Method for detecting pivalic acid in tebipenem pivoxil and tebipenem pivoxil granules

Abstract

The invention belongs to the field of pharmaceutical analysis, and discloses a method for detecting pivalic acid in tebipenem pivoxil and tebipenem pivoxil granules, which is characterized in that high performance liquid chromatography is adopted for detection, octadecylsilane chemically bonded silica is used as a chromatographic column of a filler, 0.015-0.035 mol / L phosphate buffered solution with the pH value of 2.5-3.5 is used as a mobile phase A, the mobile phase B is used as a mobile phase C, the mobile phase C is used as a mobile phase D, the mobile phase D is used as a mobile phase C, and the mobile phase D is used as a mobile phase D; an organic solvent is used as a mobile phase B, gradient elution is carried out, a detector is an ultraviolet detector, the detection wavelength is 210-230 nm, and the column temperature is 25-35 DEG C. The detection method provided by the invention can effectively detect the content of the pivalic acid in the tebipenem pivoxil and the tebipenem pivoxil granules, and the method has the advantages of strong specificity, high sensitivity and accuracy, good repeatability and durability, simple operation and reliable result, and is suitable for detection and quantitative control of the pivalic acid in the tebipenem pivoxil and the tebipenem pivoxil granules.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a method for detecting pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules. Background Art

[0002] Tipipenam pivoxil, chemical name is (+)-(4R, 5S, 6S)-6-[(lR)-hydroxyethyl]-4-methyl-7-oxo-3[[1-(2-thiazolin-2-yl)-3-azetidinyl]sulfur]-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (2,2-dimethyl-1-oxopropoxy)methyl ester, belongs to carbapenem antibiotics, tipipenam pivoxil was first developed by Pfizer of the United States, tipipenam pivoxil granules were developed by Meiji Co., Ltd. of Japan, approved by Japan in February 2009, and launched in April 2009. Tipipenam pivoxil granules are composed of tipipenam pivoxil as main ingredient, and microcrystalline cellulose, hydroxypropyl cellulose, sucrose, pigments, etc. as auxiliary materials. Tipopenem has a broad antibacterial spectrum. For most clinically isolated strains, Tipopenem shows stronger antibacterial activity than penicillin series and cephalosporin series. Compared with other injectable carbapenem antibiotics, Tipopenem also shows the same or stronger antibacterial effect. In particular, it shows a very strong antibacterial effect against penicillin-resistant Streptococcus pneumoniae, erythromycin-resistant Streptococcus pneumoniae and Haemophilus influenzae, which are the main causes of childhood infection.

[0003] China attaches great importance to the control of antibiotic impurities. Pivalic acid is one of the degradation impurities of tebipenem pivoxil granules. In order to ensure the safety and effectiveness of tebipenem granules, pivalic acid needs to be tested. The determination method of pivalic acid in tebipenem pivoxil granules has not been reported in domestic and foreign literature.

[0004] Therefore, in order to accurately monitor the amount of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, it is urgent to find a simple, specific and accurate high performance liquid analysis method that can achieve qualitative and quantitative analysis of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules. Summary of the invention

[0005] The purpose of the present invention is to establish a high performance liquid phase analysis method for determining pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, which can achieve qualitative and accurate quantitative analysis of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules. The method has strong specificity, high sensitivity and accuracy, good repeatability and durability, and stable method.

[0006] To achieve the purpose of the present invention, the inventors conducted a large number of experiments and finally obtained the following specific scheme:

[0007] A method for detecting pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules by high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are:

[0008] Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler;

[0009] Mobile phase: composed of mobile phase A and mobile phase B, mobile phase A is phosphate buffer, mobile phase B is acetonitrile, methanol or one or more; wherein the concentration of phosphate in the buffer is 0.015-0.035 mol / L, and the pH is adjusted to 2.5-3.5; gradient elution, the volume ratio of mobile phase A to mobile phase B is 40-90:10-60;

[0010] Column temperature: 25℃~35℃;

[0011] Flow rate: 0.8ml / min~1.2ml / min;

[0012] Detection wavelength: 210nm~230nm;

[0013] Injection volume: 30~70μl.

