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    • 专利摘要:
    The present invention relates to a method for the detection and quantification of fosfomycin and impurities and/or degradation products thereof in samples of fosfomycin or a pharmaceutically acceptable salt thereof or in pharmaceutical compositions comprising fosfomycin or a pharmaceutically acceptable salt thereof. The present invention further relates to a process for manufacturing fosfomycin, or a pharmaceutically acceptable salt thereof having a specified purity degree, as well as to the fosfomycin or a pharmaceutically acceptable salt thereof as obtained.The present invention further relates to a process for manufacturing a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified purity degree, as well as to the pharmaceutical composition as obtained.
    公开号:ES2872674A2
    申请号:ES202190053
    申请日:2020-03-10
    公开日:2021-11-02
    发明作者:Robert Divasto；Marta Marino
    申请人:Laboratorios Ern S A；Nabriva Therapeutics Ireland Dac；
    IPC主号:
    专利说明:

    [0002] METHOD FOR THE DETECTION AND QUANTIFICATION OF PHOSPHOMYCIN AND ITS IMPURITIES AND / OR DEGRADATION PRODUCTS
    [0004] Technique field
    [0005] The present invention relates to a method for detecting and quantifying the antibiotic fosfomycin and its impurities and / or degradation products, both in samples of the active principle alone, and in pharmaceutical compositions containing it.
    [0007] Prior state of the art
    [0008] Fosfomycin is a broad-spectrum antibiotic with activity against gram-positive bacteria, including multidrug resistant (MDR) pathogens associated with life-threatening infections. Its mechanism of action is related to the inhibition of the synthesis of the bacterial cell wall through the inhibition of the enzyme phosphoenolpyruvate transferase.
    [0010] Fosfomycin can be administered both orally and by intramuscular or intravenous injection.
    [0012] When administered orally, fosfomycin is generally used in the form of calcium salt or trometamol salt and is available in pharmaceutical forms such as capsules, or granules or powder to be reconstituted with water, and is indicated primarily for the treatment of infections. urinary.
    [0014] Injectable compositions generally comprise the disodium salt of fosfomycin and are available as a sterile powder, usually with succinic acid as an excipient, to be reconstituted prior to injection.
    [0016] By intramuscular route, fosfomycin is mainly indicated for the treatment of infections of the genitourinary tract, respiratory tract and soft tissue infections, among others, while the main indications for intravenous fosfomycin are complicated urinary tract infections, osteomyelitis, infections nosocomials of the lower respiratory tract and bacterial meningitis, among others.
    [0018] As for any other active pharmaceutical ingredient, it is essential to have effective analytical methods for the quantitative determination of fosfomycin and its impurities and / or degradation products, both in samples of the active principle and in pharmaceutical compositions, in order to ensure that the necessary purity levels are met.
    [0020] The determination of fosfomycin is problematic because it is a small, acidic, hydrophilic, and highly polar molecule, so the most common techniques, such as reversed-phase or normal-phase liquid chromatography, are inadequate. Another added difficulty is that the fosfomycin molecule lacks a chromophore, so the use of a standard ultraviolet (UV) detector is also ineffective.
    [0022] Consequently, there are few descriptions in the prior art of effective methods for determining fosfomycin in pharmaceutical samples, much less for determining fosfomycin and its impurities and degradation products.
    [0024] In the article by Liu et al., Determined of fosfomycin tmmetamol and its related substances in the bulk drug by ion-pair HPLC with evaporative light scattering detection, J. Liq. Chromatogr. Relat. Technol., 2006, 29, 15-24, an ion pair reversed phase HPLC method is described to determine fosfomycin and only one impurity thereof (impurity A) in a fosfomycin trometamol bulk drug sample. Using a C18 column, the optimized mobile phase was a 15 mM octylamine solution (adjusted to pH 5.2 with glacial acetic acid) with acetonitrile (92: 8). An evaporative light scattering detector (ELSD) was used.
    [0026] Therefore, there is still a need for an effective analytical method for the determination of fosfomycin and its impurities and / or degradation products, both in samples of the active ingredient and in pharmaceutical compositions, which allows the identification and quantification of essentially all impurities and / or degradation products present in the samples.
    [0028] Object of the invention
    [0029] The object of the present invention is a method for the detection and quantification of fosfomycin and its impurities and / or degradation products.
    [0031] Another aspect of the invention is a process for making fosfomycin or a pharmaceutically acceptable salt thereof having a specified degree of purity comprising the use of said method.
    [0032] Another aspect of the invention is a process for manufacturing a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity comprising the use of said method.
    [0034] Another aspect of the invention is fosfomycin or a pharmaceutically acceptable salt thereof having a specified degree of purity prepared by the processes described herein.
    [0036] Another aspect of the invention is a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity prepared by the processes described herein.
    [0038] Description of Figures
    [0039] - Figure 1 shows a representative chromatogram using HILIC high performance liquid chromatography and CAD detection (HILIC-HPLC-CAD) of the diluent solution (25 mM ammonium acetate in acetonitrile, 50:50, v / v) (Example 1) . The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0041] - Figure 2 shows a representative chromatogram by HILIC-HPLC-CAD of the succinic carrier solution (0.040 mg / ml of succinic acid) (Example 1). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0043] - Figure 3 shows a representative chromatogram by HILIC-HPLC-CAD of the reference standard of Fosfomycin disodium (Example 1). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0045] - Figure 4 shows a representative chromatogram by HILIC-HPLC-CAD of a sample of fosfomycin pharmaceutical product for injection in vials containing a sterile dry powder mix of 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin disodium) and 150 mg of succinic acid (1.86% by weight) (Example 1). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0047] - Figure 5 shows a representative chromatogram by HILIC-HPLC-CAD of a sample of fosfomycin pharmaceutical product for injection in vials containing a sterile dry powder mixture of 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin disodium) and 150 mg of succinic acid (1.86% by weight) (Example 2 ). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0049] - Figure 6 shows a representative chromatogram by HILIC-HPLC-CAD of the reference standard of impurity A (Example 2). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0051] - Figure 7 shows a representative chromatogram by HILIC-HPLC-CAD of an aged sample of fosfomycin pharmaceutical product for injection (mixture of fosfomycin disodium and succinic acid 1.86% by weight) and non-sterile phosphomycin disodium active substance alone ( Example 2). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0053] - Figure 8 shows a representative chromatogram by HILIC-HPLC-CAD of the active substance fosfomycin disodium and a bulk sample of the pharmaceutical product fosfomycin disodium for injection (mixture of fosfomycin disodium and succinic acid at 1.86% by weight) (Example 2). The x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area).
    [0055] - Figure 9 shows a schematic representation of the coupling of the HPLC column with a charged aerosol detector (CAD) and with a mass spectrometer (MS), according to an LC-CAD-MS combination procedure.
