Method for determining related substances in fosfomycin sodium by using hydrogen nuclear magnetic resonance spectrum

Abstract

The present invention discloses a method for determining related substances in fosfomycin sodium by using nuclear magnetic resonance hydrogen spectroscopy. The method adopted by the present invention is nuclear magnetic resonance hydrogen spectroscopy, preferably using 4-bromopyridine hydrochloride as an internal standard, more preferably using 4-bromopyridine hydrochloride with a mass fraction of 90% as an internal standard, and selecting D 2 O is used as a deuterated solvent. The specific steps of determining the related substances in fosfomycin sodium in the present invention include: (1) accurately weighing a certified batch sample of fosfomycin sodium and an internal standard, dissolving them in a deuterated solvent, and obtaining a test solution; (2) accurately measuring the test solution and transferring it to an NMR tube, performing a nuclear magnetic resonance hydrogen spectrum test, and recording a spectrum. The conditions of the nuclear magnetic resonance hydrogen spectrum test include: a Bruker DRX300 nuclear magnetic resonance instrument equipped with a 5 mm multi-nuclear "QNP" probe; a spectrum width of 3500 Hz; a temperature of 296 K; a pulse of 30°; an acquisition time of 4.7 s; a deuterated solvent: D 2 The determination method of the present invention is simple, easy to implement, highly accurate, and highly specific, and can better control impurities in the sample and ensure the sample quality.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining related substances in fosfomycin sodium by utilizing nuclear magnetic resonance hydrogen spectrum. Background Art

[0002] Fosfomycin is a phosphate derivative in structure. It was discovered and developed by Merck & Co., Inc. of the United States and CEPA of Spain in 1967 from Streptomyces in Spanish soil. It is mainly used for Gram-negative and Gram-positive bacterial infections.

[0003] Fosfomycin sodium and calcium salts were launched in Europe in 1975; Meiji products were launched in Japan in 1980; fosfomycin butane triol was launched in Europe in 1988; and in the United States in 1996. The main dosage form of fosfomycin is fosfomycin sodium powder injection, which is suitable for drip and intravenous injection. Other dosage forms include fosfomycin calcium tablets, capsules, syrup, etc. At present, there are four main varieties of fosfomycin, namely fosfomycin sodium, fosfomycin calcium, fosfomycin tromethamine, and fosfomycin benzylamine salt.

[0004] The patent application with publication number CN110887925A discloses a method for determining related substances of fosfomycin sodium. The method uses a chromatographic column with octadecylsilane bonded silica gel as a filler and a UV detector to determine four impurities in fosfomycin sodium: sodium propylene acid, sodium cis-propylene phosphate, 2,3-allyl phenylethylamine phosphate and sodium trans-propylene phosphate.

[0005] The patent with authorization announcement number CN102841155B discloses a method for detecting related substances in fosfomycin sodium by high performance liquid chromatography. The method uses an aminopropyl-bonded silica gel column and a differential refractometer to determine (1,2-dihydroxypropyl)phosphonic acid and other impurities in fosfomycin sodium.

[0006] The above two HPLC methods for determining fosfomycin sodium-related substances have good versatility and specificity for specific impurities and are easy to operate, but still have the following defects: ① Chromatographic separation loss: the commonly used ODS chromatographic column loses water-soluble components with high polarity during the separation process; ② The analysis time is relatively long.

[0007] Therefore, it is necessary to develop an analytical method that is convenient, time-saving, low-loss, and highly accurate for effectively detecting fosfomycin impurities.

[0008] In recent years, nuclear magnetic resonance is the most powerful spectroscopic tool for determining unknown structures of organic and biological samples. Today, industrial chemical research and quality control require the participation of nuclear magnetic resonance. Nuclear magnetic resonance has a fast detection speed and is not limited by the state of the sample. Gas, liquid and solid can all be analyzed by nuclear magnetic resonance. NMR can not only make a qualitative assessment of the sample, but also quickly quantify all the components present. Excellent experimental precision, accuracy, specificity, linearity, low detection limit and durability can be easily achieved with extremely short sample preparation time. Therefore, NMR is obviously superior to chromatography in terms of sensitivity, speed, precision and accuracy, and there is no need to establish a reference substance for each analytical analyte. Summary of the invention

[0009] The technical problem that the invention can solve is: quickly and efficiently determining the content of related substances in fosfomycin sodium.