[0014] Preferably, in the above chromatographic conditions, the octadecylsilane bonded silica gel chromatographic column is a Themo Hypersil GoldaQ, 4.6×100 mm, 3.5 μm or an Agilent TC-C18, 4.6×150 mm, 5 μm chromatographic column.

[0015] Preferably, in the above chromatographic conditions, the mobile phase A buffer is sodium dihydrogen phosphate buffer, the concentration of the sodium dihydrogen phosphate buffer is 0.025 mol / L, and phosphoric acid is used to adjust the pH of the solution to 3.0.

[0016] Preferably, in the above chromatographic conditions, mobile phase B is acetonitrile.

[0017] Preferably, the mobile phase gradient elution condition in the above chromatographic conditions is: by volume ratio

[0018] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0019] Preferably, the column temperature in the above chromatographic conditions is 30°C.

[0020] Preferably, the flow rate of the mobile phase in the above chromatographic conditions is 1.0 ml / min.

[0021] Preferably, the detection wavelength in the above chromatographic conditions is 220 nm.

[0022] Preferably, the injection volume in the above chromatographic conditions is 50 μl.

[0023] Specifically, the chromatographic conditions of the liquid chromatography method of the present invention are:

[0024] Chromatographic column: chromatographic column with octadecylsilane bonded silica as filler (Themo Hypersil Gold aQC18, 4.6×100mm, 3.5μm);

[0025] Mobile phase: Mobile phase A (0.025 mol / L sodium dihydrogen phosphate buffer solution, pH adjusted to 3.0 with phosphoric acid) and mobile phase B (acetonitrile), gradient elution. Gradient elution conditions: by volume ratio,

[0026] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0027] Column temperature: 30°C;

[0028] Flow rate: 1.0ml / min;

[0029] Detection wavelength: 220nm;

[0030] Injection volume: 50 μl

[0031] The method of the present invention is a method for detecting pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules by high performance liquid chromatography, and the specific operating steps are as follows:

[0032] a. Take an appropriate amount of pivalic acid reference substance, weigh it accurately, dissolve it in 30% acetonitrile water and quantitatively dilute it to make a solution with a concentration of about 10 μg / ml as the reference substance solution;

[0033] b. Take an appropriate amount of tebipenem pivoxil or tebipenem pivoxil granules (equivalent to 40 mg of tebipenem), accurately weigh, place in a 10 ml volumetric flask, add an appropriate amount of acetonitrile, dissolve by ultrasonication under ice bath, dilute to scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, and take the filtrate as the test solution;

[0034] c. Take 50 μl of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph to complete the analysis and detection of pivalic acid in the tipipenam pivoxil granules according to the following conditions:

[0035] The chromatographic conditions are:

[0036] The chromatographic column was Themo Hypersil Gold aQ C18, 4.6 × 100 mm, 3.5 μm;

[0037] The mobile phase was a 0.025 mol / L sodium dihydrogen phosphate buffer solution (pH adjusted to 3.0 with phosphoric acid) and acetonitrile for gradient elution;

[0038] The column temperature was 30°C;

[0039] Flow rate: 1.0 ml / min;

[0040] The detection wavelength is 220nm;

[0041] In terms of volume ratio, the gradient elution was set as follows:

[0042]

[0043]

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The analytical detection method of the present invention can effectively detect the content of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, and the method has good repeatability and durability, low detection limit, simple operation, stable and reliable results, and can be used for controlling pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, providing effective guarantee for the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 HPLC chromatogram of blank solvent

[0047] Figure 2 HPLC chromatogram of blank excipient

[0048] Figure 3 HPLC chromatogram of pivalic acid reference solution

[0049] Figure 4 HPLC chromatogram of the mixed solution of pivalic acid reference substance and tebipenem ester granules

[0050] Figure 5 Example 2 Pivalic acid reference solution HPLC chromatogram

[0051] Figure 6 Example 2 HPLC chromatogram of a mixed solution of pivalic acid reference substance and tipipenam ester granules

[0052] Figure 7 Comparative Example 1 Pivalic Acid Reference Solution HPLC Chromatogram

[0053] Figure 8 HPLC chromatogram of blank auxiliary material solution of comparative example 1

[0054] Fig. 9 Comparative Example 2 Pivalic Acid Reference Solution HPLC Chromatogram DETAILED DESCRIPTION

[0055] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention in detail, but it should not be understood that the present invention is limited to the following implementation cases within the scope of the above-mentioned subject. Without departing from the above-mentioned technical premise of the present invention, the corresponding replacement or modification made according to the common technical knowledge and customary means in the field are all included in the present invention.

