TW201522364A - Characterization of colistimethate sodium (CMS) - Google Patents

Abstract

The present invention regards a HPLC method useful for the analyzing CMS or separation of CMS components.

Description

Qualitative method of colistin sodium methanesulfonate (CMS)

The present invention is capable of characterization of colistin sodium methanesulfonate (CMS). The object of the present invention is to develop a method for chromatographically characterizing CMS.

CMS is made by chemically modifying the antibiotic mixture "colistin" (also known as polymyxin E). The conversion of colistin to CMS involves the chemical modification of the primary amine group of colistin with a methylsulfonate group (Barnette et al, Brit. J. Pharmacol. (1964), 23, 552). According to the United States Pharmacopoeia (USP), CMS consists only of two components with a clear molar mass derived from colistin A (1749.82 DA) and colistin B (1735.80 Da).

The European Pharmacopoeia (E.P.) claims that colistin contains five polymyxin components (E1, E2, E1-i, E1-7MOA and E3). According to E.P., the sum of the above five components in colistin should be at least 77%, and E1-i, E3 or E1-7MOA should not exceed 10% individually. Therefore, the major part of colistin is polymyxin E1 and polymyxin E2 (colistin A and B, respectively). The ratio of polymyxin E1 to polymyxin E2 in colistin varies depending on the production conditions and the production strain.

Colistin comprises a cyclic heptapeptide and a tripeptide side chain which is deuterated by a fatty acid at the N-terminus. In addition to the ingredients already mentioned, at least 30 different components of colistin have been detected (Orwa et al., J. Antibio. (2001). 51, 433) and The ingredients are colistin A (polymyxin E1) and B (polymyxin E2). The difference between colistin A and colistin B lies in the fatty acid side chain, see polymyxin E1 in Figure 1.

The prior art is insufficient in providing analytical methods to separate and identify various components of the CMS. The literature has previously described a high performance liquid chromatography (HPLC) method for the analysis of CMS in water (Li et al, Antimicro. Agents. Chemo (2003) 47, 4). Despite the limited quality of HPLC reported by Li et al., it is still possible to study how CMS degrades in aqueous solutions in vivo. Li et al. indicate that CMS degrades rapidly in solution, resulting in a partially sulfomethylated colistin mixture. Although Li et al. did not successfully analyze the chromatographic peaks by mass spectrometry (MS) analysis, the functional groups showed that all chromatographic peaks were derived from colistin A and B.

To the best of the inventors' knowledge, there has been no reliable analytical method to determine the quality and composition of CMS. According to the requirements of the USP, IR is used to identify the CMS. However, analysis of the sample by IR does not provide any information about the degree of modification, degree of substitution, position of the substituent, degradation, etc. or the actual composition of the CMS. In addition, microbiological assays are used to ensure that the sample exhibits the desired degree of antimicrobial efficacy. However, colistin also exerts antibacterial activity, which is generally said to be more potent than CMS. Free colistin is generally referred to as more toxic and lacks prodrug properties of CMS. Therefore, there is limited knowledge of the actual quality, stability and composition of the CMS.

The complex composition of colistin and the different substitution possibilities lead to qualitative difficulties in CMS. In the analytical development of the HPLC method, it was found that the fresh CMS solution consisted of more than 100 different components (see Figure 4). Therefore, the HPLC distribution described by Li et al. in 2003 appears to be incomplete and shows only a small fraction of the actual composition and CMS composition.

Due to the lack of appropriate analytical tools to characterize the quality and composition of CMS preparations, the dosage and safety of some CMS products are of great concern (see, for example, "The NAN Alert, "Risk of serious or fatal medication error", 29 ^th June 2011" ). Therefore, an analytical method for determining the content and quality of CMS components is highly desirable.

The present invention relates to a method of characterizing CMS using HPLC. The present invention also provides an analytical method for determining the quality and stability of CMS preparation to ensure that CMS does not degrade during manufacture, handling, and storage, and that the manufacturing conditions employed provide CMS preparation with predetermined prodrug characteristics.

The present invention relates to a chromatographic method for separating components of a CMS.

In a preferred embodiment, the method uses an HPLC method of a binary mobile phase system and a hydrophobic resin.