    [0057] Detailed description of the invention
    [0058] The object of the present invention is a method for the detection and quantification of fosfomycin and its impurities and / or degradation products in samples of fosfomycin or of a pharmaceutically acceptable salt thereof or of pharmaceutical compositions comprising fosfomycin or a pharmaceutically salt acceptable thereof, characterized in that it comprises:
    [0059] (a) subjecting the sample to hydrophilic interaction liquid chromatography (HILIC) with gradient elution using as mobile phase a mixture of acetonitrile (mobile phase A) and an aqueous solution of ammonium acetate (mobile phase B); and
    [0060] (b) detect and quantify fosfomycin, impurities and / or degradation products separated in step a) using a charged aerosol detector (CAD).
    [0062] After unsuccessfully evaluating many analytical options, the present inventors have developed a method based on hydrophilic interaction liquid chromatography (HILIC) combined with a charged aerosol detector (CAD) which, surprisingly, allows the identification of many more impurities or products of degradation of fosfomycin than with the methods described so far in the state of the art. That is, more than 10 different substances or degradation products were successfully identified and quantified.
    [0064] Throughout the present description, as well as in the claims, the expressions in the singular, generally preceded by the articles "the", "the", "a", "an" which are intended to include also the plural forms, unless the context clearly indicates otherwise. Furthermore, numerical values preceded by the term "about" are intended to include the exact indicated value and also some variation around said value, ie, a variation of ± 5% from the indicated amount. The numeric ranges defined by lower and upper end points are intended to include such indicated end points as well. Unless otherwise indicated, percentages (%) are expressed as weight / weight percentages. It is part of the general knowledge of a person skilled in the art that a pharmaceutical composition comprises at least one pharmaceutically acceptable excipient (see below where explicitly described).
    [0066] Fosfomycin
    [0067] Fosfomycin is the International Non-proprietary Name (INN) for [(2R, 3S) -3-methyloxiran-2-yl] phosphonic acid (CAS number 23155-02-4, molecular formula C3H7O4P, molecular weight 138.06 g / mol ) and has the following structure:
    [0072] It is a broad spectrum antibiotic that acts by inhibiting the synthesis of the bacterial cell wall. Chemically, it is an acidic substance, specifically a phosphonic acid, and is commonly used in therapy in the form of a salt, for example, as a disodium salt, calcium salt or salt with tromethamine.
    [0074] In a preferred embodiment, the method of the invention is for the detection and quantification of fosfomycin and its impurities and / or degradation products in samples containing fosfomycin in the form of fosfomycin disodium salt.
    [0076] Fosfomycin disodium, in particular, has the chemical formula C3H5Na3O4P and the molecular weight is 182.02 g / mol.
    [0078] In the monographs of the disodium, calcium and tromethamine salts of fosfomycin of the European Pharmacopoeia, only one impurity is identified, called "impurity A", which corresponds to (1,2-dihydroxypropyl) phosphonate, that is, to the opening of the ring epoxy. For example, for the case of fosfomycin disodium, impurity A has the following structure:
    [0083] In one embodiment of the invention, the method refers to the detection and quantification of fosfomycin and its impurities and / or degradation products in a sample of fosfomycin or of a pharmaceutically acceptable salt of the same active principle (API sample), preferably in a sample of fosfomycin disodium API.
    [0085] In another embodiment, the method refers to the detection and quantification of fosfomycin and its impurities and / or degradation products in a sample of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof, preferably fosfomycin disodium, as an active ingredient. and at least one pharmaceutically acceptable excipient.
    [0087] The pharmaceutical composition may be in the form of different dosage forms, for example, as a powder, granulate, capsule or tablet for oral administration, as a powder for reconstitution to prepare an injectable solution or as an injectable solution.
    [0089] Among the various commercially available fosfomycin dosage forms, there is an injectable form that contains fosfomycin disodium as the active ingredient and succinic acid. as an excipient.
    [0091] In one embodiment, the pharmaceutical composition is a powder for reconstitution for injection.
    [0093] In another embodiment, the pharmaceutical composition is a capsule or tablet.
    [0095] In one embodiment, the pharmaceutical composition comprises succinic acid as an excipient.
    [0097] In a preferred embodiment of the invention, the pharmaceutical composition consists essentially of fosfomycin or a pharmaceutically acceptable salt thereof, preferably fosfomycin disodium, and succinic acid, preferably wherein the amount of succinic acid in the composition is between 1-5% (w / w), in relation to the total weight of the composition.
    [0099] The analytical method of the invention
    [0100] Charged Aerosol Detector ( CAD)
    [0101] Charged Aerosol Detectors (CAD), also known as "Corona", are well known as universal detectors for use in conjunction with liquid chromatography. In summary, its action is based on nebulizing the eluent containing the analytes into droplets when it leaves the column, subsequently evaporating the solvent to form particles, loading the particles in a reaction chamber by collision with ionized nitrogen, which is formed when passing nitrogen over a corona wire, and measure the charge of the particles with a sensitive electrometer. This generates a signal directly proportional to the amount of analyte present. Any analyte, as long as it forms a particle, can be measured by charged aerosol detection, regardless of its chemical structure.
    [0103] The principles and uses of charged aerosol detection (CAD) are well known in the art and are widely described in the literature, for example, in the book Charged Aerosol Detection for Liquid Chromatography and Related Separation Techniques, PH Gamache Editor, John Wiley & Sons, 2017.
    [0105] CAD detectors are commercially available for use in conjunction with liquid chromatography columns, for example from Thermo Fisher Scientific.
    [0106] Hydrophilic Interaction Liquid Chromatography ( HILIC)
    [0107] Hydrophilic Interaction Liquid Chromatography (HILIC), as it is well known in the field of analytical chemistry, is a type of high-performance liquid chromatography characterized by using mixtures of water or buffer solution and organic solvents as mobile phase, while that stationary phases are highly hydrophilic polar adsorbents (see, for example, Hydropilic Interaction Chromatography. A Guide for Practitioners. BA Olsen & BW Pack, Editors, John Wiley & Sons, 2013).
    [0109] Examples of stationary phases are unmodified silica or silica with some polar group attached, such as amines, amides, polyols, cyano or zwitterions. Typically, in zwitterionic stationary phases the bonded polar group combines a quaternary ammonium group and a sulfonic group (as described, for example, in García-Gómez et al., Stationary phases for separation of nucleosides and nucleotides by hydrophilic interaction liquid chromatography , Trends Anal Chem., 2013, 47, 111-128 or in A practical guide to HILIC, including ZIC®-HILIC applications, Editor: T. Jonsson, Merck SeQuant, 2009).
    [0111] In a preferred embodiment of the invention a zwitterionic stationary phase is used.
    [0113] These types of columns, suitable for HILIC, are widely available commercially.
    [0115] For example, the commercially available zwitterionic HPLC column ZIC®-pHILIC (Merck SeQuant AB) can be used.
    [0117] The mobile phase is a mixture of acetonitrile (mobile phase A) and an aqueous solution of ammonium acetate (mobile phase B).
    [0119] Generally, the concentration of ammonium acetate in mobile phase B is between 10 and 40 mM, preferably between 15 and 35 mM, more preferably between 20 and 30 mM, even more preferably it is about 25 mM and even more preferably it is 25 mM .