[0056] Example 1

[0057] 1. Instrument and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18, (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer solution (adjusted to pH 3.0 with phosphoric acid) and acetonitrile as mobile phase, gradient elution; column temperature 30°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0058] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0059] 2. Test steps:

[0060] 2.1 Exclusivity

[0061] 1) Blank solvent interference test

[0062] Take an appropriate amount of 30% acetonitrile water, filter, take the filtrate as the blank solution, measure according to the above chromatographic conditions, record the chromatogram, HPLC spectrum see Figure 1 .

[0063] Depend on Figure 1 It can be seen that there is no impurity peak in the blank solvent that interferes with the detection of pivalic acid.

[0064] 2) Blank excipient solution interference test

[0065] Take an appropriate amount of the excipient of the tebipenem pivoxil granules (equivalent to about 40 mg of tebipenem), accurately weigh it, place it in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve it by ultrasonication under an ice bath, dilute it to the scale with 30% acetonitrile water, shake it well, centrifuge it, take the supernatant, filter it, and take the filtrate as the blank excipient solution. Determine it according to the above chromatographic conditions, record the chromatogram, and the HPLC spectrum is shown in Figure 2 .

[0066] Depend on Figure 2 It can be seen that there is no impurity peak in the blank excipient solution that interferes with the detection of pivalic acid.

[0067] 3) Pivalic acid reference solution

[0068] Take an appropriate amount of pivalic acid reference substance and quantitatively dilute it with 30% acetonitrile water to make a solution containing about 20 μg / ml as the reference substance solution. Measure according to the above chromatographic conditions and record the chromatogram. The HPLC spectrum is shown in Figure 3 .

[0069] Depend on Figure 3 It can be seen that the retention time of pivalic acid is 12.057 min.

[0070] 2.2 Mixed solution of pivalic acid reference substance and tipipenam ester granules

[0071] Take an appropriate amount of tebipenem pivoxil granules (equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve by ultrasound under ice bath, add pivalic acid reference solution, dilute to scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, take the filtrate as a mixed solution of pivalic acid reference and tebipenem pivoxil granules. Accurately measure 50 μl and inject into liquid chromatograph, record the chromatogram, HPLC spectrum is attached Figure 4 .

[0072] Depend on Figure 4 It can be seen that the retention time of pivalic acid is 12.029min, the separation degree between pivalic acid and adjacent impurity peaks is 4.37, and the retention time of tebipenem pivoxil is 21.207, which meets the detection requirements.

[0073] 2.3 Limit of quantification and limit of detection

[0074] 1) Quantitative limit solution: Take an appropriate amount of pivalic acid reference substance, weigh accurately, dissolve in 30% acetonitrile water and quantitatively dilute to make a solution with a concentration of about 1.50 μg / ml as the quantitative limit solution. Measure according to the above chromatographic conditions and record the chromatogram.

[0075] The retention time of pivalic acid is 12.150 min, and the S / N is 12.9.

[0076] 2) Detection limit solution: Take an appropriate amount of pivalic acid reference substance, weigh accurately, dissolve in 30% acetonitrile water and quantitatively dilute to make a solution with a concentration of about 0.60 μg / ml as the detection limit solution. Measure according to the above chromatographic conditions and record the chromatogram.

[0077] The retention time of pivalic acid is 12.080 min, and the S / N is 4.9.

[0078] 2.4 Linearity and range tests

[0079] Take an appropriate amount of pivalic acid reference substance, quantitatively dilute with 30% acetonitrile water to prepare 1.5μg / ml, 4.0μg / ml, 8.0μg / ml, 10.0μg / ml, 20.0μg / ml, 50.1μg / ml, 100.2μg / ml series of solutions, inject respectively, measure according to the above chromatographic conditions, record the chromatogram, and obtain the linear equation. The results are shown in Table 1.

[0080] Table 1 Pivalic acid linearity and range test results

[0081]

[0082] It can be seen from Table 1 that the linear relationship between the concentration and the peak area of ​​pivalic acid is good within the range of 1.5 to 100.2 μg / ml.