According to an embodiment, an HPLC method for separating a CMS component comprises loading a sample comprising CMS into a stationary phase and eluting with a mobile phase, wherein: - the mobile phase comprises a mixture of solvent A and solvent B, wherein solvent A comprises 90 %-99% v/v phosphate buffer and 1%-10% v/v acetonitrile, solvent B contains 40%-60% v/v phosphate buffer and 40%-60% v/v Acetonitrile; and - wherein the mobile phase initially comprises 70%-90% v/v of solvent A and 10%-30% v/v of solvent B; and - wherein the composition of the mobile phase gradually changes to 40%-60% v/ Solvent A of v and 40%-60% v/v of solvent B; and - wherein the stationary phase is a hydrophobic resin.

According to an aspect of this embodiment, solvent A comprises 92%-97% v/v phosphate buffer and 3%-8% v/v acetonitrile.

According to another aspect of this embodiment, solvent A comprises 95% v/v phosphate buffer and 5% v/v acetonitrile.

According to still another aspect of this embodiment, solvent B comprises 45%-55% v/v phosphate buffer and 45%-55% v/v acetonitrile.

According to still another aspect of this embodiment, solvent B comprises about 50% v/v phosphate buffer and about 50% v/v acetonitrile.

According to another aspect of this embodiment, the concentration of the phosphate buffer is from 0.01 M to 0.1 M.

According to still another aspect of this embodiment, the concentration of the phosphate buffer is from 0.03 M to 0.07 M.

According to still another aspect of this embodiment, the phosphate buffer has a concentration of 0.05M.

According to another aspect of this embodiment, the phosphate buffer has a pH of 6-7.

According to still another aspect of this embodiment, the phosphate buffer has a pH of from 6.3 to 6.8.

According to still another aspect of this embodiment, the pH of the phosphate buffer is 6.5.

According to another aspect of this embodiment, the stationary phase is a hydrophobic C8-C24 resin.

According to still another aspect of this embodiment, the stationary phase is a hydrophobic C15-C20 resin.

According to still another aspect of this embodiment, the stationary phase is a hydrophobic C18 resin.

According to another aspect of this embodiment, the detection wavelength is from 190 nanometers (nm) to 230 nm.

According to still another aspect of this embodiment, the detection wavelength is from 200 nm to 220 nm.

According to still another aspect of this embodiment, the detection wavelength is 210 nm.

According to another aspect of this embodiment, the column temperature is between 20 ° C and 40 ° C.

According to still another aspect of this embodiment, the column temperature is from 25 ° C to 35 ° C.

According to still another aspect of this embodiment, the column temperature is about 30 °C.

According to another aspect of this embodiment, the flow rate is from 0.1 ml/min to 0.60 ml/min.

According to still another aspect of this embodiment, the flow rate is from 0.2 ml/min to 0.40 ml/min.

According to still another aspect of this embodiment, the flow rate is about 0.30 ml/min.

According to an embodiment of the invention, the stationary phase has a particle size of from 0.5 micron to 5 microns.

According to a preferred embodiment of the invention, the stationary phase has a particle size of from about 1 micron to about 2 microns.

According to a preferred embodiment of the invention, the stationary phase has a particle size of about 1.7 microns.

According to an embodiment of the invention, the length of the column comprising the stationary phase is greater than 5 centimeters (cm) to 30 cm.

According to a preferred embodiment of the invention, the length of the column comprising the stationary phase is from about 10 cm to about 20 cm.

According to a preferred embodiment of the invention, the length of the column comprising the stationary phase is about 15 cm.

According to an embodiment of the invention, the column comprising the stationary phase has a diameter of from 1 millimeter (mm) to 5 mm.

According to an embodiment of the invention, the column comprising the stationary phase has a diameter of from 2.1 mm to 4.6 mm.

According to an embodiment of the invention, the diameter of the column comprising the stationary phase is greater than 2.1 mm.

According to another aspect of the invention, the loaded sample comprises CMS dissolved in a solution comprising 90%-99% v/v methanol and 1%-10% v/v water.

According to yet another aspect of the invention, the loaded sample comprises CMS dissolved in a solution comprising 95% v/v methanol and 5% v/v water.

According to another embodiment of the invention, an HPLC method is provided for analyzing a CMS comprising a binary gradient system, wherein the mobile phase comprises a mixture of A) and B): A) about 95% v/v and a pH of 6-7 Phosphate buffer and about 5% v/v acetonitrile; B) about 50% v/v phosphate buffer at pH 6-7 and about 50% v/v acetonitrile; and the mobile phase initially contained About 80% v/v A) and about 20% v/v B); and the composition of the mobile phase thereof is gradually changed to A) of about 50% v/v and B) of about 50% v/v; and the stationary phase thereof is a hydrophobic C18 resin.

