information on Linearity[10].

 

Procedure:

An accurately measured aliquot portion of working standard solution of Vaborbactam and Meropenem were transferred into a five separate 10ml volumetric flasks. The volume was made up to the mark using methanol to obtain concentrations (20-50µg/ml). Absorbance of these solutions was measured at 270nm. Calibration curve was plotted, absorbance vs concentration as shown in fig 5. The results of linearity data were given in table 2.

 

Table 2: Linearity data of Vaborbactam and Meropenem

S. No

Parameter

Vaborbactam

Meropenem

1

Correlation coefficient

0.999

0.999

2

Slope

9069

20547

3

Intercept

101920

26213

 

Specificity:

Specificity is the ability to assess accurately the analyte in the presence of components which may be expected to be present in the sample matrix. Typically these might include impurities, degradants, matrix etc. It is a measure of the degree of interference from such other things such as other active ingredients, excipients, impurities, and degradation products, ensuring that a peak response is due to a single component only[11,12].

 

 

Fig. 5: Linearity graph of Vaborbactam and Meropenem

 

Specificity is divided into two separate categories like identification and assay and impurity tests. For identification purpose, specificity is demonstrated by the ability to discriminate between compounds of closely related structures or comparison to a known reference standard1. For assay/impurity tests, specificity is demonstrated by the resolution of the two closely eluting compounds. These compounds are usually the major component or the active ingredient and an impurity. Blank solution was injected and the chromatogram was recorded for the same as Placebo solution was prepared and it was injected and the chromatogram was recorded. Interference with the peak and mobile phase is does not interfere with the two drugs. The results are specified within the limits[13,14].

 

Accuracy:

The accuracy of a measurement is defined as the closeness of the measured value to the true value. In a method with high accuracy, a sample (whose “true value” is known) is analyzed and the measured value is identical to the true value. Typically, Accuracy is represented and determined by recovery studies, but there are three ways to determine accuracy.

 

Procedure:

Accuracy of the method was determined by recovery studies. To the formulation (preanalysed sample), the reference standards of the drugs were added at the level of 50%, 100%, 150%. The recovery studies were carried out three times and the percentage recovery and percentage mean recovery were calculated for each drug. The results of accuracy were given in table 3.

 

Limit of detection and limit of quantification:

These limits are normally applied to related substances in the drug substance or drug product. Specifications on these limits are submitted with the regulatory impurities method relating to release and stability of both drug substance and drug product[13,14].

 

Limit of detection is the lowest concentration of analyte in a sample that can be detected, but not necessarily Quantified, under the stated experimental conditions. With UV detectors, it is difficult to assure the detection precision of low-level compounds due to potential gradual loss of sensitivity of detector lamps with age or noise level variation by detector manufacturer.

 

LOD = 3.3 σ / S

 

Limit of quantitation is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental[15,16].

 

LOQ = 10 σ / S

 

Observation:

The lower amount of sample was quantified and detected. The vaborbactam was detected at 0.025µg/ml and meropenem was detected at 0.045µg/ml.

 

Robustness:

The concept of robustness of an analytical procedure has been defined by the ICH as “a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters.” A good practice is to vary, important parameters in the method, systematically and measure their effect on separation. The Robustness of the method was determined. The results obtained by deliberate variation in method parameters are summarized[17]. The results of robustness were summarized in table 4.


 

Table 3: Accuracy data of Vaborbactam and Meropenem

Vaborbactam

Meropenem

%

Recovery

Amount present

(µg/mL)

Amount

Found (µg/mL)*

Percent

Recovery *

%Mean Recovery

%

Recovery

Amount present (µg/mL)

Amount found

(µg/mL) *

Percent Recovery *

% Mean Recovery

50%

50

49.64

99.3

 

99.2

50%

50

49.86

99.7

 

100.4

100%

100

99.17

99.2

100%

100

99.98

100.0

150%

150

148.63

99.1

150%

150

152.08

101.4

 

 

Table 4: Robustness data for Vaborbactam and Meropenem

Chromatographic changes

Theoretical Plates

Tailing factor

Resolution

VBB

MPN

VBB

MPN

Between VBB & MPN

Flow rate

(mL/min)

0.8

11114

16033

1.27

1.27

3.08

1.2

11284

16140

1.25

1.43

2.85

1.5

11096

15870

1.25

1.43

2.64

Wavelength(nm)

268

10985

15759

1.25

1.45

2.96

272

11372

16183

1.26

1.43

2.93

273

10927

15727

1.24

1.44

3.06

 


Ruggedness:

Degree of reproducibility of test results obtained by the analysis of the same samples under a variety of condition such as different laboratories, different analysts, different instruments etc, normally expressed as the lack of influence on test results of operational and environmental variable of the analytical method[18,19]. Ruggedness is a measurement of reproducibility of test results under the variation in condition normally expected from laboratory to laboratory and from analyst to analyst[20]. The ruggedness of the method was studied by the determining the analyst to analyst variation by performing the Assay by two different analysts.