[0083] 2.5 Repeatability test

[0084] Take an appropriate amount of pivalic acid reference substance, quantitatively dilute with 30% acetonitrile water to prepare solutions with concentrations of 4.0 μg / ml, 10.0 μg / ml, and 50.0 μg / ml, prepare 3 portions for each concentration, measure according to the above chromatographic conditions, record the chromatogram, measure the peak area, and calculate the relative standard deviation. The results are shown in Table 2.

[0085] Table 2 Pivalic acid precision test results

[0086]

[0087]

[0088] It can be seen from Table 2 that at low, medium and high concentrations, the RSD of the peak area of ​​pivalic acid was less than 2.0%, indicating that the method had good precision.

[0089] 2.6 Accuracy test

[0090] Take an appropriate amount of tebipenem pivoxil granules (equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve by ultrasonication under ice bath, add about 40 μg of pivalic acid reference substance, shake well, dilute to scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, take the filtrate as the low concentration solution of recovery rate. Prepare three copies in the same way.

[0091] Take an appropriate amount of tebipenem pivoxil granules (equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve by ultrasonication in an ice bath, add about 100 μg of pivalic acid reference substance, shake well, dilute to the scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, take the filtrate as the medium concentration solution of recovery rate. Prepare three copies in the same way.

[0092] Take an appropriate amount of tebipenem pivoxil granules (equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve by ultrasonication in an ice bath, add about 500 μg of pivalic acid reference substance, shake well, dilute to the scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, take the filtrate as a high-concentration solution with a recovery rate. Prepare three copies in the same way.

[0093] Take an appropriate amount of tebipenem pivoxil granules (approximately equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric flask, add an appropriate amount of acetonitrile, sonicate in an ice bath to dissolve, dilute to the scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, and take the filtrate as the blank correction solution.

[0094] Take an appropriate amount of pivalic acid reference substance, weigh it accurately, dissolve it in 30% acetonitrile water and quantitatively dilute it to make a solution with a concentration of about 10 μg / ml as the reference substance solution.

[0095] The above chromatographic conditions were used for determination, the chromatogram was recorded, the peak area was measured, and the recovery was calculated by the external standard method. The results are shown in Table 3.

[0096] Table 3 Pivalic acid test accuracy test results

[0097]

[0098] It can be seen from Table 3 that this method has high accuracy.

[0099] 2.7 Durability test

[0100] Durability test solution:

[0101] Durability test reference solution: Take an appropriate amount of pivalic acid reference substance and quantitatively dilute it with 30% acetonitrile water to prepare a solution containing about 10 μg / ml as the reference solution.

[0102] Durability test test solution: Take an appropriate amount of tipipenam pivoxil granules (equivalent to 40 mg of tipipenam pivoxil), accurately weigh, put into a 10 ml volumetric flask, add an appropriate amount of acetonitrile, dissolve by ultrasonic treatment under ice bath, shake well, dilute to the scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, and take the filtrate as the test solution.

[0103] 1) Durability flow rate change

[0104] ① Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 30°C, flow rate 0.9ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0105] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0106] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0107] Under this condition, the retention time of pivalic acid was about 13.181 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.024%.

[0108] ②Instrument and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 30°C, flow rate 1.1ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0109] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0110] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0111] Under this condition, the retention time of pivalic acid was about 12.838 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.025%.

[0112] 2) Durability column temperature change

[0113] ① Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 25°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0114]

[0115]

[0116] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0117] Under this condition, the retention time of pivalic acid was about 13.399 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.025%.

[0118] ②Instrument and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 35°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0119] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0120] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0121] Under this condition, the retention time of pivalic acid was about 12.634 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.025%.

[0122] 3) Durability of mobile phase buffer pH changes

[0123] ① Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 2.8 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 30°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0124]

[0125]

[0126] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0127] Under this condition, the retention time of pivalic acid was about 12.912 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.025%.

[0128] ②Instrument and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ C18 (4.6×100mm, 3.5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.2 with phosphoric acid) and acetonitrile as mobile phase, gradient elution, column temperature 30°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0129] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0130] Determination: Accurately measure 50 μl of the durability test reference solution and the durability test test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the pivalic acid content detected in the test sample by the external standard method.

