Method for estimation of compounds in a mixture

Abstract

METHOD FOR ESTIMATION OF COMPOUNDS IN A MIXTURE A method for simultaneous estimation of Sulbactam and Durlobactam in a mixture using ultra-performance liquid chromatography (UPLC) is described. The method involves preparing a sample of Sulbactam and Durlobactam in a solvent and buffer solution, followed by analysis using UPLC. The chromatographic separation is achieved using a HSS column. The analysis is conducted at a wavelength between 240 nm and 260 nm to determine the recovery of Sulbactam and Durlobactam in the mixture.

Description

[0001] FIELD OF THE INVENTION

[0002] The present invention relates to a method for simultaneous estimation of pharmaceutical compounds in combination using ultra-performance liquid chromatography (UPLC).

[0003] BACKGROUND OF THE INVENTION

[0004] In the pharmaceutical industry, having efficient and reliable testing methods is very important, especially for checking the quality of complex medicines. Chromatography, especially liquid chromatography, is widely used to separate and measure different substances in a mixture. A modem and improved version of this is ultra-performance liquid chromatography (UPLC), which offers faster, more accurate, and more detailed results than traditional HPLC. This technique is especially useful for testing multiple active ingredients in one go, which is important for combination medicines. For example, Sulbactam and Durlobactam are two ingredients being studied for the treatment of serious bacterial infections and measuring them together in tablets can be difficult due to their different chemical properties and the presence of other ingredients. Having a reliable method to separate and measure both accurately is necessary to make sure the medicines are safe, effective, and meet quality standards.

[0005] CN111735880A discloses a method for detecting antibacterial drugs in serum using ultra- high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS), targeting drugs like sulbactam, imipenem, linezolid, and others. The method uses isotope internal standards, establishes calibration curves based on concentration and peak area ratios, and enables rapid, sensitive, and specific detection within 5 minutes for clinical monitoring of drug concentrations.

[0006] CN115144505B discloses a method for simultaneously measuring the concentration of valsartan, sha Kuba koji, and metabolite LBQ657 in human urine using UPLC-MS / MS. The method uses acetonitrile for pretreatment, requires a small sample volume, reduces instrument contamination, adopts gradient elution to prevent residue, and is suitable for patients with end-stage renal failure due to its minimal sample requirement.

[0007] CN-113820430-B discloses a reagent and its preparation method for detecting antibacterial drugs using liquid chromatography tandem mass spectrometry (LC-MS / MS). The calibrator and quality control product enable simultaneous detection of at least 16 drugs, improving detection efficiency with high responsiveness and good repeatability. The above-mentioned applications disclose a method and reagents for the detection of pharmaceutical substances through liquid chromatography and mass spectrometry techniques, are primarily directed towards bioanalytical testing in biological matrices such as serum and urine. These disclosures focus on enhancing detection sensitivity, improving calibration accuracy, and minimizing sample volumes for clinical or diagnostic purposes. However, they do not address the analytical requirements necessary for pharmaceutical formulations, such as simultaneous estimation in dosage forms, impurity profiling, stabilityindicating capabilities, or compliance with regulatory guidelines for pharmaceutical quality control.

[0008] Therefore, there is a clear need for a more robust and precise analytical method specifically designed for pharmaceutical formulations. Such a method should provide accurate quantification, reliable impurity separation, and stability assessment under various stress conditions, all while meeting stringent regulatory expectations. This would ensure reliable quality control during formulation development and manufacturing processes, addressing limitations present in the cited disclosures.

[0009] OBJECT OF THE INVENTION

[0010] An object of the present invention is to provide a method for simultaneous estimation of pharmaceutical compounds in combination using ultra-performance liquid chromatography (UPLC).

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides a method for simultaneous estimation of pharmaceutical compounds in fix dose combination using UPLC. The method comprising: a) preparing a sample of the mixture of Sulbactam and Durlobactam in a solution of a solvent and a buffer; b) analysing said sample using ultra-performance liquid chromatography (UPLC) by: i. setting parameters including flow rate, retention time, temperature, LOD (limit of detection), LOQ (limit of quantification), RSD (relative standard deviation); ii. using a chromatographic column selected from a group consisting of HSS Cl 8, Cl 8, C8, phenyl, and cyano columns, preferably C18 or C8 columns, more preferably a HSS Cl 8 column, with specific dimension and particle size of 1.7 pm to 2.5 pm for chromatographic separation; and iii. carrying out analysis of the sample at a wavelength in the range of 240 nm to 260 nm, preferably 245 to 250 nm, more preferably at 248 nm, for determining recovery of Sulbactam and Durlobactum in the mixture.