[0010] In view of this, the object of the present invention is to provide a method for determining related substances in fosfomycin sodium using hydrogen nuclear magnetic resonance spectroscopy.

[0011] Among them, the chemical structural formula of fosfomycin sodium is shown as (i):

[0012]

[0013] To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0014] A method for determining related substances in fosfomycin sodium using hydrogen nuclear magnetic resonance spectroscopy, the method comprising the following steps:

[0015] Step 1: Preparation of test solution: accurately weigh the certified batch sample of fosfomycin sodium and the internal standard, dissolve them in a deuterated solvent to obtain the test solution;

[0016] Step 2: Nuclear magnetic resonance hydrogen spectrum test: accurately measure the test solution and transfer it to an NMR tube, perform nuclear magnetic resonance hydrogen spectrum test, and integrate the characteristic peaks of the internal standard proton and each component respectively;

[0017] Step 3: Use the formula Calculate the content of the related substances in fosfomycin sodium in mg;

[0018] Among them, m A : unknown mass number of the substance A; m B : known mass of the internal standard;

[0019] P B : abundance of internal standard;

[0020] M A : molecular weight of the analyte A; M B : Molecular weight of the internal standard;

[0021] I A : The integrated area of ​​the selected signal of the object A under test; I B : integrated area of ​​the selected signal of the internal standard;

[0022] N A : The number of protons corresponding to the integral signal of the analyte A; N B : The number of protons corresponding to the integrated signal of the internal standard;

[0023] Preferably, the related substances include sodium (1Z)-prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate, and sodium (1,2-dihydroxypropyl)phosphonate.

[0024] Preferably, the internal standard is 4-bromopyridine hydrochloride, more preferably, the internal standard is 4-bromopyridine hydrochloride with a mass fraction of 90%, and the 4-bromopyridine hydrochloride with a mass fraction of 90% is purchased from Aldrich.

[0025] Preferably, the deuterated solvent is D2O. The D2O is purchased from Cambridge Isotope Laboratories.

[0026] Preferably, the conditions for the nuclear magnetic resonance hydrogen spectrum test include: BrukerDRX300 nuclear magnetic resonance instrument equipped with a 5mm multi-nuclear "QNP" probe; spectrum width: 3500Hz; temperature: 296K; pulse: 30°; acquisition time: 4.7s.

[0027] In the present invention, the chemical structural formula of the sodium (1Z)-prop-1-en-1-ylphosphonate is shown in (ii), the chemical structural formula of the sodium propylphosphonate is shown in (iii), the chemical structural formula of the sodium 2-propenylphosphonate is shown in (iv), and the chemical structural formula of the sodium (1,2-dihydroxypropyl)phosphonate is shown in (v):

[0028]

[0029] In the present invention, in order to ensure the specificity of the related substances in each analyte, i.e., fosfomycin sodium, during the determination process, 4-bromopyridine hydrochloride is preferably used as the internal standard, and 4-bromopyridine hydrochloride with a mass fraction of 90% is further preferably used as the internal standard to ensure that each analyte has a clear structural attribution.

[0030] In the present invention, in order to avoid the interference of hydrogen atoms in common solvents such as water, methanol, etc. on the nuclear magnetic resonance hydrogen spectrum measurement, a deuterated solvent is used as the solvent used in the present invention, and the preferred deuterated solvent is D2O or CD3OD.

[0031] In the present invention, the abundance of the internal standard is determined by glycine (NMR frequency: 300 MHz, deuterated solvent: D2O) (3.5 ppm singlet peak) with a mass fraction of 98%. 1 H NMR spectrum Figure 1 As shown. Applicability of internal standards: Figure 1 The mass fraction of 4-bromopyridine hydrochloride as internal standard is shown at 90%. 1 H-NMR, whose resonance occurs in the chemical shift range of 7.75-8.75 ppm, includes 13 C satellite peaks, this region does not interfere with possible impurities of fosfomycin sodium, including aromatic compounds such as toluene.

