Mayani Karina*, Basareddy Chandrasekhar, Dhirendra Kumar Tarai, Santosh Kirtane

1.1   Introduction to disease (Urinary Tract Infection)

5. Urinary tract infections (UTIs) are among the most common bacterial infections in humans, representing a major public health concern worldwide.
5. They account for approximately 40% of all hospital-acquired infections and are a leading cause of outpatient visits and antibiotic prescriptions.
5. Globally, UTIs affect an estimated 150 million people annually, resulting in significant healthcare costs and morbidity.

1.2 Introduction of Drug

5. Sulopenem Etzadroxil

Fig : 1.1 Structure of Sulopenem Etzadroxil

• It is a prodrug of sulopenem (a penem β-lactam antibiotic).
• Molecular formula : C16H17N5O6S2
• Molecular weight : ~443.5 g/mol
• Mechanism:

• Binds to penicillin-binding proteins (PBPs)
• Inhibits bacterial cell wall synthesis
• Leads to bacterial death (bactericidal action).

5. Probenecid

Fig : 1.2 Structure of Probenecid

• A renal tubular transport inhibitor
• Molecular formula : C13H19NO4S
• Molecular weight : 285.36 g/mol
• Mechanism:

• Blocks organic anion transporters (OAT1/OAT3) in the kidney
• Reduces excretion of sulopenem
• Increases drug concentration in blood

2.Material and methods

Materials

• Drug Substance: Sulopenem Etzadroxil and Probenecid
• Brand name: Orlynvah
• Product name: Sulopenem etzadroxil and Probenecid Tablet Manufacturer: Iterum Therapeutics

Instrumentation:

Table 2.1 Apparatus and instrument for High Performance Liquid Chromatography

Table: 2.2 Instrument specification for melting point apparatus

Table: 2.3 Instrument specification for UV-visible spectrophotometry

Table: 2.4 Reagents and materials for HPLC

2.1Preparation of solutions Preparation of Mobile Phase

5. The mobile phase, consisting of Methanol:ammonium formate buffer (pH 3.2 with formic acid) (40:60), was prepared, filtered, and degassed. This composition was chosen as the optimal mobile phase for both the drugs, as it exhibited excellent resolution and accurate peak characteristics.
5. Ammonium formate buffer (10 mM, pH 3.2) was prepared by dissolving 0.77 g of ammonium formate in purified water, adjusting the pH to 3.0 with formic acid, and making up the volume to 1000 mL with water. The buffer was filtered through a 0.45 µm membrane filter prior to use.

Preparation of Standard Stock Solution

5. Accurately weighed 10 mg of sulopenem etzadroxil and probenecid were separately transferred into volumetric flasks and dissolved in methanol 10 ml to obtain stock solutions. Pipette out 2 ml of this solution and dilute upto 10 ml to obtain 200 ppm for both the drugs.

Preparation of sample solution

5. A synthetic mixture containing sulopenem etzadroxil and probenecid in the 1:1 ratio
5. was prepared by accurately weighing the drugs (10 mg each), transferring them into a volumetric flask, and dissolving them in the methanol.
5. The solution was sonicated to ensure complete dissolution and then filtered before analysis. Appropriate dilution with mobile phase was made to obtain final concentrations within the working range of the method.

Chromatographic condition

The chromatographic separation of sulopenem etzadroxil and probenecid was achieved on Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm) by using mobile phase composed of Methanol: ammonium formate buffer (pH 3.2 with formic acid) (40:60), at flow rate 1 ml/min with run time of 10 minutes. Detection of drug was carried out at 272 nm by using diluent as mobile phase.