[0131] Under this condition, the retention time of pivalic acid was about 13.235 min, and there was no impurity peak interfering with the detection of pivalic acid in the chromatogram of the test solution. The content of pivalic acid in the test sample was 0.022%.

[0132] The durability results are summarized in Table 4.

[0133] Table 4 Pivalic acid durability test results

[0134]

[0135]

[0136] It can be seen from Table 4 that this method has good durability.

[0137] Example 2

[0138] Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Agilent TC-C18 (4.6×150mm, 5μm); 0.025mol / L sodium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid)-acetonitrile as mobile phase, gradient elution, column temperature 30°C, flow rate 1.0ml / min, detection wavelength 220nm, injection volume 50μl. In terms of volume ratio, the gradient elution settings are:

[0139] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 70 30 3 89 11 14 89 11 16 40 60 26 40 60 27 70 30 40 70 30

[0140] 2. Test steps:

[0141] Experiment: 1) Take an appropriate amount of pivalic acid reference substance and quantitatively dilute it with 30% acetonitrile water to make a solution containing about 20μg / ml as the reference substance solution. Accurately measure 50μl and inject it into the liquid chromatograph to record the chromatogram. The HPLC spectrum is attached. Figure 5 .

[0142] Depend on Figure 5 It can be seen that the retention time of pivalic acid is 12.755 min, and there is no impurity peak that interferes with the detection of pivalic acid.

[0143] 2) Take an appropriate amount of tebipenem pivoxil granules (equivalent to 40 mg of tebipenem pivoxil), accurately weigh, place in a 10 ml volumetric flask, add an appropriate amount of acetonitrile, dissolve by ultrasound under ice bath, add pivalic acid reference solution, dilute to scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, take the filtrate as a mixed solution of pivalic acid reference and tebipenem pivoxil granules. Accurately measure 50 μl and inject into liquid chromatograph, record the chromatogram, HPLC spectrum is attached Figure 6 .

[0144] Depend on Figure 6 It can be seen that the retention time of pivalic acid is 12.906 min, the separation degree between pivalic acid and adjacent impurity peaks is 4.02, and the retention time of tipiperam pivoxil is 22.075 min, which meets the detection requirements.

[0145] From the above test results, it can be seen that the detection method of the present invention can effectively detect the content of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, and the blank solvent and blank excipients have no interference in its detection. Therefore, this method can be used for the quantitative analysis of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules.

[0146] Comparative Example 1

[0147] 1. Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Agilent TC-C18 (4.6×250 mm, 5 μm); mobile phase A is 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B is acetonitrile, gradient elution according to the table below; flow rate is 1.0 ml per minute; column temperature is 30°C; detection wavelength is 220 nm; injection volume is 20 μL.

[0148] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 75 25 15 75 25 15.1 40 60 49 40 60 49.1 75 25 60 75 25

[0149] 2. Test steps:

[0150] experiment:

[0151] 1) Pivalic acid reference solution

[0152] Take an appropriate amount of pivalic acid reference substance and quantitatively dilute it with 30% acetonitrile water to make a solution containing about 20 μg / ml as the reference substance solution. Measure according to the above chromatographic conditions and record the chromatogram. The HPLC spectrum is shown in Figure 7 .

[0153] Depend on Figure 7 It can be seen that the retention time of pivalic acid is 11.752 min.

[0154] 2) Blank excipient solution interference test

[0155] Take an appropriate amount of the excipient of the tebipenem pivoxil granules (equivalent to about 40 mg of tebipenem), accurately weigh it, place it in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve it by ultrasonication under an ice bath, dilute it to the scale with 30% acetonitrile water, shake it well, centrifuge it, take the supernatant, filter it, and take the filtrate as the blank excipient solution. Determine it according to the above chromatographic conditions, record the chromatogram, and the HPLC spectrum is shown in Figure 8 .

[0156] Depend on Figure 8 It can be seen that the blank excipient solution has a large excipient peak at 11.755min, which interferes with the detection of pivalic acid. This method is not suitable for the determination of pivalic acid content in tebipenem pivoxil and tebipenem pivoxil granules.

[0157] Comparative Example 2

[0158] 1. Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Themo Hypersil GoldaQ (4.6×250mm, 5μm); mobile phase A is 0.05mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B is acetonitrile, gradient elution according to the table below; flow rate is 1.0ml per minute; column temperature is 30℃; detection wavelength is 210nm; injection volume is 20μL.