    [0121] The elution time of the mobile phase is generally between 45 and 60 minutes, preferably it is about 50 minutes and more preferably it is 50 minutes.
    [0123] The flow rate of the mobile phase is generally between 0.7 and 1 ml / min, preferably it is about 0.8 ml / min and more preferably it is 0.8 ml / min.
    [0125] The mobile phase travels through the stationary phase according to a gradient elution mode, that is, the composition of the mobile phase changes during the journey, that is, the ratio between mobile phase A (acetonitrile) and mobile phase B (aqueous solution of ammonium acetate). (A: B) in the solvent mixture changes during elution.
    [0127] Preferably, such a gradient involves changing from an initial A: B ratio of about 85:15 (t = 0) to an A: B ratio of about 40:60 at about 35 minutes, and then back again to about 85:15. at the end of the elution time.
    [0129] In a preferred embodiment, the initial and final A: B ratio, which is preferably about 85:15, is kept constant for a short period of time, for example between 3 and 15 minutes, at the beginning and / or at the end of elution; more preferably, the 85:15 ratio is kept constant:
    [0131] - at the beginning of the elution for about 3 to 8 minutes, preferably for about 4 to 6 minutes, more preferably for about 5 minutes and even more preferably for 5.0 minutes; me
    [0133] - at the end of the elution for about 8 to 15 minutes, preferably for about 10 to 12.5 minutes, more preferably for about 12 minutes and even more preferably for 11.9 minutes.
    [0135] In another preferred embodiment, the lowest A: B ratio, which is preferably about 40:60, and is preferably reached at about 35 minutes, is held constant for a short period of time, typically between 2 and 5 minutes. , preferably for about 3 minutes, before returning to the higher A: B ratio, which is preferably about 85:15. More preferably, the A: B ratio of about 40:60 is held constant for about 3 minutes from about 35 minutes to 38 minutes.
    [0137] In a particularly preferred embodiment of the invention, the elution time is 50 minutes, and the gradient expressed as the ratio between mobile phase A and mobile phase B in each time point is: 85:15 (0 min), 85:15 (5.0 min), 40:60 (35.0 min), 40:60 (38.0 min), 85:15 (38, 1 min) and 85:15 (50.0 min).
    [0139] The only known disclosed substances that are expected to be determined by the analytical method of the present invention are fosfomycin, impurity A, as well as possible excipients when the sample is a fosfomycin pharmaceutical composition.
    [0141] However, the present inventors observed that, surprisingly, using the analytical method of the invention, an optimal separation between fosfomycin and its impurities and / or degradation products is achieved. Thus, as described in the Examples, it is possible to separate more than 10 different substances (see Table 9), including "impurity A".
    [0143] To identify and quantify fosfomycin, impurity A, and known excipients, pure reference substances can be used, and a suitable calibration is generally performed from known reference standard samples. Alternatively, calibration can be performed using mixtures of reference standards, to avoid a possible matrix effect. For example, the calibration of impurity A can be performed with a sample containing a mixture of reference standards of impurity A and fosfomycin.
    [0145] To quantify other substances, it can be assumed that all impurities and degradation products have the same response factor as impurity A.
    [0147] The substances that are used as reference standards in the method can be obtained commercially, and it is preferred that their purity is at least 94%, more preferably at least 95%, even more preferably at least 96%, still more preferably of at least 97%, even more preferably of at least 98% and even more preferably of at least 99%.
    [0149] The analytical method developed allows to determine concentrations of impurities or degradation products in fosfomycin samples as low as 0.10%.
    [0151] Mass Spectrometry ( MS)
    [0152] To identify unknown substances detected by HILIC-CAD, mass spectrometry (MS) was used, using a combined strategy LC-CAD-MS (liquid chromatography-detection of charged aerosols-mass spectrometry).
    [0153] Mass spectrometry is a well known analytical technique that is essentially based on the generation of ions from compounds by any suitable method, the separation of these ions by their mass / charge ratio (m / z) and their detection. Mass spectrometers generally consist of an ion source, a mass analyzer, and a detector that operate under high vacuum conditions.
    [0155] Tandem mass spectrometry (MS / MS) is used advantageously. MS / MS subjects the selected mass ions to a second mass spectrometric analysis, according to two combined steps of mass spectrometry (MS1 and MS2).
    [0157] The principles and practicalities of mass spectrometry are well known to the person skilled in chemical analysis and are also described in many reference books, for example in Mass Spectrometry. To textbook. J H. Gross, Second Edition, Springer, 2011.
    [0159] Typically, a mass spectrum of a substance is the two-dimensional representation of signal intensity (ordinate) versus m / z (abscissa). The intensity of the peak correlates with the abundance of that ion. Often times, the highest m / z peak results from the detection of the intact ionized molecule, the molecular ion. The peak of the molecular ion is usually accompanied by several peaks of smaller m / z generated by the fragmentation of the molecular ion, resulting in fragmented ions.
    [0161] In the present method, mass spectrometry is used in association with liquid chromatography, as a chromatographic detector, to identify each eluted substance through its mass spectrum. More specifically, liquid chromatography is simultaneously coupled with a charged aerosol detector and a mass spectrometer (LC-CAD-MS) in order to identify the different substances eluted and detected in CAD. A schematic of the LC-CAD-MS apparatus is shown in Figure 9.
    [0163] Therefore, the analyzed sample passes through the HILIC HPLC column, a T-shaped mixer is applied as a diverter valve immediately after the column, so the post-column flow is divided into the MS and the CAD. Generally, the flow ratio between MS and CAD is approximately 1: 2.
    [0165] Uses of the analytical method of the invention
    [0166] The analytical method of the invention can be included in a process to manufacture fosfomycin or a pharmaceutically acceptable salt thereof, to ensure that a pharmaceutical active ingredient of the required purity is obtained.
    [0168] Therefore, another aspect of the present invention refers to a process for the manufacture of fosfomycin or a pharmaceutically acceptable salt thereof that has a specified degree of purity, characterized in that it comprises the following steps:
    [0169] (i) providing a batch of fosfomycin or a pharmaceutically acceptable salt thereof; (ii) quantifying fosfomycin impurities and / or degradation products in a sample from the batch of step (i) using the method of the invention; and
    [0170] (iii) validate the batch only if the percentage of impurities in the sample meets the specified degree of purity.
    [0172] Another aspect of the present invention relates to fosfomycin or a pharmaceutically acceptable salt thereof having a specified degree of purity, prepared by the following steps:
    [0173] (i) providing a batch of fosfomycin or a pharmaceutically acceptable salt thereof; (ii) quantifying the fosfomycin impurities and or degradation products in a sample from the batch of step (i) using the method of the invention; and
    [0174] (iii) validate the batch only if the percentage of impurities in the sample meets the specified degree of purity.
    [0176] The specified degree of purity of fosfomycin or a pharmaceutically acceptable salt thereof may vary depending on the intended use or applicable regulatory requirements. The degree of purity can typically be expressed as a maximum allowable percentage of impurities, either referred to total impurities, or referred to a particular specific impurity.