[0032] In the present invention, the integrated signals of fosfomycin sodium and related substances in fosfomycin sodium are assigned as follows:

[0033] Fosfomycin sodium shows three multiplets at 1.41, 2.76 and 3.21 ppm with relative intensities of 3:1:1. The doublet at a chemical shift of 1.41 ppm corresponds to a methyl group and has a coupling constant with a neighboring proton of 5.56 Hz; the doublet at a chemical shift of 2.76 ppm represents protons generated from phosphorus and implies two coupling constants, one with its neighboring protons (5.17 Hz) and the other with phosphorus atom (18.54 Hz); the third multiplet at a chemical shift of 3.21 ppm corresponds to protons generated from methyl group and is an obvious sextet involving three coupling constants, among which: ①: neighboring protons of methyl group (5.56 Hz); ②: ortho-epoxy protons (5.17 Hz); ③: phosphorus atom (5.40 Hz). The integral numbers corresponding to fosfomycin sodium in Table 1 are 3 and 7.

[0034] Although fosfomycin sodium is 99.3% pure, NMR technology can detect the following impurities:

[0035] (1): Sodium propylphosphonate (PP). The certified sodium fosfomycin sample already contains a small amount of sodium propylphosphonate, which shows a triplet peak at a chemical shift of 0.88 ppm and a triplet peak at a chemical shift of about 1.40 ppm. The latter is indistinguishable because it coincides with the signal of the main component at 1.41 ppm. Therefore, the triplet peak with a chemical shift of 0.88 ppm (integral number 1 in Table 1) is used to quantify sodium propylphosphonate.

[0036] (2): Sodium (1Z)-prop-1-en-1-ylphosphonate (i.e., cis-olefin acid). Multiple peaks at chemical shifts of 1.82, 5.65, and 6.00 ppm (relative intensities of 3:1:1, respectively) are attributed to this component and correspond to ①: methyl; ②: olefin protons paired with phosphorus atoms; ③: olefin protons protonated by methyl groups, respectively. The signal at chemical shift of 1.82 ppm is a doublet-doublet-doublet with coupling constants of 1.59, 3.41, and 7.04 Hz; the signal at chemical shift of 5.65 ppm is a doublet-doublet-quartet with coupling constants of 1.59, 13.04, and 18.88 Hz; the last signal at chemical shift of 6.00 ppm is a distinct doublet-sextet with coupling constants of 7.04, 13.04, and 47.04 Hz.

[0037] The coupling constant of 13.04 Hz maintained by the two olefin protons ensures the Z or cis configuration of the double bond. The integral numbers in Table 1 corresponding to sodium (1Z)-prop-1-en-1-ylphosphonate (ie, cis-olefin acid) are 4, 11, and 12.

[0038] (3): Sodium 2-propenylphosphonate (i.e., terminal olefin). This compound shows multiplets at chemical shifts of 2.35 (doublet-doublet), 5.09, and 6.10 ppm, which coincide with the lowest field signal of sodium (1Z)-prop-1-en-1-ylphosphonate at chemical shift of 6.10 ppm. Therefore, the first two multiplets with integral numbers 6 and 10 in Table 1 were used for quantification. It should be noted that the multiplet at chemical shift of 2.35 ppm is close to the 13 The C satellite peak is to the right but can be discerned quite well.

[0039] (4): Sodium (1,2-dihydroxypropyl)phosphonate (ie, diol). The diol has multiple peaks at chemical shifts of 1.40 and 4.00 ppm, the latter being the only identifiable polyol in the region of integral number 9 in Table 1.

[0040] Beneficial effects of the present invention:

[0041] (1) The related substances in fosfomycin sodium do not need to be separated during the determination process, which simplifies the pretreatment time of fosfomycin sodium samples before determination.