[0159]

[0160]

[0161] 2. Test steps:

[0162] experiment:

[0163] Take an appropriate amount of pivalic acid reference substance and quantitatively dilute it with 30% acetonitrile water to make a solution containing about 20 μg / ml as the reference substance solution. Measure according to the above chromatographic conditions and record the chromatogram. The HPLC spectrum is shown in Fig. 9 .

[0164] Depend on Fig. 9 It can be seen that the retention time of pivalic acid is 8.474min, which is located at the inflection point of the mobile phase gradient, and pivalic acid cannot be accurately integrated. This method is not suitable for the determination of pivalic acid content in tebipenem pivoxil and tebipenem pivoxil granules.

[0165] The experimental results show that the detection method of the present invention can effectively detect the content of pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, and the method has high separation, good repeatability and durability, simple operation, stable and reliable results, and can be used for the quantitative analysis of pivalic acid in tipipenam pivoxil and tipipenam granules.

Claims

1. A method for detecting pivalic acid in tipipenam pivoxil and tipipenam pivoxil granules, characterized in that: The detection method is to use high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase: consists of mobile phase A and mobile phase B, mobile phase A is phosphate buffer, mobile phase B is acetonitrile, methanol or one or more; wherein the concentration of the buffer substance in the buffer is 0.015-0.035 mol / L, and the pH is adjusted to 2.5-3.

5.

2. The detection method according to claim 1, characterized in that The mobile phase A buffer is sodium dihydrogen phosphate buffer.

3. The detection method according to claim 2, characterized in that: The mobile phase B is acetonitrile.

4. The detection method according to claim 3, characterized in that: The mobile phase gradient elution conditions are: by volume ratio, 。 5. The detection method according to claim 4, characterized in that: The chromatographic conditions of the described high performance liquid chromatography are: Column temperature: 25℃~35℃; Flow rate: 0.8ml / min~1.2ml / min; Detection wavelength: 210nm~230nm; Injection volume: 30~70μl.

6. The detection method according to claim 1, characterized in that: The octadecylsilane bonded silica gel chromatographic column is a Themo Hypersil Gold aQ C18 or Agilent TC-C18 chromatographic column.

7. The detection method according to claim 1, characterized in that: The concentration of the sodium dihydrogen phosphate buffer solution is 0.025 mol / L, and phosphoric acid is used to adjust the pH of the solution to 3.

0.

8. The detection method according to claim 4, characterized in that: The chromatographic conditions of the described high performance liquid chromatography are: Column temperature: 30°C; Flow rate: 1.0ml / min; Detection wavelength: 220nm; Injection volume: 50 μl.

9. The detection method according to any one of claims 1 to 8, characterized in that: The operating steps of the described high performance liquid phase method are as follows: a. Take an appropriate amount of pivalic acid reference substance, accurately weigh it, dissolve it and quantitatively dilute it to make a solution as the reference substance solution; b. Take an appropriate amount of tebipenem pivoxil or tebipenem pivoxil granules, accurately weigh them, put them in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve them by ultrasonic treatment under ice bath, dilute to the scale with acetonitrile water, shake well, centrifuge, take the supernatant, filter, and take the filtrate as the test solution; c. Separately inject the reference solution and the test solution into the high performance liquid chromatograph to complete the analysis and detection of pivalic acid in tipipenam pivoxil granules.

10. The detection method according to claim 9, characterized in that: The operating steps of the described high performance liquid phase method are as follows: a. Take an appropriate amount of pivalic acid reference substance, weigh it accurately, dissolve it in 30% acetonitrile and quantitatively dilute it to make a solution with a concentration of about 10 μg / ml as the reference substance solution; b. Take an appropriate amount of tebipenem pivoxil or tebipenem pivoxil granules, weigh accurately, place in a 10 ml volumetric bottle, add an appropriate amount of acetonitrile, dissolve by ultrasonication under ice bath, dilute to scale with 30% acetonitrile water, shake well, centrifuge, take the supernatant, filter, and take the filtrate as the test solution; c. Take 50 μl of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph to complete the analysis and detection of pivalic acid in the tipipenam pivoxil granules according to the following conditions.

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