    [0178] To provide the batch of fosfomycin or a pharmaceutically acceptable salt thereof, according to step (i), any source can be used, in particular, any suitable synthetic process can be used to prepare fosfomycin or a pharmaceutically acceptable salt of the same, optionally including a purification step.
    [0180] Subsequently, in stage (ii), a sample of the active principle is taken from the batch and subjected to to the analytical method of the invention, described above, to quantify the amount of impurities.
    [0182] Finally, the percentage of impurities in the sample is compared with the degree of impurity required; normally, it is evaluated if the percentage of impurities does not exceed the maximum degree of impurity allowed. If this requirement is met, the batch of fosfomycin or a pharmaceutically acceptable salt thereof is validated, that is, it is classified as suitable for subsequent use, that is, to be used as a pharmaceutical active ingredient to be incorporated into pharmaceutical compositions. .
    [0184] If the sample does not meet the purity requirements, it is discarded, that is, it is considered unsuitable for use as an active pharmaceutical ingredient. Discarded batches can undergo a purification process, for example.
    [0186] Similarly, the analytical method of the invention can be included in a process for manufacturing a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, to ensure that a pharmaceutical composition is obtained from it. required purity.
    [0188] Therefore, another aspect of the present invention refers to a process for manufacturing a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity, characterized in that it comprises the following stages:
    [0189] (i) providing a batch of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient;
    [0190] (ii) quantifying fosfomycin impurities and / or degradation products in a sample from the batch of step (i) using the method of the invention; and
    [0191] (iii) validate the batch only if the percentage of impurities meets the required purity requirements.
    [0193] A further aspect of the present invention relates to a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity, prepared by the following steps:
    [0194] (i) providing a batch of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient;
    [0195] (ii) quantifying fosfomycin impurities and / or degradation products in a sample from the batch of step (i) using the method of the invention; and
    [0196] (iii) validate the batch only if the percentage of impurities meets the required purity requirements.
    [0198] The batch of the pharmaceutical composition of step (i) can be bulk, that is, typically unpackaged powder, tablets or capsules, or it can be a finished pharmaceutical composition suitably packaged, for example, powder for oral use in single-dose sachets, or powder for injection in vials, or tablets or capsules in blisters or in bottles or other containers.
    [0200] To provide the batch of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, according to step (i), any source can be used, in particular, said batch can be obtained using any standard manufacturing process, as are well known in the field of pharmaceutical technology, as described in reference books well known in the field, eg Aulton's Pharmaceutics book. The design and manufacture of medicines, ME Aulton and KMG Taylor, editors, Churchill Livingstone Elsevier, 4th edition, 2013; or in the book Remington Essentials of Pharmaceutics, L. Felton, editor, Pharmaceutical Press, 2013; or in the Pharmaceutics. Basic principles and application to pharmacy practice. AK Dash, S. Singh and J. Tolman, editors, Academic Press, Elsevier, 2014.
    [0202] Subsequently, in step (ii), a sample is taken from the batch of the pharmaceutical composition and subjected to the analytical method of the invention, described above, to quantify the amount of impurities.
    [0204] Finally, the percentage of impurities in the sample is compared with the degree of impurity required; Typically, it is evaluated whether the percentage of impurities does not exceed the maximum degree of impurity allowed, whether it is related to the total amount of impurities or to a specific impurity in particular. If this requirement is met, the batch of the composition is validated, it is that is, it is classified as suitable for safe use in therapy.
    [0206] If the sample does not meet the purity requirements, it is discarded.
    [0208] Examples
    [0209] Example 1: HILIC-HPLC method with CAD detection: quantification of fosfomycin in fosfomycin samples
    [0210] The following fosfomycin samples were tested:
    [0211] - Fosfomycin injectable finished product ("Fosfomycin DP"): vials containing a sterile dry powder mixture of 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin disodium) and 150 mg of succinic acid (1.86% by weight ).
    [0212] - Fosfomycin for injection in bulk (“Fosfomycin BDP”): same mixture of dry powder of fosfomycin disodium and succinic acid, in the same proportion (1.86% by weight of succinic acid), but preserved in drums, not yet packed in vials sterile.
    [0213] - Pharmaceutical active ingredient of fosfomycin disodium ("Fosfomycin API")
    [0215] Equipment used
    [0217]
    [0220] HPLC method operating parameters:
    [0221] Mobile phase A: Acetonitrile
    [0222] Mobile phase B: Ammonium acetate 25 mM
    [0223] Flow rate: 0.8 ml / min
    [0224] Injection volume: 12 ^ l
    [0225] Column temperature: 50 ° C
    [0226] Autosampler temperature: 5 ° C
    [0227] EXP: Nebulizer temperature: 30 ° C
    [0228] Collection rate: 10 Hz
    [0229] Elution time: 50 min
    [0230] Gradient:
    [0233] Reference reagents / standards
    [0234] • High purity water (Milli-Q)
    [0235] • Acetonitrile, Fisher, Optima LC / MS grade
    [0236] • Ammonium acetate, Fisher, HPLC / ACS grade
    [0237] • Sample diluent: 1: 1 (v / v) mixture of mobile phase A: mobile phase B
    [0238] • Succinic acid, Sigma-Aldrich, BioXtra> 99.0%
    [0239] • Reference standard of fosfomycin disodium salt ("Fosfomycin RS") (Ercros) (total purity 98.8% by weight)
    [0241] Preparation of mobile phase B
    [0242] Mobile phase B is 25 mM ammonium acetate, pH is approximately 6.8, although pH measurement is not required. 3.9 g of ammonium acetate was added to a 2000 ml volumetric flask and water was added to a total volume of 2000 ml.
    [0244] Preparation of succinic acid solutions.
    [0245] A succinic acid stock solution ("20x succinic acid") was prepared as follows: 80 ± 1 mg of succinic acid was weighed into a 100 ml volumetric flask; Approximately 40 ml of mobile phase B were added and mixed until dissolved; then approximately 40 ml of mobile phase A (acetonitrile) was added and mixed; and finally a sufficient amount of sample diluent was added to a volume of 100 ml.
    [0247] The "succinic carrier" solution was prepared by transferring 0.5 ml of the 20x succinic acid solution to a 10 ml volumetric flask and adding sample diluent to a volume of 10 ml. The concentration of the succinic carrier solution was 0.04 mg / ml. The solution was stored at 5 ° C.
    [0249] Preparation and calibration of the fosfomycin disodium standard solution
    [0250] A standard solution of fosfomycin disodium ("Stock Solution") at 2.75mg / ml was prepared as follows: 55 ± 1mg of Fosfomycin RS were weighed into a 20 ml volumetric flask; 10 ml of mobile phase B were added and mixed until dissolved, then 10 ml of mobile phase A was added and mixed and finally a sufficient amount of sample diluent (1: 1 mixture of mobile phases A and B) was added until a volume of 20 ml. The solution was stored at 5 ° C.