 

Forced degradation studies:

Forced degradation or stress studies are undertaken to deliberately degrade the sample. These studies are used to evaluate an analytical method ability to measure an active ingredient and its degradation products, without interference by generating potential degradation product. During validation of the method, drug substance are exposed to acid, base, heat, light and oxidizing agent to produce approximately 10% to 30% degradation of active substance[21,22]. The studies can also provide information about the degradation pathways and degradation products that could from during storage. These studies may also help in the formulation development, manufacturing and packaging to improve a drug product. Reasons for carrying out forced degradation studies include development and validation of stability-indicating methodology, determination of degradation pathways of drug substances and products, discernment of degradation products in formulations that are related to drug substances versus those that are related to non-drug substances[23,24]. The results of forced degradation studies were summarized in table 5.

 

Peroxide degradation:

Sample solution of Vaborbactam and Meropenem (10µg/ml) and 1 ml of 20% hydrogen peroxide (H2O2) was mixed. For UHPLC study, 10µl were injected into the system and the chromatogram was recorded to assess the stability of sample.

 

Photolytic degradation:

The photochemical stability of the drug was studied by exposing the 100µg/ml solution to UV light by keeping the beaker in UV chamber for 7 days. For UHPLC study, the resultant solution 10µl was injected into the system and the chromatogram were recorded to assess the stability of sample.

 

Acid degradation:

Take 1 tablet, powdered and place in a 50ml volumetric flask and dissolve in mobile phase up to 75% then sonicate it for 10 minutes then add 1 ml of 0.1N HCl then kept in oven at 600c for 1 hour then cool and add 1 ml of 0.1N NaOH it then make up the volume up to 50ml with mobile phase, then place the sample in the vial and measure the chromatogram.

 

Alkaline degradation:

Take 1 tablet, powdered and place in a 50ml volumetric flask and dissolve in mobile phase up to 75% then sonicate it for 10 minutes then add 1 ml of 0.1N NaOH then kept in oven at 600°C for 1 hour then cool it and add 1ml of 0.1N HCl then make up the volume up to 50ml with mobile phase, then place the sample in the vial and measure the chromatogram.

 

Thermal degradation:

Sample solution of Vaborbactam and Meropenem (10µg/ml) was placed in oven at 105°C for 6hr to study dry heat degradation. for UHPLC study, the resultant solution was injected into the system and the chromatograms were recorded to assess the stability of the sample.


 

Table 5: Degradation data for Vaborbactam and Meropenem

Vaborbactam

Meropenem

Method

std area

Degradation area

% Obtained

% Degraded

std area

Degradation area

% Obtained

% Degraded

Peroxide

559559

1041210

101.488

0.412

282306

2046738

101.493

0.407

Photolytic

178620

1041237

101.491

0.409

863520

2046725

101.492

0.408

Acidic

561133

1041221

101.489

0.411

271334

2046742

101.493

0.407

Alkaline

178620

1041238

101.491

0.409

863520

2046731

101.492

0.408

Thermal

267582

1041241

101.491

0.409

267582

2046729

101.492

0.408

 


Acceptance criteria:

The % Degraded for Vaborbactam and Meropenam from these stability methods should be not more than 1.0 %.

 

Observation:

Degradation studies were carried out with acid, base, peroxide, thermal. It was observed that the response of peak area and retention time of Vaborbactam and Meropenem were nearly same as obtained in control samples of Vaborbactam and Meropenem. Degradation was found in acid, base, peroxide, thermal and UV conditions because extra peaks were observed.

 

CONCLUSION:

A new precise, accurate, rapid method has been developed for the estimation of Meropenam and Vaborbactam pharmaceutical drug by UHPLC. From the above experimental results and parameters it was concluded that, this newly developed method for the estimation Meropenem and Vaborbactam was found to be simple, precise, accurate and high resolution and shorter retention time makes this method more acceptable. The present recovery was found to be 98.0-101.5% was linear and precise over the same range. Both system and method precision was found to be accurate and within range. Detection and quantification limit was found to be 0.025 Vaborbactam and 0.045 Meropenem. The analytical method was found linearity over the range of 20-60ppm of the target concentration for both the drugs. The analytical method passed both robustness and ruggedness tests. On both cases, relative standard deviation was well satisfactory.

 

CONFICT OF INTEREST:

No conflict of interest.