[0042] (2) The determination of related substances in fosfomycin sodium by hydrogen nuclear magnetic resonance spectroscopy has the advantages of rapid determination and simple operation. It will not cause damage to the fosfomycin sodium sample during the determination, and the determination process can be completed without the need for its own reference substance.

[0043] (3) In the process of determining related substances in fosfomycin sodium by hydrogen nuclear magnetic resonance spectroscopy, various substances in fosfomycin sodium can be determined simultaneously, and the reproducibility of each substance is good and the specificity is strong. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : The internal standard is 90% 4-bromopyridine hydrochloride and 98% glycine. 1 HNMR spectrum;

[0045] Figure 2 : The blank control example in the present invention 1 H NMR spectrum;

[0046] Figure 3 : Embodiment 1 of the present invention 1 H NMR spectrum;

[0047] Figure 4 : Embodiment 2 of the present invention 1 H NMR spectrum;

[0048] Figure 5 : Embodiment 3 of the present invention 1 H NMR spectrum;

[0049] Figure 6 : Embodiment 4 of the present invention 1 H NMR spectrum;

[0050] Figure 7 : Embodiment 5 of the present invention 1 H NMR spectrum;

[0051] Figure 8 : Embodiment 6 of the present invention 1 H NMR spectrum;

[0052] Fig. 9 :Propylphosphonic acid (400MHz, CD3OD) 1 H NMR spectrum;

[0053] Fig.10 :(1Z)-prop-1-en-1-ylphosphonic acid (300MHz, CDCl3) 1 H NMR spectrum;

[0054] Fig.11 :2-Propylenephosphonic acid (400MHz, CD3OD) 1 H NMR spectrum;

[0055] Fig.12 :Sodium (1,2-dihydroxypropyl)phosphonate (300MHz, D2O) 1 H NMR spectrum. DETAILED DESCRIPTION

[0056] The present invention can be further understood by the specific examples of the present invention given below, but they are not intended to limit the present invention.

[0057] Blank control example

[0058] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample I of fosfomycin sodium comprises the following steps:

[0059] Step 1: Preparation of test solution: accurately weigh 15 mg of fosfomycin sodium certified batch sample I and dissolve it in 0.5 ml D2O to obtain the test solution;

[0060] Step 2: H NMR spectrum test: 0.5 ml of the test solution was transferred to an NMR tube, and a Bruker DRX300 NMR instrument equipped with a 5 mm multi-nuclear "QNP" probe was used. The spectrum width was 3500 Hz, and the offset relative to the residual HDO was about 0 Hz. At 296 K, 30° pulse and 4.7 s acquisition time, 16K data points of 264 transient numbers were collected, and the spectrum was recorded. The characteristic peaks of each component were integrated and the relative percentage was calculated.

[0061] This blank control example 1 H NMR spectrum Figure 2 shown.

[0062] Embodiment 1

[0063] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample II of fosfomycin sodium comprises the following steps:

[0064] Step 1: Preparation of the first mixed solution: accurately weigh 470.5 mg of fosfomycin sodium sample II with a mass fraction of 99.3% and 190.1 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%, and dissolve them in 10 ml of D2O to obtain the first mixed solution;

[0065] Step 2: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution, add 10 ml of D2O to dilute, and obtain the test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0066] Step 3: Nuclear magnetic resonance hydrogen spectrum test: The test steps are the same as the blank control example. Record the spectrum. Integrate the characteristic peaks of the internal standard and each component respectively.

[0067] Step 4: Use the formula Calculate the content of related substances in fosfomycin sodium in mg;

[0068] Among them, m A : unknown mass number of the substance A; m B : known mass of the internal standard;

[0069] P B : abundance of internal standard;

[0070] M A : molecular weight of the analyte A; M B : Molecular weight of the internal standard;

[0071] I A : The integrated area of ​​the selected signal of the object A under test; I B : integrated area of ​​the selected signal of the internal standard;

[0072] N A : The number of protons corresponding to the integral signal of the analyte A; N B : The number of protons corresponding to the integrated signal of the internal standard;

[0073] The test substance A includes sodium (1Z)-prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate, and sodium (1,2-dihydroxypropyl)phosphonate.