    [0252] The molar concentration of fosfomycin disodium in the stock solution was 14.92 mM and was calculated taking into account the water content of Fosfomycin RS (0.03% by weight) and the total purity of Fosfomycin RS (98.8% by weight ).
    [0254] Calibration of the standard solution was performed in the range of 75% to 125% of the test concentration. Calibration was used to quantify the content of fosfomycin in the samples.
    [0256] A 100% linearity standard solution was prepared by diluting 4 ml of "Stock Solution" with sample diluent to 5 ml (final concentration 11.9 mM). The solution was stored at 5 ° C. Additional solutions were prepared as described in Table 1 (at 125%, the stock solution is used directly to prepare an HPLC sample):
    [0260]
    [0263] A calibration curve was created by plotting the peak area against the theoretical amount (mM) of active principle (fosfomycin) and impurity A. A second order polynomial equation was used to fit the data:
    [0264] F ( x) = c bx ax2
    [0265] Where a, b and c are calibration coefficients.
    [0266] Sample preparations
    [0267] To prepare the API sample of fosfomycin disodium (Fosfomycin API), 44 ± 1 mg of Fosfomycin API were weighed into a 20 ml volumetric flask, 10 ml of mobile phase B were added and stirred until dissolved, then 10 ml of mobile phase A and mixed, and finally a sufficient amount of sample diluent (1: 1 mixture of mobile phases A and B) was added to a volume of 20 ml. The solution was labeled "API Working Sample" and stored at 5 ° C.
    [0269] To prepare the sample of the pharmaceutical product fosfomycin (Fosfomycin DP), the contents of a vial of the powder drug, containing 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin disodium) and 150 mg of succinic acid (1 , 86% w / w) in mobile phase B up to a volume of 250 ml. 10 ml of this solution was transferred to a 150 ml volumetric flask, 10 ml of mobile phase A (acetonitrile) was added and the diluent solution was added to 150 ml. The sample solution was approximately 11.8 mM as pure fosfomycin free acid. It was labeled "DP Working Sample" and stored at 5 ° C.
    [0271] To prepare the bulk pharmaceutical product (BDP) (Fosfomycin BDP) sample, first 45 ± 1 mg of fosfomycin bulk pharmaceutical product was dissolved with 10 ml of mobile phase B in a 20 ml volumetric flask, then 10 ml of mobile phase A, and finally a sufficient amount of sample diluent was added to a volume of 20 ml. The solution was labeled "BDP Working Sample" and stored at 5 ° C.
    [0273] Results
    [0274] Fosfomycin was identified in the sample chromatograms when the retention time was within ± 5% of the average retention time of the fosfomycin peaks (n = 6) in the standard solution.
    [0276] Based on the calibration with the fosfomycin standard, the concentration in mM of the drug in unknown samples was calculated from known equations, starting from the area of the main peak, and using the dilution factor (DF) to determine the concentration (mM ) of fosfomycin in the original sample (sample stock solution):
    [0277] Equation 1: Fosfomycin concentration
    [0279] Where:
    [0280] a, b, c Coefficients defined by second-order polynomial calibration
    [0281] DF 1 for API
    [0282] DF 1 for Bulk Pharmaceutical Products (BDP)
    [0283] DF 15 for pharmaceutical product (DP)
    [0285] Equation 2: Amount of fosfomycin in finished pharmaceutical products
    [0286] . . / mg Free acid phosphomycin ( md) = Conc ( mM) x ( Vol.) x ( MW ------- -I v mmoi- ' Where:
    [0287] Conc. Concentration (mM) of fosfomycin free acid equivalents according to Equation 1
    [0288] Volume Volume of original DP working sample solutions (0.250 μl for DP)
    [0289] PM 138.06 mg / mmol (fosfomycin as free acid in DP)
    [0291] Equation 3: Amount of fosfomycin in bulk pharmaceuticals and API
    [0292] . . / mg Phosphomycin sodium ( md) = Conc ( mM) x ( Vol.) x ( PM ------- -I v mmoi- ' Where:
    [0293] Conc. = Concentration (mM) of fosfomycin disodium according to Equation 1
    [0294] Vol. = Volume of API and BDP working sample solutions (0.020 l for BDP and API)
    [0295] MW = 182.02 mg / mmol (fosfomycin as disodium salt in API and BDP)
    [0297] Equation 4: Percentage declared on the labeling of the finished pharmaceutical product Pure Observed Quantity Fosfomycinanion ( mg)
    [0298] % Labeled Declared x 100 Labeled Quantity Fosfomycin Anion ( mg)
    [0300] Equation 5: Expected amount of fosfomycin in BDP and API (corrected with% moisture and% purity)
    [0301] Expected phosphomycin disodium ( mg ) = Sample weight ( mg ) x / 100 - % moisture / 100 - % ac. succinic /% purity 100 ) X i 100 ) X i 100 ) Where% succinic acid is 1.86% for BDP and 0% for API
    [0303] Equation 6: Percentage of content for API and BDP
    [0305] % Content = - C - a - n - t - i - d - a - d --- P - u - r - a - O --- b - s - e - r - v - a - d - a --- F - o - s - f - o - m - i - c - inad a¡isqoóda¡icrna k ( m u q) j x 100
    [0306] Expected Disodium Phosphomyeline ( mg)
    [0307] Where:
    [0308] Observed Fosfomycin = Observed amount (mg) of fosfomycin disodium according to Equation 3
    [0309] Expected Fosfomycin = Expected Amount (mg) of fosfomycin disodium according to Equation 5
    [0311] For illustrative purposes, Figures 1 to 4 show some chromatograms obtained, in which the x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area):
    [0312] - Diluent (25 mM ammonium acetate in acetonitrile, 50:50, v / v) (Figure 1) The following peaks are shown: 15,968 and 26,779 (trimer).
    [0313] - Succinic vehicle solution (0.040 mg / ml of succinic acid) (Figure 2). The following peaks are shown: 14,593 (pre-peak 1), 19,460 (succinic acid) and 26,767 (trimer).
    [0314] - 100% Fosfomycin RS (Figure 3). The following peaks are shown: 13,618 (pre-peak 2), 14,594 (pre-peak 1), 15,336 (fosfomycin), 18,199 (impurity A), 20,984 (post-peak 1) and 21,250 (post-peak 2) and 26,746 (trimer).
    [0315] - 100% Fosfomycin DP (Figure 4). The following peaks are shown: 10,447 (pre-peak_10RT), 11,124, 11,633 (pre-peak_11RT), 13,105 (pre-peak 3), 13,606 (pre-peak 2), 14,554 (pre-peak 1), 15,365 (fosfomycin) , 18.165 (impurity A), 19.389, 19.854, 21.005, 21.238 (impurity_21RT), 21.859, 22.969, 23.778 and 26.757 (trimer).
    [0317] Example 2: HILIC-HPLC method with CAD detection: quantification of impurities and degradation products in fosfomycin samples
    [0318] Equipment, HPLC operating parameters, reagents, reference standards, mobile phase A and B, and sample diluent were the same as described in Example 1. The only differences were that, in this case, the CAD detector used was specifically Corona Veo and the injection volume was 25 ^ l, instead of 12 ^ l.