Figure 1 illustrates the colistin A structure according to the teachings of the prior art, thereby suggesting the compositional structure of colistin sodium methanesulfonate (CMS) according to the Coly-Mycin® label.

Figure 2 illustrates a blank chromatogram.

Figure 3 illustrates a chromatogram of the quantitative limit solution.

Figure 4 illustrates a chromatogram of the CMS USP standard.

CMS here is any composition produced by the treatment of colistin with formaldehyde and bisulfite. Therefore, regardless of the method used, the CMS is intended to contain a mixture of compounds that occurs when sulfomethylated colistin is present. The chemical abstract has specified this composition as number 8068-28-8.

Colistin is used herein as any composition comprising polymyxin E1 and polymyxin E2. The chemical abstract has designated colistin as number 1066-17-7.

Polymyxin E1 is used herein as a compound having the CAS number 7722-44-3. Polymyxin E1 is synonymous with colistin A.

Polymyxin E2 is used herein as a compound having the CAS number 7739-48-7. Polymyxin E2 is synonymous with colistin B.

"Substantially pure" is used herein as a composition contained in HPLC chromatography in which the composition of the integrated area exceeds 90%.

"HPLC" (High Performance Liquid Chromatography) is used herein as any chromatographic technique for separating mixtures of compounds to identify, quantify or purify individual components of the mixture. HPLC typically utilizes pressure to cause the mobile phase to leak through the column including the stationary phase.

The "binary gradient system" is used herein as a chromatographic method which performs gradient elution using a mobile phase containing two different solvents.

According to an embodiment, a sample comprising CMS is loaded to the stationary phase. The sample is soluble in organic solvents, including methanol, ethanol and acetonitrile, but not limited thereto. The sample preferably contains from 1% to 10% water to promote dissolution of the CMS. The best loaded sample contained CMS dissolved in 95% v/v methanol and 5% v/v water.

According to an embodiment, the two different solvents of the mobile phase comprise different amounts of acetonitrile and phosphate buffer, for example wherein the first solvent (solvent A) comprises 90%-99% v/v phosphate buffer and 1%- 10% v/v acetonitrile wherein the second solvent (solvent B) comprises 40% to 60% v/v phosphate buffer and 40% to 60% v/v acetonitrile.

In order to obtain a binary gradient system, the mobile phase is gradually changed, for example, the mobile phase initially contains 70%-90% v/v of solvent A and 10%-30% v/v of solvent B, wherein the mobile phase composition gradually changes to 40%-60% v/v solvent A and 40%-60% v/v solvent B.

The solvent used in the binary gradient system can be graded. Acetonitrile is preferably used as one of the two solvents of the binary gradient system. The acetonitrile-based HPLC gradient grade used should be understood. Gradient grade acetonitrile can be obtained from various manufacturers, such as the LiChrosolv® gradient grade (Merck number: 1.00030).

The phosphate buffer is preferably also of the HPLC gradient grade. Gradient grade phosphoric acid Salt buffers can be obtained from various manufacturers, such as sodium dihydrogen phosphate dihydrate (Merck number: 1.06498).

The hydrophobic resin suitable as the stationary phase may be a hydrophobic C8-C24 resin such as a hydrophobic C15-C20 resin such as a hydrophobic C18 resin. According to one embodiment, the hydrophobic resin corresponds to Acquity UPLC CSH C18 from Waters Corporation, 1.7 micrometers (μm), 150 x 2.1 mm. Other useful resins include bridged ethyl blends (BEH) from Waters Corporation.

The CMS comprises a mixture of compounds derived from polymyxin E1 and E2. E1 and E2 CMS account for about 80% of the CMS content, which is roughly the same as colistin.

It is reported that colistin can be stabilized in aqueous solution for more than 120 hours at 37 ° C (Li et al, Antimicro. A and Chemoth, 2003, vol 47, No. 4, p1364). However, CMS is easily hydrolyzed into various derivatives. Even the newly dissolved CMS is a complex mixture of various derivatives. Li et al. also proved multi-component composition in 2003. Previous techniques have concluded that CMS is a colistin prodrug, which will eventually be hydrolyzed to colistin in vitro/in vivo (Bergen et al, Antimicr. Agents and Chemoth. 50 (6), 2006, 1953).

The HPLC method according to the present invention will now be described below. It is to be understood that the examples provided are not intended to limit the scope of the invention.

Instance 5.1 equipment

A binary gradient HPLC system with a pressure limit of at least 11,000 psi and an ultraviolet (UV) detector capable of measuring at 210 nm.