[0013] Flow rate is maintained in the range of 0.1 mL / min to 0.5 mL / min, preferably 0.2mL / min to 0.4 mL / min, more preferably 0.3 mL / min. Retention times for Sulbactam and Durlobactum are in the range of 1.2 min to 2.0 min, preferably 1.3 min to 1.8 min, more preferably 1.77 min and 1.33 min. The %RSD values are in the range of 0.1 to 2.0, preferably 0.2 to 1.0, more preferably below 0.5 for both, indicating high precision. Recovery studies yield results of 99.49% for Sulbactam and 99.90% for Durlobactum, confirming the accuracy of the method. Limit of detection (LOD) and limit of quantification (LOQ) for Sulbactam are in the range of 0.1 to 0.4 pg / mL and 0.5 to 0.8pg / mL, respectively, preferably 0.3 pg / mL and 0.6 pg / mL, and more preferably 0.19 pg / mL and 0.56 pg / mL. Limit of detection (LOD) and limit of quantification (LOQ) for Durlobactam are in the range of 0.05 to 0.1 pg / mL and 0.2 to 0.5 pg / mL, respectively, preferably 0.09 pg / mL and 0.3 pg / mL, and more preferably 0.08 pg / mL and 0.26 pg / mL. The method effectively separates degradation products from active pharmaceutical ingredients under stress conditions including acidic, basic, oxidative, thermal, neutral, and photolytic environments; utilizing the freshly prepared mobile phase prior to each use to ensure consistency and reproducibility. The method is validated in accordance with ICH guidelines, being simple, robust, and economical, thereby supporting regulatory submissions with reliable data and being suitable for large-scale production and routine quality control applications.

[0014] BRIEF DESCRIPTION OF FIGURES

[0015] Figure 1 depicts the optimized Chromatogram of Sulbactam and Durlobactam DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0016] The present invention provides a method for simultaneous estimation of pharmaceutical compounds in fix dose combination using UPLC. The method comprising: a) preparing a sample of the mixture of Sulbactam and Durlobactam in a solution of a solvent and a buffer; b) analysing said sample using ultra-performance liquid chromatography (UPLC) by: i. setting parameters including flow rate, retention time, temperature, LOD (limit of detection), LOQ (limit of quantification), RSD (relative standard deviation); ii. using a chromatographic column selected from a group consisting of HSS Cl 8, Cl 8, C8, phenyl, and cyano columns, preferably C18 or C8 columns, more preferably a HSS Cl 8 column, with specific dimension and particle size of 1.7 pm to 2.5 pm for chromatographic separation; and iii. carrying out analysis of the sample at a wavelength in the range of 240 nm to 260 nm, preferably 245 to 250 nm, more preferably at 248 nm, for determining recovery of Sulbactam and Durlobactum in the mixture.

[0017] Flow rate is maintained in the range of 0.1 mL / min to 0.5 mL / min, preferably 0.2mL / min to 0.4 mL / min, more preferably 0.3 mL / min. Retention times for Sulbactam and Durlobactum are in the range of 1.2 min to 2.0 min, preferably 1.3 min to 1.8 min, more preferably 1.77 min and 1.33 min. The %RSD values are in the range of 0.1 to 2.0, preferably 0.2 to 1.0, more preferably below 0.5 for both, indicating high precision. Recovery studies yield results of 99.49% for Sulbactam and 99.90% for Durlobactum, confirming the accuracy of the method. Limit of detection (LOD) and limit of quantification (LOQ) for Sulbactam are in the range of 0.1 to 0.4 pg / mL and 0.5 to 0.8pg / mL, respectively, preferably 0.3 pg / mL and 0.6 pg / mL, and more preferably 0.19 pg / mL and 0.56 pg / mL. Limit of detection (LOD) and limit of quantification (LOQ) for Durlobactam are in the range of 0.05 to 0.1 pg / mL and 0.2 to 0.5 pg / mL, respectively, preferably 0.09 pg / mL and 0.3 pg / mL, and more preferably 0.08 pg / mL and 0.26 pg / mL. The method effectively separates degradation products from active pharmaceutical ingredients under stress conditions including acidic, basic, oxidative, thermal, neutral, and photolytic environments; utilizing the freshly prepared mobile phase prior to each use to ensure consistency and reproducibility. The method is validated in accordance with ICH guidelines, being simple, robust, and economical, thereby supporting regulatory submissions with reliable data and being suitable for large-scale production and routine quality control applications.

[0018] According to the present invention, for estimating a fix dose combination of Sulbactam and Durlobactam method comprises; i. using a ultra-performance liquid chromatography (UPLC) method; characterized in that the method comprises; ii. using a chromatographic column HSS Cl 8; iii. using a mobile phase consisting of buffer and acetonitrile in a ratio of 70:30 (v / v); iv. carrying out detection at a wavelength of 248 nm; v. flow rate is maintained in the range of 0.3 mL / min; vi. the retention times for Sulbactam and Durlobactam are found to be 1.77 min and 1.33 min; vii. the %RSD values are below 0.5 for both Sulbactam and Durlobactam, indicating high precision; viii. recovery studies yield results of 99.49% for Sulbactam and 99.90% for Durlobactam, confirming the accuracy of the method; ix. the limit of detection (LOD) and limit of quantification (LOQ) for Sulbactam are 0.19 pg / mL and 0.56 pg / mL; x. the limit of detection (LOD) and limit of quantification (LOQ) for Durlobactam are 0.08 pg / mL and 0.26 pg / Ml