    [0320] Additionally, in this example, the following additional reference standard was used:
    [0321] • Reference standard for impurity A (Impurity A RS): P - [(1R, 2R) -1,2-dihydroxypropyl)] - phosphonic acid, ammonium salt (Toronto Research Chemicals). C3H7O5P (NH3) 2; MW = 190.14 (total purity 94.6% by weight).
    [0323] Succinic acid solutions ("20x succinic acid" and "succinic vehicle" solutions) were prepared analogously to that described in Example 1.
    [0325] Thus, for the preparation of the "20x succinic acid" solution, 84 ± 1 mg of succinic acid were weighed into a 100 ml volumetric flask, 40 ml of mobile phase B were added and mixed until dissolved, then 40 ml of mobile phase A and mixed, and finally a sufficient amount of sample diluent was added up to 100 ml.
    [0327] To prepare the "succinic carrier" solution, 0.5 ml of "20x succinic acid" solution was added to a 10 ml volumetric flask and sufficient amount of sample diluent was added to 10 ml.
    [0329] The fosfomycin samples analyzed were:
    [0330] - Injectable Fosfomycin ("Fosfomycin DP"): vials containing a sterile dry powder mixture of 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin disodium) and 150 mg of succinic acid (1.86% by weight).
    [0331] - Fosfomycin for injection in bulk ("Fosfomycin BDP"): same mixture of dry powder of fosfomycin disodium and succinic acid, with the same proportion (1.86% by weight of succinic acid), but without packaging in sterile vials.
    [0332] - Pharmaceutical active ingredient of fosfomycin disodium ("Fosfomycin API")
    [0333] In this Example, non-sterile fosfomycin API samples, which had a higher content of impurities, were also analyzed as a fingerprint for the impurity peak mapping.
    [0335] Preparation and Calibration of Impurity A Standard Solution
    [0336] A stock standard solution of Impurity A ("Impurity Stock A") was prepared at 11 mM. In a 20 ml volumetric flask, 47 ± 1 mg of ammonium salt of impurity A were dissolved of fosfomycin, first adding 10 ml of mobile phase B until dissolved, then adding 10 ml of mobile phase A and finally adding a sufficient amount of sample diluent to a volume of 20 ml. The solution was labeled "Impurity Stock A" and stored at 5 ° C.
    [0338] The molar concentration of impurity A in the stock standard solution of impurity A was 10.98 mM, and it was calculated taking into account the water content of the impurity A RS (6.1% by weight) and the total purity of the impurity A RS (94.6% by weight).
    [0340] A working standard solution of impurity A ("LL Estd. 2") was prepared by diluting 2.0 ml of the stock solution of impurity A with sample diluent to 100 ml (final concentration 0.230 mM). The solution was stored at 5 ° C.
    [0342] From the Impurity A working standard ("LL Estd. 2"), low concentration linearity standards (LL Estd.) Were prepared using sample diluent as described in Table 2. A calibration was used. based on these standards to quantify the content of impurities in the samples, both as impurity A and other unknown impurities.
    [0346]
    [0349] LL Estd were used. 0.5, 0.10, 0.20, and 0.50 for five-point calibration on Impurity A peak responses at low concentration, representing peak area versus theoretical concentration (mM).
    [0351] Sample preparations
    [0352] API (Fosfomycin API) samples were prepared analogously to that described in Example 1, labeled "API Working Sample" and stored at 5 ° C.
    [0353] Analogously, an "API resolution sample" solution was prepared, but using non-sterile API, which had higher levels of impurities and was used to select columns based on resolution and as a fingerprint for peak assignment. of impurities.
    [0355] To prepare the sample of the pharmaceutical product fosfomycin (Fosfomycin DP), a vial containing 6 g of fosfomycin (corresponding to 7.92 g of fosfomycin sodium) and 150 mg of succinic acid (1.86% w / w) was opened and They dissolved 45 ± 1 mg of Fosfomycin DP by first adding 10 ml of mobile phase B to a 20 ml volumetric flask, then 10 ml of mobile phase A, and finally adding sample diluent to 20 ml. It was labeled "DP Working Sample" and stored at 5 ° C.
    [0357] Fosphomycin BDP samples were prepared as described in Example 1, and the solution was labeled "BDP Working Sample" and stored at 5 ° C.
    [0359] Denomination of the peaks
    [0360] The naming of the peaks was based on the LC / MS / MS impurity peaks identification study of Example 3. The naming of the peaks was slightly different for API and for DP and BDP because the latter contained succinic acid, therefore that there were some impurity peaks related to succinic acid in DP and BDP, but not in API.
    [0362] The non-sterile API samples were high in most impurities (see Figure 7) and could be used as a fingerprint for additional guidance for the assignment of impurity peaks.
    [0364] The peaks that could not be identified were labeled "unknown" with RT and RRT.
    [0366] Results
    [0367] The identification / designation of the impurity peaks and other important peaks for the Fosfomycin API sample are shown in Table 3.
    [0368]
    [0370] The identification / designation of the impurity peaks and other main peaks of Fosfomycin DP and Fosfomycin BDP are shown in Table 4:
    [0371]
    [0372]
    [0373] The retention time of impurity A in the sample solutions was ± 5% of the mean retention time of the impurity A peaks (n = 6) in the standard solutions.
    [0375] A calibration curve was created by plotting the peak area versus the theoretical concentration (mM) of impurity A in the low concentration calibration (levels include 0%, 0.05%, 0.10%, 0.20% and 0.50%). A second order polynomial equation was used:
    [0376] F ( x) = c bx ax2
    [0377] Where a, b and c are calibration coefficients.
    [0378] Based on calibration with the impurity A standard, the concentration in mM of each impurity in an unknown sample could be calculated from the peak area of each impurity, taking into account the dilution factor (DF) to determine the concentration ( mM) of each impurity in the original sample (sample stock solution), using the following formula:
    [0382] Where a, b, c are coefficients defined by the second order polynomial calibration.
    [0384] The content of each unknown impurity in the original sample could be calculated using the following formula (this calculation assumed that all impurities have the same response factor as impurity A):
    [0385] . . / mg
    [0386] Impurity Amount ( mq) = Conc ( mM) x ( Vol.) X I PM ------- -)
    [0387] v mmo or b V '
    [0388] Where:
    [0389] Conc. Concentration (mM) of each impurity from the calibration of the Impurity A standard
    [0390] Volume Volume of original working sample solutions (0.020 μl for DP, BDP and API samples)
    [0391] PM 154.06 mg / mmol (Impurity A as free acid)
    [0393] For API samples, the amount of dry fosfomycin is calculated based on the measured water content, according to the following formula:
    [0394] Amount Fosfomycin dry ( mg) = API sample weight ( mg) x
    [0395] 100 Water sample to pi ! .38.06
    [0397] For DP and BDP samples, the amount of dry fosfomycin can be calculated according to the following formula:
    [0398] Amount Fosfomycin dry ( mg ) = Sample weight DP ( mg ) x
    [0399] 100 - Water sample AP¡ 100 - 1.86 138.06
    [0401] Where 1.86 is the percentage of succinic acid excipient in DP / BDP formulations and 138.06 / 182.02 is the weight fraction of fosfomycin free acid in the fosfomycin disodium sample.