5.2 Chemicals and reagents

Sodium dihydrogen phosphate dihydrate (Merck number: 1.06345) or equivalent.

Sodium hydroxide pellets (Merck No.: 1.06498) or equivalent.

Acetonitrile, LiChrosolv gradient grade (Merck number: 1.00030) or equivalent.

CMS control sample, USP reference standard or equivalent.

1M sodium hydroxide solution

4.0 g (g) of sodium hydroxide was added to 100 ml (ml) of ultrapure water, and the mixture was stirred until all of the sodium hydroxide was dissolved.

0.05M phosphate buffer, pH 6.5

7.8 g of sodium dihydrogen phosphate dihydrate was dissolved in about 980 ml of ultrapure water. The pH was adjusted to 6.5 with a 1 M sodium hydroxide solution, and ultrapure water was added to 1000 ml.

LC Solvent A

475 ml, 0.05 M, pH 6.5 phosphate buffer was added to 25 ml of acetonitrile. The mixture was filtered through a 0.22 μm membrane filter and degassed in an ultrasonic bath for 10 minutes.

LC solvent B

250 ml, 0.05 M, pH 6.5 phosphate buffer was added to 250 ml of acetonitrile. The mixture was filtered through a 0.22 μm membrane filter and degassed in an ultrasonic bath for 10 minutes.

5.3 System and method parameters

Column: Waters Acquity UPLC CSH C18, 1.7μm, 150×2.1mm or equivalent

Front column: Waters VanGuard UPLC CSH C18, 1.7μm, 150×2.1mm or equivalent

Column temperature: 30 ° C

Flow rate: 0.30 ml / min

Flow type: gradient

LC solvent A: 0.05 M phosphate buffer, pH 6.5 / acetonitrile; 95/5 v / v

LC solvent B: 0.05 M phosphate buffer, pH 6.5 / acetonitrile; 50 / 50 v / v

Gradient: initially, 20% B; 0-10 minutes, linearity becomes 32% B; 10-35 minutes, linearity becomes 47% B; 35-36 minutes, linearity becomes 20% B; 36-44 minutes, 20% B

Balance: at least 30 minutes. At least 2 control sample injections were performed to check that the residence time was constant.

Wavelength: 210nm

Injection volume: 2.0 microliters

Execution time: 44 minutes

Automatic sampling: 5 ° C

5.4 Test sample solution preparation

In the case of 1 million unit bottles: 2.00 ml of ultrapure water (about 21 ° C) and dissolved (about 37.5 mg / ml of CMS) were added. After solubilization, 1 ml was transferred to a 20 ml volumetric flask, filled and labeled with methanol, and the solution was stored at 2 ° C to 8 ° C (about 1.875 mg / ml of CMS).

In the case of 2 million unit bottles: 4.00 ml of sterile water (about 21 ° C) and dissolved (about 37.5 mg / ml of CMS) were added. After dissolution, it will be 1 The ml was transferred to a 20 ml volumetric flask and filled and labeled with methanol, and the solution was stored at 2 ° C to 8 ° C (about 1.875 mg / ml of CMS).

In the finished dosage form (corresponding to 150 mg of colistin): 2.0 ml of water for injection (about 21 ° C) or equivalent and dissolved. After dissolution, 200 microliters (μl) of the solution was transferred to a 20 ml volumetric flask, filled and labeled with methanol, and the solution was stored at 2 ° C to 8 ° C. The final CMS concentration in the test sample solution was about 2 mg/ml.

5.5 Preparation of control sample solution

Accurately weigh 10 mg of CMS control sample (USP reference standard or equivalent) into a 5 mL measuring flask (2.0 mg/ml). Add 0.25 ml of ultrapure water and dissolve the CMS. After dissolution, it was diluted to the mark with methanol, and the solution was stored at 2 ° C to 8 ° C.

5.6 Quantitative Limit (QL) Sample Solution Preparation

The 1.5 ml control sample solution prepared in 5.5 was transferred to a 25 ml volumetric flask (2.0 mg/ml). Dilute to the mark (0.12 mg/ml) with methanol.

5.7 HPLC analysis 5.7.1 Analysis Settings

Sample analysis is shown in Table 1.

5.7.2 Acceptance criteria for system suitability

Blank: There should be no peaks that would interfere with the blank CMS peak.

Quantitative Limit (QL): The S/N of the most abundant peak (RRT 1.00) in the QL sample should not be less than 10 times that of the noise.