[0019] EXAMPLES

[0020] Example 1: A mixture of Sulbactam and Durlobactam was prepared using a mobile phase comprising a phosphate buffer and acetonitrile in a 70:30 (v / v) ratio. Analysis was performed using Ultra-Performance Liquid Chromatography (UPLC) with an HSS Cl 8 column having a particle size of 1.8 pm. The flow rate was maintained at 0.3 mL / min, and detection was carried out at a wavelength of 248 nm. The retention times observed were 1.77 min for Sulbactam and 1.33 min for Durlobactam, which are within the optimal range for effective separation. The relative standard deviation (RSD) values for both Sulbactam and Durlobactam were below 0.5, demonstrating the high precision of the method. The recovery values for both compounds were found to be 99.49% for Sulbactam and 99.90% for Durlobactam, confirming the accuracy of the method. The limit of detection (LOD) and limit of quantification (LOQ) for Sulbactam were 0.19 pg / mL and 0.56 pg / mL, respectively, while the LOD and LOQ for Durlobactam were 0.08 pg / mL and 0.26 pg / mL, respectively. These results confirm that the method provides optimum sensitivity, reproducibility, and accuracy within the preferred ranges disclosed in the present invention.

Claims

We Claim:

1. A method for simultaneous estimation of Sulbactam and Durlobactam in a mixture comprising: a) preparing a sample of the mixture of Sulbactam and Durlobactam in a solution of a solvent and a buffer; b) analysing said sample using ultra-performance liquid chromatography (UPLC) by: i. setting parameters including flow rate, retention time, temperature, LOD (limit of detection), LOQ (limit of quantification), RSD (relative standard deviation); ii. using a chromatographic column selected from a group consisting of HSS C18, Cl 8, C8, phenyl, and cyano columns, preferably Cl 8 or C8 columns, more preferably a HSS Cl 8 column, with specific dimension and particle size of 1.7 pm to 2.5 pm for chromatographic separation; and iii. carrying out analysis of the sample at a wavelength in the range of 240 nm to 260 nm, preferably 245 to 250 nm, more preferably at 248 nm, for determining recovery of Sulbactam and Durlobactum in the mixture.

2. The method as claimed in claim 1, wherein the buffer is selected from a group comprising Potassium dihydrogen phosphate, Ammonium acetate, Ammonium formate buffer, preferably Potassium dihydrogen phosphate, Ammonium acetate, more preferably Potassium dihydrogen phosphate, and the solvent is selected from a group comprising Methanol, Isopropanol, Acetonitrile, Tetrahydrofuran, preferably Methanol and Isopropanol, more preferably Methanol or combinations thereof.

3. The method as claimed in claim 1, wherein ratio of the buffer and the solvent in the solution is 90: 10 (v / v), preferably 80:20 (v / v), more preferably 70:30 (v / v).

4. The method as claimed in claim 1, wherein the flow rate is maintained in a range of 0.1 to 0.5 mL / min, preferably 0.2 to 0.4 mL / min, more preferably 0.3 mL / min.

5. The method as claimed in claim 1, wherein the injection volume is in a range of 1 to 5 pL, preferably 2 to 4 pL, more preferably 3 pL.

6. The method as claimed in claim 1, wherein the retention times for the Sulbactam and the Durlobactam are in a range of 1.2 to 2.0 min, preferably 1.3 to 1.8 min, more preferably 1.77 min for Sulbactam and 1.33 min for Durlobactam.

7. The method as claimed in claim 1, wherein the RSD values are in a range of 0.1 to 2.0, preferably 0.2 to 1.0, more preferably below 0.5 for both the Sulbactam and Durlobactam, indicating high precision.

8. The method as claimed in claim 1, wherein the recovery of the Sulbactam and Durlobactam individually, is in a range of 98 to 101%, preferably 99 to 100%, more preferably 99.49% and 99.90% for Sulbactam and Durlobactam, respectively, confirming the accuracy of the method.

9. The method as claimed in claim 1, wherein the LOD and LOQ for the Sulbactam are in a range of 0.1 to 0.4 pg / mL and 0.5 to 0.8pg / mL, respectively, preferably 0.3 pg / mL and 0.6 pg / mL, and more preferably 0.19 pg / mL and 0.56 pg / mL.

10. The method as claimed in claim 1, wherein the LOD and LOQ for the Durlobactam are in a range of 0.05 to 0.1 pg / mL and 0.2 to 0.5 pg / mL, respectively, preferably 0.09 pg / mL and 0.3 pg / mL, and more preferably 0.08 pg / mL and 0.26 pg / mL.

11. The method as claimed in claim 1, effectively separates degradation products from active pharmaceutical ingredients under stress conditions including acidic, basic, oxidative, thermal, neutral, and photolytic environments; utilizing the freshly prepared mobile phase prior to each use to ensure consistency and reproducibility.

12. The method as claimed in claim 1, is validated in accordance with ICH guidelines, being simple, robust, and economical, thereby supporting regulatory submissions with reliable data and being suitable for large-scale production and routine quality control applications.

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