[0074] In this Example 1, the amount of propylphosphonic acid with a mass fraction of 95% in the test solution is 0.00 mg.

[0075] This embodiment 1 1 H NMR spectrum Figure 3 shown.

[0076] Embodiment 2

[0077] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample III of fosfomycin sodium comprises the following steps:

[0078] Step 1: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution in Example 1, add 10 ml of D2O to dilute, and obtain a second mixed solution; add 0.1 ml of 20 ml of D2O solution containing 10.1 mg of propylphosphonic acid with a mass fraction of 95% to the second mixed solution to obtain a test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0079] The remaining steps are the same as those in the first embodiment.

[0080] In the test solution of Example 2, the amount of propylphosphonic acid with a mass fraction of 95% is 0.05 mg.

[0081] This embodiment 21 H NMR spectrum Figure 4 shown.

[0082] Embodiment 3

[0083] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample IV of fosfomycin sodium comprises the following steps:

[0084] Step 1: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution in Example 1, add 10 ml of D2O to dilute, and obtain a second mixed solution; add 0.2 ml of 20 ml of D2O solution containing 10.1 mg of propylphosphonic acid with a mass fraction of 95% to the second mixed solution to obtain a test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0085] The remaining steps are the same as those in the first embodiment.

[0086] In the test solution of Example 3, the amount of propylphosphonic acid with a mass fraction of 95% is 0.10 mg.

[0087] In this embodiment three 1 H NMR spectrum Figure 5 shown.

[0088] Embodiment 4

[0089] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample V of fosfomycin sodium comprises the following steps:

[0090] Step 1: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution in Example 1, add 10 ml of D2O to dilute, and obtain a second mixed solution; add 0.3 ml of 20 ml of D2O solution containing 10.1 mg of propylphosphonic acid with a mass fraction of 95% to the second mixed solution to obtain a test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0091] The remaining steps are the same as those in the first embodiment.

[0092] In the test solution of Example 4, the amount of propylphosphonic acid with a mass fraction of 95% is 0.14 mg.

[0093] The fourth embodiment of 1 H NMR spectrum Figure 6 shown.

[0094] Embodiment 5

[0095] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample VI of fosfomycin sodium comprises the following steps:

[0096] Step 1: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution in Example 1, add 10 ml of D2O to dilute, to obtain a second mixed solution, add 0.4 ml of 20 ml of D2O solution containing 10.1 mg of propylphosphonic acid with a mass fraction of 95% to the second mixed solution, to obtain a test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0097] The remaining steps are the same as those in the first embodiment.

[0098] In the test solution of Example 5, the amount of propylphosphonic acid with a mass fraction of 95% is 0.19 mg.

[0099] This embodiment 5 1 H NMR spectrum Figure 7 shown.

[0100] Embodiment 6

[0101] The determination of (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate and sodium (1,2-dihydroxypropyl)phosphonate in the certified batch sample VII of fosfomycin sodium comprises the following steps:

[0102] Step 1: Preparation of test solution: accurately measure 0.5 ml of the first mixed solution in Example 1, add 10 ml of D2O to dilute, and obtain a second mixed solution; add 0.5 ml of 20 ml of D2O solution containing 10.1 mg of propylphosphonic acid with a mass fraction of 95% to the second mixed solution to obtain a test solution (the test solution contains 23.36 mg of fosfomycin sodium and 8.55 mg of 4-bromopyridine hydrochloride with a mass fraction of 90%);

[0103] The remaining steps are the same as those in the first embodiment.

[0104] In the test solution of Example 6, the amount of propylphosphonic acid with a mass fraction of 95% is 0.24 mg.

[0105] This embodiment six 1 H NMR spectrum Figure 8 shown.

[0106] The spectrum integration results of the blank control example and the fosfomycin sodium samples of Examples 1 to 6 of the present invention are shown in Table 1.