2.2   Identification of drugs

Identification by Melting Point Determination

Melting point of drugs has been determined. The melting points of the compounds were taken by open capillary method

Table 2.1 Melting Point of Drugs

IR characterization and interpretation is shown in Figure 2.1 and 2.2 for Sulopenem Etzadroxil and Probenecid respectivel

Figure 2.1 IR Characterization of Sulopenem Etzadroxil

Table 2.2 IR Characterization of Sulopenem Etzadroxil

Figure 2.2 IR characterization of Probenecid

Table 2.3 IR Characterization of Probenecid

Solution Stability

5. The solubility of both the drugs practically was determined separately by taking 100 mg of the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes.
5. Solubility data for each study was observed and recorded in Table 6.8.

Table 2.4 Solubility Table

Table 2.5 Solubility Table of Sulopenem Etzadroxil

Table 2.6 Solubility Table of Probenecid

2.4 Development and Optimization of RP-HPLC Method

Selection of Wavelength

5. A µg/mL solution of both the drugs in methanol was scanned in the UV range of 200–400 nm using a UV-Visible spectrophotometer. The wavelength showing maximum absorbance (λmax) was selected as the detection wavelength.
5. The wavelength selected was 272nm because isobastic point was obtained for both the drugs at this wavelength.

Selection of Chromatographic Conditions

5. Proper selection of the HPLC method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, pKa and solubility. RP-HPLC was selected for the initial separation based on literature survey and its simplicity and suitability.
5. To optimize the chromatographic conditions the effect of chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. Finally, the chromatographic condition was chosen that give the best resolution, symmetry and capacity factor for estimation of both drugs.

Selection of column

5. For RP-HPLC Method, various columns are available but based on literature survey Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)) was selected.

Preparation of Mobile Phase

5. The mobile phase, consisting of Methanol:ammonium formate buffer (pH 3.2 with formic acid) (40:60), was prepared, filtered, and degassed.
5. This composition was chosen as the optimal mobile phase for both the drugs, as it exhibited excellent resolution and accurate peak characteristics.
5. Ammonium formate buffer (10 mM, pH 3.2) was prepared by dissolving 0.77 g of ammonium formate in purified water, adjusting the pH to 3.0 with formic acid and making up the volume to 1000 mL with water.
5. The buffer was filtered through a 0.45 µm membrane filter prior to use.

3.Method validation

3.1 Linearity and range

5. Linearity was determined by regression analysis, which involves recording average areas of triplicate injections of 25-150 µg/mL for Sulopenem Etzadroxil and 25-150. µg/mL for Probenecid. Plot a linearity graph with concentration against peak area and for Sulopenem Etzadroxil and Probenecid correlation coefficient values were acceptable fits for the data of regression line.

3.2 Repeatability (Precision

5. Six separate assays of standard preparation were conducted to assess the assay method's precision in terms of repeatability and the assay's percentage RSD was computed. Under the identical experimental settings, a different analyst completed the procedure with intermediate precision.
5. For intraday precision, six replicates of each concentration of both standard and sample solutions were consecutively administered on the same day.
5. To ensure interday precision, the same standard and sample solutions were injected on three different days.
5. The precision results are reported as the percentage relative standard deviation (% RSD). This statistical measure provides insights into the variability of the results, helping to gauge the precision of the analytical method across different concentrations and time intervals.
6. 3. System Suitability Parameters
7. This test serves to verify the operational capability of the analytical system and its ability to generate precise and accurate results. In a 10 ml volumetric flask, a 1.0 ml portion of the 100 µg/ml Sulopenem Etzadroxil standard solution was added, andthe volume was adjusted to 10 ml with mobile phase to achieve a concentration of 10 µg/ml.
5. The solution was then sonicated for 15 min. Subsequently, 20 µl of this standard solution was injected into the HPLC system, and the chromatogram was analyzed for the drug retention time, peak area, and peak resolution. These observations contribute to the assessment of the system's performance and its ability to generate reliable analytical data.
5. The same procedure was followed for probenecid.
6. 4. Accuracy
7. Samples were arranged in triplicates by sample solution at known concentrations of Sulopenem Etzadroxil and Probenecid at 50%, 100%, and 150% spike levels. The peak area values observed in each analysis were compared with the corresponding standard and % recovery of Sulopenem Etzadroxil and Probenecid was calculated.
5. %Recovery=Amount found−Amount in sample/ Amount added×100% Acceptance criterion: Mean recovery 98.0–102.0%
5. %RSD ≤ 2.0% at each level.