    [0402] The individual weight percent of each impurity and the weight percent of all impurities in the API, BDP, and DP samples were calculated relative to the amount of dry fosfomycin free acid, using the following formulas:
    [0403] Amount Impurity ( mg)
    [0404] % Impurity = -------------------- --------------------- - ---- - x 100
    [0405] Amount Fosfomycin, dry (mg)
    [0407] y Amount of Impurity ( mg)
    [0408] % Total Impurities = -------- -—----------------------------- - ---- - x 100 Amount Fosfomycin, dry (mg)
    [0410] For illustrative purposes, some representative chromatograms are shown in the attached figures 5 to 8, in which the x-axis represents the retention time (in minutes) and the y-axis indicates the detector response (peak area):
    [0411] - Fosfomycin disodium DP (Figure 5). The following peaks are shown: 10,763 (ethanol adduct), 11,021 (saturated olefinic acid ethanol adduct), 13,652 (olefinic acid), 14,480 (methanol adduct), 15,402 (fosfomycin), 18,060 (impurity A), 19,519 (succinic 1), 19.974 (succinic 2), 20.707 (dimer 1), 20.936 (dimer 2), 22.589 (sodium), and 26.319 (trimer).
    [0412] - Impurity A RS at 2% (Figure 6). The following peaks are shown: 14,316, 15,757 (fosfomycin) and 17,927 (impurity A).
    [0413] - API (non-sterile) and DP (aged) fosfomycin disodium samples (Figure 7). The following peaks are shown in the first chromatogram: 10,132, 10,426 (ethanol adduct), 11,450 (saturated olefinic acid ethanol adduct), 11,913 (saturated olefinic acid), 13,316 (related to olefinic acid), 13,798 (olefinic acid) , 14,727 (methanol adduct), 15,357 (fosfomycin), 18,196 (impurity A), 20,270 (olefinic acid adduct methanol adduct), 21,363 (dimer 1), 21,533 (dimer 2), 22,167 (dimer 3), 22,413 ( dimer 4), 23,264 (dimer 5), 23,872 (dimer 6), 24,460, 26,532 (trimer 1), 27,431 (trimer 2), 27,721 (trimer 3), and 28,382 (trimer 4). In the second chromatogram, the following peaks are shown: 10,515 (ethanol adduct), 13,431 (related to olefinic acid), 13,833 (olefinic acid), 14,742 (methanol adduct), 15,357 (fosfomycin), 18,297 (impurity A), 19,073 (succinic acid 1), 19,342 (succinic acid 2), 20,315 (methanol adduct olefinic acid adduct), 21,250 (dimer 1), 21,530 (dimer 2), 22,361 (dimer 3 / dimer 4), 23,262 (dimer 5 succinic acid adduct), 24,027 (dimer 6) , 24,460, 26,896 (trimer 1).
    [0414] - Samples of Fosfomycin disodium API and BDP (Figure 8). In the first chromatogram, the following peaks are shown: 10,768 (ethanol adduct), 10,993 (saturated olefinic acid ethanol adduct), 13,583 (olefinic acid), 14,392 (methanol adduct), 15,368 (fosfomycin), 17,942 (impurity A ), 20,544 (dimer 1), 20,760 (dimer 2), 22,488 (succinic acid adduct dimer 5), 23,137 (dimer 6), 23,628, 26,095 (trimer 1). In the second chromatogram, the following peaks are shown: 10,777 (ethanol adduct), 10,994 (ethanol adduct), 13,575 (olefinic acid), 14,434 (methanol adduct), 15,368 (fosfomycin), 17,933 (impurity A), 19,442 (succinic acid) 1), 19,720 (succinic acid 2), 20,524 (dimer 1), 20,765 (dimer 2), 21,636 (dimer 3), 22,490 (dimer 5 succinic acid adduct), 23,135 (dimer 6), 23,628 and 26.057 (trimer 1).
    [0416] Example 3: HPLC-CAD-MS for peak identification
    [0417] Identification of impurity peaks was performed using HPLC in conjunction with a charged aerosol detector (CAD) and mass spectrometry (MS).
    [0419] The samples analyzed were:
    [0420] - Pharmaceutical product fosfomycin (Fosfomycin DP) after 48 months of accelerated stability at 30 ° C;
    [0421] - Aged sample of fosfomycin API (manufactured in April 2013)
    [0423] These samples were expected to provide a complete profile of impurity peaks.
    [0425] A Waters Q-Tof Premier mass spectrometer was used.
    [0427] Reagents, reference standards, and HPLC equipment and conditions were the same as described in Example 2.
    [0429] In addition, the following mass spectrometer calibration solutions were used: - Leucine enkephalin (1 ng / ^ l)
    [0430] - Sodium formate (10% formic acid: 0.1 M NaOH: acetonitrile in a ratio of 1: 1: 8)
    [0432] A T-shaped mixer was applied as a bypass valve immediately after the HPLC column. The post-column flow was then divided 1: 2 towards the MS and the CAD, respectively. The ratio was adjusted using different lengths of PEEK (polyether ether ketone) tubing. A longer length resulted in a lower flow velocity towards the MS, and a shorter length resulted in a higher flow velocity towards the CAD. Although these different tube lengths result in different retention times in the two detectors, the relative retention time (RRT) of each peak relative to fosfomycin was used to compare the MS and CAD results.
    [0434] The ionization and fragmentation conditions were developed using direct injection of the pharmaceutical product fosfomycin. The cone, capillary and collision voltages were varied at 30 second intervals and the spectra were examined for each condition. The final values for the cone and capillary voltages were chosen to achieve the highest intensity without fragmenting the original molecules. Final method parameters are detailed in Table 5 (setting page parameters) and Table 6 (MS method parameters):
    [0442] The mass spectrometer was calibrated in negative resolution mode for fosfomycin analysis. Leucine enkephalin was used as the standard and sodium formate as the calibration solution.
    [0444] Sample preparations
    [0445] For the preparation of the API sample, 44 ± 1 mg of fosfomycin API were dissolved in a 20 ml volumetric flask with 10 ml of mobile phase B, then 10 ml of mobile phase A were added and finally a sufficient amount of diluent was added. up to 20 ml.
    [0447] To prepare the sample of the pharmaceutical product Fosfomycin (DP), 22.5 ± 0.5 mg of fosfomycin DP was dissolved in a 10 ml volumetric flask using 5 ml of diluent under sonication and adding additional diluent up to 10 ml.
    [0449] Results
    [0450] The CAD and TIC chromatograms were performed with an aged sample of the pharmaceutical product Fosfomycin (DP) and a sample of Fosfomycin API and were labeled in their absolute retention times. Based on the relative retention times and the chromatographic profiles, the peaks observed in the CAD chromatogram matched the peaks observed in MS TIC.