[0107] As can be seen from the determination steps of the blank control example and Examples 1 to 6, the blank control example did not add 4-bromopyridine hydrochloride and propylphosphonic acid with a mass fraction of 90% of the internal standard, Example 1 did not add propylphosphonic acid, and Examples 2 to 6 added propylphosphonic acid with a mass fraction of 95%.

[0108] Propylphosphonic acid (NMR frequency: 400 MHz, deuterated solvent: CD3OD) 1 H NMR spectrum Fig. 9 As shown, (1Z)-prop-1-en-1-ylphosphonic acid (NMR frequency: 300 MHz, deuterated solvent: CDCl3) 1 H NMR spectrum Fig.10 As shown, 2-propenylphosphonic acid (NMR frequency: 400 MHz, deuterated solvent: CD3OD) 1 H NMR spectrum Fig.11 As shown, sodium (1,2-dihydroxypropyl)phosphonate (NMR frequency: 300 MHz, deuterated solvent: D2O) 1 H NMR spectrum Fig.12 shown.

[0109] The results of absolute quantitative determination (mg) of the components to be tested in the certified batch samples of fosfomycin sodium to which propylphosphonic acid with a mass fraction of 95% is added are shown in Table 2.

[0110] The results of absolute quantification (w / w%) of the components to be tested in the certified batch samples of fosfomycin sodium to which propylphosphonic acid with a mass fraction of 95% is added are shown in Table 3.

[0111] Table 1 Spectral integration results of fosfomycin sodium samples

[0112]

[0113] Table 2 Absolute quantification of the components to be tested in the certified batch sample of fosfomycin sodium with 95% propylphosphonic acid added (mg)

[0114]

[0115]

[0116] a Fosfomycin Sodium Certification Batch II was not considered. b 4-Bromopyridine hydrochloride with a mass fraction of 90% of the internal standard was not considered.

[0117] Table 3: Absolute quantification (w / w%) of the components to be tested in the certified batch of fosfomycin sodium spiked with 95% propylphosphonic acid and relative quantification of sample I

[0118]

[0119] a Fosfomycin sodium certified batch sample I and fosfomycin sodium certified batch sample II were not considered.

[0120] In Table 2 and Table 3: the cis-olefin acid is sodium (1Z)-prop-1-en-1-ylphosphonate, the terminal olefin is sodium prop-2-en-1-ylphosphonate, and the diol is sodium (1,2-dihydroxypropyl)phosphonate.

[0121] Table 2 lists the necessary parameters, which together with the integration of Table 1 and the equation, allow for the absolute quantification of the analyte (mg).

[0122] The lack of 4-bromopyridine hydrochloride with a mass fraction of 90% of the internal standard in the certified batch sample I of fosfomycin sodium made it impossible to absolutely quantify the different components, but relative quantification could be achieved. Table 3 contains the results and the absolute quantification of the certified batch samples II to VII of fosfomycin sodium. In this case, the results are expressed in w / w% instead of mg.

[0123] Table 2 shows that the measured absolute amount of fosfomycin sodium is 23.38±0.14 mg, which is consistent with the actual dosage of 23.36 mg in each sample. On the other hand, it can be seen from Table 3 that the average absolute amount w / w% of fosfomycin sodium certified batch samples II to VII containing 4-bromopyridine hydrochloride with an internal standard mass fraction of 90% is very similar to the relative amount w / w% of fosfomycin sodium certified batch sample I lacking 4-bromopyridine hydrochloride with an internal standard mass fraction of 90%, ensuring the applicability of 4-bromopyridine hydrochloride with an internal standard mass fraction of 90%.

[0124] Exclusive

[0125] Compared to other analytical techniques, QHNMR (Quantitative H Nuclear Magnetic Resonance) fully ensures specificity because the analyte exhibits specific resonances, giving it a clear structural assignment.

[0126] Accurate results from QHNMR (quantitative hydrogen nuclear magnetic resonance) spectroscopy depend on careful selection of analyte resonances to avoid overlapping resonances with impurities, and accurate measurement of NMR peak areas.