 

REFERENCES:

1.      Geetha Susmitha A, Rajitha G. Development and validation of stability indicating UPLC method for simultaneous estimation of sofosbuvir and velpatasvir in tablet dosage forms. International Journal of Pharmaceutical Science and Research. 2018; 9(11): 4764-4769.

2.      Vipul Negi et.al. Method development and validation of meropenem in pharmaceutical dosage form by RP-HPLC. Journal of Chemical Technology. 2017; 24(5): 441-446.

3.      https://www.drugbank.ca/drugs/DB00983.

4.      https://www.drugbank.ca/drugs/DB00986

5.      ICH Text on validation of analytical procedures, ICH –Q2A, International Conference on Harmonization. IFPMA, Geneva. 1995.

6.      ICH Guidelines, Q2 (R1)- Validation of Analytical Procedures: Text and Methodology, 2005.

7.      Mudassara HE Madhkhan Pathan, Ajay Kshirsagar. Development of validated stability indicating method by RP-HPLC for simultaneous estimation of meropenem and vaborbactam in bulk and pharmaceutical formulation. International Journal of Pharmacy and Pharmaceutical Sciences. 2019; 11(3): 102-108.

8.      Ramonakhanum et.al. Development and validation of a RP-HPLC method for the detection of meropenem as a pure compound in a pharmaceutical dosage form and post thermal induced degradation. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(4): 149-152.

9.      Sudha Rani Bollimuntha et.al. Validation of simple isocratic RP-UPLC method for meropenem and vaborbactam determination and its application in the study of stress degradation. Journal of Scientific Research in Pharmacy. 2018; 7(2): 150-156.

10.   Sree Lakshmi M et.al. HPLC method for simultaneous estimation of meropenem and vaborbactam in bulk samples. International Journal of Medical Science and Innovative Research. 2017; 2(5): 361-367.

11.   Przemyslaw Zalewski. et.al. Development and validation of stability indicating HPLC method for simultaneous determination of meropenem and potassium clavulanate. Acta Poloniae Pharmaceutica-Drug Research. 2014; 71(2): 255-260.

12.   Bikash Ranjanjena et.al. UPLC analytical method development and validation for the simultaneous estimation of paracetamol and caffeine capsules dosage form. Pharmaceutical Regulatory Affairs. 2017; 6(1): 1-9.

13.   Upasana K Patel MS, Jaymin Ganshyambhai Patel Sharda MR. Analytical method development and validation of stability indicating RP-HPLC method for estimation of meropenem and vaborbactam in synthetic mixture. International Journal of Advanced Research. 2019; 7(3): 865-871.

14.   Urukundu V et.al. Simultaneous estimation of new analytical method development and validation of meropenem and vaborbactam by HPLC in bulk and marketed formulation. Innovat International Journal of Medical and Pharmaceutical Sciences. 2018; 3(1): 29-32.

15.   Ramya Kuber B, Sravanthi PSK. Analytical method development and validation of ceftazidime pentahydrate and tazobactam sodium by RP-HPLC method in bulk and dosage form. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2017; 8(3): 2447-2457.

16.   Ram Garg et.al UPLC method development and validation for cefditoren pivoxil in active pharmaceutical ingredient. Journal of Applied Pharmaceutical Science. 2011; 01(07): 149-153.

17.   International conference on the harmonization. ICH Harmonized Tripartite Guideline. Stability Testing of New Drug Substances and Products Q1A (R2), 2003.

18.   International conference on the harmonization. ICH Harmonized Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2 (R1), 2005.

19.   Swartz M, Ira Krull S. Analytical method development. Analytical method. development and validation. Marcel Dekker, Inc: New York. 2009; 1st ed: pp.17-80.

20.   Satinder A, Dong MW. Method Development and Validation. Pharmaceutical Analysis with HPLC, 15th ed; New York. 2005; 16-70.

21.   Ngwa G. Forced degradation studies. Forced degradation as an integral part of HPLC stability indicating method development. Drug Delivery Technology. 2010; 10(5): 55-62.

22.   Vibha G et.al. Development and validation of HPLC method. International Research Journal of Pharmaceutical and Applied Science. 2012; 2(4): 17-25.

23.   Navyaja K, Ramya Kuber B. Analysis of pesticide residue in coconut water and tomatoes around Tirupathi region by using reverse phase ultra-high performance liquid chromatography. Chemical Science Review and Letters. 2017; 6(24): 2616-2621.

24.   Neethu Mathew, Jane T Jacob. Stability profiling of amodiaquine under stress degradation conditions. Research Journal of Pharmacy and Technology. 2019; 12(8): 3807-3810.

 

 

 

 

 

Received on 06.02.2020           Modified on 01.04.2020

Accepted on 26.05.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):363-368.

DOI: 10.5958/0974-360X.2021.00066.4