3.5 Limit of detection and Limit of Quantification

5. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ = 10 σ/s

LOD = 3.3 σ/s Where,

σ = the standard deviation of the response S = the slope of the calibration curve

3.6 Robustness

5. It is evaluated to know the change in system suitability parameters in response to variations in flow rate, mobile phase, and temperature.
5. % RSD was calculated for evaluation of the parameters and found to be less than 2% which satisfies the acceptance criteria.

3.7 Assay

5. To determine the concentration of the drug, inject 10 µL of both the standard and sample solutions into the RP HPLC system and measure the peak areas for Probenecid and Sulopenem Etzadroxil.
5. The sample concentration is determined by comparing it to the standard peak area and employing an appropriate calculation formula.

%Assay=AT/AS×WS/WT×DT/DS×P×100

where:

AT = peak area of sample, AS = peak area of standard WS = weight of standard, WT = weight of sample

DT and DS = dilution factors of sample and standard

P = purity of reference standard (as decimal)

3.8 Selection of Wavelength

To determine wavelength for measurement, standard spectra of Sulopenem Etzadroxil and Probenecid was scanned between 200-400 nm against diluents. The wavelength selected was 272nm because isobastic point was obtained for both the drugs at this wavelength.

Fig. 3.1 Overlain spectra of Sulopenem etzadroxil and probenecid

Selection of mobile phase

Trial 1

• Column: Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)
• Mobile Phase: MeOH:Water (60:40) + 0.1% FA
• Detection: 272 nm
• Flow rate: 1 ml/min
• Run Time: 5 minutes
• Observations: peak of Sulopenem etzadroxil was broad and there was tailing in peak of probenecidFig 3.2 Trial 1 – Chromatogram

Trial 2

• Column: Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)
• Mobile Phase: MeOH:Water (70:30) + 0.1% FA
• Detection: 272 nm
• Flow rate: 1 ml/min Run Time: 5 minutes
• Observations: peak of Sulopenem etzadroxil was with fronting and there was slight tailing in peak of probenecid

Fig 3.3 Trial 2 – Chromatogram

Trial 3

• Column: Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)
• Mobile Phase: MeOH: Ammonium formate (65:35)
• Detection: 272 nm
• Flow rate: 1 ml/min
• Run Time: 5 minutes
• Observations: peak of Sulopenem etzadroxil was sharp and there was slight tailing in
• peak of probenecid

Fig 3.4 Trial 3 – Chromatogram

Trial 4

• Column: Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)
• Mobile Phase: Methanol : ammonium formate buffer (40:60), pH (2.8)
• Detection: 272 nm
• Flow rate: 1 ml/min Run Time: 5 minutes
• Observations: peak of Sulopenem etzadroxil was good and there was broadening in peak of probenecid

Fig 3.5 Trial 4 – Chromatogram

3.9 Chromatographic conditions for optimized mobile phase trial

• Stationary phase: Hypersil BDS C18 130 A? (250 × 4.6 mm, 5 µm)
• Mobile Phase: Methanol : ammonium formate buffer (40:60), pH 3.2
• Detection: 272 nm
• Flow rate: 1ml/min
• Run Time: 5 minutes
• Detector: UV detector
• Injection volume: 20 μl
• Mode: Isocretic

  

  Fig 3.6: Optimized mobile phase trial for optimized chromatogram Retention time for Sulopenem Etzadroxil: 2.403 min

Retention time for Probenecid: 3.336 min

Fig 3.7 : Chromatogram of blank

4. Method Validation

4.1.1 Linearity and range

Preparation of Solution for linearity studies: Linearity was determined by regression analysis, which involves recording average areas of triplicate injections of 25-150 µg/mL for Sulopenem Etzadroxil and 25-150 µg/mL for Probenecid. Plot a linearity graph with concentration against peak area and for Sulopenem Etzadroxil and Probenecid correlation coefficient values were acceptable fits for the data of regression line.