[0127] To obtain higher confidence, the purity assessment of fosfomycin sodium was based on its three resonances. When the data in Table 1 were converted to Tables 2 and 3, the signal at the chemical shift of 1.41 ppm overlapped with the multiplets corresponding to the four PP protons, and necessary area subtraction was performed.

[0128] According to the determination of Example 1 to Example 6, the NMR data of each analyte in fosfomycin sodium can be obtained, wherein the proton NMR data of (1Z)-prop-1-en-1-ylphosphonic acid is shown in Table 4, the proton NMR data of propylphosphonic acid is shown in Table 5, the proton NMR data of 2-propenylphosphonic acid is shown in Table 6, and the proton NMR data of sodium (1,2-dihydroxypropyl)phosphonate is shown in Table 7. Tables 4 to 7 are shown below:

[0129] (1Z)-Prop-1-en-1-ylphosphonic acid:

[0130] Table 4 Proton NMR data of (1Z)-prop-1-en-1-ylphosphonic acid

[0131] <![CDATA[δ 1 H(ppm)]]> Group <![CDATA[6.45( 3 J H,H =14.2Hz)( 2 J H,P =60.0Hz)]]> <![CDATA[Olefin CH attached to CH3]]> <![CDATA[5.60( 3 J H,H =14.2Hz)( 3 J H,P =20.0Hz)]]> Olefin CH attached to P atom 1.60 <![CDATA[CH3]]>

[0132] Propylphosphonic acid:

[0133] Table 5 Proton NMR data of propylphosphonic acid

[0134]

[0135] 2-Propylenephosphonic acid:

[0136] Table 6 Proton NMR data of 2-propenylphosphonic acid

[0137]

[0138] Sodium (1,2-dihydroxypropyl)phosphonate:

[0139] Table 7 Proton NMR data of sodium (1,2-dihydroxypropyl)phosphonate

[0140]

[0141] The above is only an implementation method of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining related substances in fosfomycin sodium using hydrogen nuclear magnetic resonance spectroscopy, characterized in that: The method comprises the following steps: Step 1: Preparation of test solution: accurately weigh the certified batch sample of fosfomycin sodium and the internal standard, dissolve them in a deuterated solvent to obtain a test solution; wherein the internal standard is 4-bromopyridine hydrochloride, and the deuterated solvent is D2O; Step 2: Nuclear magnetic resonance hydrogen spectrum test: accurately measure the test solution and transfer it to an NMR tube, perform nuclear magnetic resonance hydrogen spectrum test, and integrate the characteristic peaks of the internal standard proton and each component respectively; Step 3: Use the formula Calculate the content of the related substances in fosfomycin sodium, in mg; wherein the related substances include (1Z)-sodium prop-1-en-1-ylphosphonate, sodium propylphosphonate, sodium 2-propenylphosphonate, and sodium (1,2-dihydroxypropyl)phosphonate; Among them, m A : unknown mass number of the substance A; m B : known mass of the internal standard; P B : abundance of internal standard; M A : molecular weight of the analyte A; M B : Molecular weight of the internal standard; I A : The integrated area of ​​the selected signal of the object under test A; I B : integrated area of ​​the selected signal of the internal standard; N A : The number of protons corresponding to the integral signal of the analyte A; N B : The proton number corresponding to the integrated signal of the internal standard.

2. The method for determining related substances in fosfomycin sodium by hydrogen nuclear magnetic resonance spectroscopy according to claim 1, characterized in that: The internal standard substance is 4-bromopyridine hydrochloride with a mass fraction of 90%.

3. The method for determining related substances in fosfomycin sodium by hydrogen nuclear magnetic resonance spectroscopy according to any one of claims 1 to 2, characterized in that: The conditions for the nuclear magnetic resonance hydrogen spectrum test include: BrukerDRX300 nuclear magnetic resonance instrument equipped with a 5mm multi-nuclear "QNP" probe; spectrum width: 3500Hz; temperature: 296K; pulse: 30°; acquisition time: 4.7s.

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