    [0451] The identification of each peak was carried out based on the data of the mass / charge ratio (m / z) and on the fragments of collision-induced dissociation (CID), according to known procedures in mass spectrometry.
    [0453] A summary of the observed peaks and the identified substances is listed in Table 7 for Fosfomycin DP and Table 8 for Fosfomycin API.
    [0455] The first column (ID) is the identification of the substance, RT is the retention time (min), RRT is the relative retention time of each peak in relation to Fosfomycin, m / z is the mass / charge ratio and the Last column shows the structure of each identified substance. In some cases (marked with an asterisk "*" in the tables) more than one isomeric structure was possible, with the same mass and the same fragments:
    [0459]
    [0460]
    [0461]
    [0462]
    [0465] Most of the impurities were considered to be process impurities from the synthesis process.
    [0467] Example 4: Stability analysis of the injectable fosfomycin pharmaceutical product using the analytical method of the invention
    [0468] As part of a stability study of the injectable fosfomycin pharmaceutical product, impurity analysis was performed according to the method of the present invention, using the HILIC-HPLC method with CAD detection, as described, for example, in Example 2 .
    [0470] The sample subjected to the stability study was injectable fosfomycin, that is, vials containing a sterile dry powder mixture of 7.92 g of fosfomycin disodium and 150 mg of succinic acid.
    [0472] The samples were stored at 25 ° C and 60% RH. The impurity analysis was performed at the following points in time: 0, 1, 3, 6, 9 and 12 months. The results of the impurity analysis are shown in Table 9. Impurities are reported in a percentage by weight> 0.05% (limit of detection of the method). "Dtd." in the table it means "detected", which means that the impurity is detected but is below the detection limit (0.05% by weight). ND means "not detected".
    [0473]
    权利要求:
    Claims (17)
    [1]
    1. Method for the detection and quantification of fosfomycin and its impurities and / or degradation products in samples of fosfomycin or a pharmaceutically acceptable salt thereof or in pharmaceutical compositions comprising fosfomycin or a pharmaceutically acceptable salt thereof, characterized in that understands:
    a) subjecting the sample to hydrophilic interaction liquid chromatography (HILIC) with gradient elution using as mobile phase a mixture of acetonitrile (mobile phase A) and an aqueous solution of ammonium acetate (mobile phase B); and
    b) detect and quantify fosfomycin, impurities and / or degradation products separated in step a) using a charged aerosol detector (CAD).
    [2]
    2. Method according to claim 1, characterized in that the samples contain fosfomycin in the form of the disodium salt of fosfomycin.
    [3]
    3. Method according to claims 1 or 2, characterized in that a stationary phase of the zwitterionic type is used.
    [4]
    4. Method according to any one of claims 1 to 3, characterized in that the mobile phase B is an aqueous solution of ammonium acetate with a concentration between 10 and 40 mM, preferably with a concentration of approximately 25 mM.
    [5]
    Method according to any one of claims 1 to 4, characterized in that the elution time of the mobile phase is between 45 and 60 minutes, and is preferably approximately 50 minutes.
    [6]
    6. Method according to any one of claims 1 to 5, characterized in that the flow rate of the mobile phase is between 0.7 and 1 ml / min, and is preferably approximately 0.8 ml / min.
    [7]
    Method according to claims 5 or 6, characterized in that the gradient elution is such that the ratio between the mobile phases A: B changes from approximately 85:15 at time 0 to approximately 40:60 at approximately 35 min and returns to change to about 85:15 at the end of the elution time.
    [8]
    Method according to claim 7, characterized in that the 85:15 ratio between the mobile phases A: B is kept constant for approximately 3 to 8 minutes at the beginning of the elution and / or for approximately 8 to 15 minutes at the end of the elution. . .
    [9]
    Method according to claims 7 or 8, characterized in that a constant ratio between the mobile phases A: B of approximately 40:60 remains constant for approximately 3 minutes from 35 minutes to 38 minutes.
    [10]
    10. Method according to any of claims 7 to 9, characterized in that the gradient expressed as the ratio of the mobile phases A: B is: 85:15 (0 min), 85:15 (5.0 min), 40:60 (35.0 min), 40:60 (38.0 min), 85:15 (38.1 min) and 85:15 (50.0 min).
    [11]
    Method according to any of claims 1 to 10, characterized in that the identification of the impurities and / or degradation products of fosfomycin is carried out by means of mass spectrometry (MS).
    [12]
    12. Method according to any of claims 1 to 11, characterized in that it refers to the detection and quantification of fosfomycin and its impurities and / or degradation products in a sample of fosfomycin or of a pharmaceutically acceptable salt of the same active principle, preferably in a sample of the active substance fosfomycin disodium.
    [13]
    13. Method according to any of claims 1 to 11, characterized in that it refers to the detection and quantification of fosfomycin and its impurities and / or degradation products in a sample of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof , preferably fosfomycin disodium, as active principle and at least one pharmaceutically acceptable excipient; preferably, the pharmaceutical composition consists essentially of fosfomycin disodium and succinic acid as an excipient.
    [14]
    14. Manufacturing process of fosfomycin or a pharmaceutically acceptable salt thereof with a specified degree of purity, characterized in that it comprises the following steps:
    (i) providing a batch of fosfomycin or a pharmaceutically acceptable salt thereof; (ii) quantifying fosfomycin impurities and / or degradation products in a sample from the batch of step (i) using the method of any of claims 1 to 11; and (iii) validate the batch only if the percentage of impurities in the sample meets the specified degree of purity.
    [15]
    15. Manufacturing process of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity, characterized in that it comprises the following steps:
    (i) providing a batch of a pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient;
    (ii) quantifying fosfomycin impurities and / or degradation products in a sample from the batch of step (i) using the method of any of claims 1 to 11; and
    (iii) validate the batch only if the percentage of impurities meets the required purity requirements.
    [16]
    16. Fosfomycin or a pharmaceutically acceptable salt thereof having a specified degree of purity, prepared by the process of claim 14.
    [17]
    17. Pharmaceutical composition comprising fosfomycin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient having a specified degree of purity, prepared by the process of claim 15.
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    同族专利:
    公开号 | 公开日
    WO2020187644A1|2020-09-24|
    ES2784076B2|2021-04-12|
    CA3132408A1|2020-09-24|
    ES2784076A1|2020-09-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US9441053B2|2013-07-11|2016-09-13|Scinopharm Taiwan, Ltd.|Analytical method for detecting sulfated oligosaccharides|
    GB201417341D0|2014-10-01|2014-11-12|Isis Innovation|Chromatography|KR102353452B1|2020-12-23|2022-01-21|한국과학기술원|Respiratory Aerosol Detection Device|
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    PCT/EP2020/056327|WO2020187644A1|2019-03-21|2020-03-10|Method for the detection and quantification of fosmomycin, impurities and degradation products thereof|
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