Fig 4.1 overlain spectra of Sulopenem Etzadroxil and Probenecid

Table 4.1 Linearity data for Sulopenem Etzadroxil

Fig 4.2 calibration curve for Sulopenem etzadroxil

Table 4.2 Linearity data for Probenecid

Fig 4.3 calibration curve for Probenecid

4.1.2 Precision

4.1.2.1 Repeatability

The data for repeatability for bothe the drugs was performed for 75 ppm is shown in table 7.3 and 7.4. The % R.S.D For Repeatability data was found to be 0.587 % and 0.50 % for Sulopenem etzadroxil and Probenecid respectively.

Table 4.3 Repeatability data for Sulopenem etzadroxil

Table 4.4 Repeatability data for Probenecid

4.1.2.2 Inter-day precision

The data for interday precision for shown Sulopenem etzadroxile in table 7.5. The%

R.S.D for intraday precision was found to be 0.64-0.77 % for Sulopenem etzadroxil

Table 4.5 Interday precision for Sulopenem etzadroxil

The data for interday precision for shown Probenecid in table 7.6.

The% R.S.D for intraday precision was found to be 0.64-0.82 % for Probenecid.

Table 4.6 Interday precision for Probenecid

4.1.2.3         Intra-day precision

The data for intra-day precision for Sulopenem etzadroxil is shown in table 7.7. The % R.S.D for intraday precision was found to be 0.49-0.61 %.

Table 4.7 Intraday precision for Sulopenem etzadroxil

The data for intraday precision for shown Probenecid in table 7.8. The% R.S.D for intraday precision was found to be 0.50-0.62 % for Probenecid.

Table 4.8 Intraday precision for Probenecid

4.1.3 Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50, 100 and 150 % of the target concentration.

Table 4.9 Accuracy results for Sulopenem etzadroxil

Table 4.10 Accuracy results for Probenecid

4.1.4 LOD and LOQ

Table 4.11 LOD for Sulopenem etzadroxil and Probenecid

Table 4.12 LOQ for Sulopenem etzadroxil and Probenecid

4.1.5 Selectivity

There is no interference in the mixture.

4.1.6 Robustness

By purposefully making tiny changes to the chromatographic settings, the method's resilience was assessed.

Table 4.13 Ronustness results for Sulopenem etzadroxil

Table 4.14 Ronustness results for Probenecid

4.1.7 Assay

To determine the concentration of the drug, inject 10 µL of both the standard and sample solutions into the RP HPLC system and measure the peak areas for Probenecid and Sulopenem Etzadroxil. The sample concentration is determined by comparing it to the standard peak area and employing an appropriate calculation formula.

Table 4.15 Assay results for Sulopenem Etzadroxil

Table 4.16 Assay results for Probenecid

4.1.8            Summary of method validation

Table 4.17 Summary of validation parameter of RP-HPLC method

Table 4.18 Summary of validation parameter

AGREE Software Evaluation

5. The AGREE assessment was performed using the desktop version of the AGREE tool.
5. Each of the 12 principles was analyzed individually by inputting method-specific data, including sampling procedure, sample preparation steps, reagents used, solvents, instrumental requirements, waste generation and operator safety considerations.
5. The resulting AGREE pictogram (Figure 7.8) displays a circular wheel divided into 12 segments, each corresponding to one GAC principle.
5. The color coding (green, yellow, orange, red) represents the degree of compliance with each principle.

Figure: 4.4 AGREE Pictogram

5. The developed analytical method achieved an overall AGREE score of: 0.75.
5. This score indicates that the method is green, meaning that it incorporates several environmentally friendly practices.