Reproducibility: In the control sample, the difference in retention time of the RRT 1.00 peak should be less than 0.5 minutes for two consecutive injections.

Number of laminates (N): In the control sample, the number of USP laminates for the RRT 1.00 peak should not be less than 20.000, and the RRT 2.61 for the control sample should not be less than 100.000.

5.7.3 Test sample analysis - peak identification and quantification

All integrals of peaks greater than 0.05% (area%) were found.

The most abundant peak within 10.5 to 12.5 was set as the identification reference peak (RRT 1.00). Calculate the RRT of all other peaks in the chromatogram.

Figure 2 illustrates a blank chromatogram, Figure 3 illustrates a chromatogram of the quantitative limit solution, and Figure 4 illustrates a chromatogram of the CMS USP standard.

The above results show that the method of the present invention provides an analytical tool that is eager for a long time to determine the content and quality of the CMS composition.

Claims (26)

An HPLC method for separating components of a CMS, the method comprising the steps of: loading a sample comprising CMS into a stationary phase and eluting with a mobile phase; wherein the mobile phase comprises a solvent A and a solvent B a mixture wherein the solvent A comprises 90%-99% v/v phosphate buffer and 1%-10% v/v acetonitrile, the solvent B comprises 40%-60% v/v phosphate buffer And 40% to 60% v/v of acetonitrile; and wherein the mobile phase initially comprises 70% to 90% v/v of the solvent A and 10% to 30% v/v of the solvent B; and the mobile phase thereof The composition is gradually changed to 40% to 60% v/v of the solvent A and 40% to 60% v/v of the solvent B; and the stationary phase is a hydrophobic resin. The method of claim 1, wherein the solvent A comprises 92%-97% v/v phosphate buffer and 3%-8% v/v acetonitrile. The method of claim 1, wherein the solvent A comprises 95% v/v phosphate buffer and 5% v/v acetonitrile. The method of claim 1, wherein the solvent B comprises 45% to 55% v/v phosphate buffer and 45% to 55% v/v acetonitrile. The method of claim 1, wherein the solvent B comprises about 50% v/v Phosphate buffer and about 50% v/v acetonitrile. The method of claim 1, wherein the phosphate buffer has a concentration of from 0.01 M to 0.1 M. The method of claim 1, wherein the phosphate buffer has a concentration of from 0.03 M to 0.07 M. The method of claim 1, wherein the phosphate buffer has a concentration of 0.05M. The method of claim 1, wherein the phosphate buffer has a pH of from 6 to 7. The method of claim 1, wherein the phosphate buffer has a pH of from 6.3 to 6.8. The method of claim 1, wherein the phosphate buffer has a pH of 6.5. The method of claim 1, wherein the stationary phase is a hydrophobic C8-C24 resin. The method of claim 1, wherein the stationary phase is hydrophobic C15-C20 resin. The method of claim 1, wherein the stationary phase is a hydrophobic C18 resin. The method of claim 1, wherein the detection wavelength is from 190 nm to 230 nm. The method of claim 1, wherein the detection wavelength is from 200 nm to 220 nm. The method of claim 1, wherein the detection wavelength is 210 nm. The method of claim 1, wherein the column temperature is from 20 ° C to 40 ° C. The method of claim 1, wherein the column temperature is from 25 ° C to 35 ° C. The method of claim 1, wherein the column temperature is about 30 °C. The method of claim 1, wherein the flow rate is from 0.1 ml/min to 0.60 ml/min. The method of claim 1, wherein the flow rate is from 0.2 ml/min to 0.40 ml/min. The method of claim 1, wherein the flow rate is about 0.30 ml/min. The method of claim 1, wherein the loaded sample comprises CMS dissolved in a solution comprising 90%-99% v/v methanol and 1%-10% v/v water. The method of claim 1, wherein the loaded sample comprises CMS dissolved in a solution comprising 95% v/v methanol and 5% v/v water. An HPLC method for analyzing a CMS comprising a binary gradient system, wherein the mobile phase comprises a mixture of A) and B): A) a phosphate buffer of about 95% v/v and a pH of 6-7 and Approximately 5% v/v acetonitrile; B) about 50% v/v and a phosphate buffer having a pH of 6-7 and about 50% v/v acetonitrile; and wherein the mobile phase initially comprises about 80% v/ A) of v and about 20% v/v of B); and the composition of the mobile phase gradually changes to about 50% v/v of A) and about 50% v/v of B); and the stationary phase thereof A hydrophobic C18 resin.