Method for simultaneously detecting taurosulfonamide and taurine in taurolidine by HPLC-CAD (high performance liquid chromatography-computer-aided design) method

Abstract

The invention provides a method for simultaneously detecting taurosulfonamide and taurine in taurolidine by an HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method, which adopts SIELC Primep 500 as a chromatographic column and ammonium formate aqueous solution (A)-acetonitrile (B) as a mobile phase, gradient elution is carried out, the concentration of the ammonium formate aqueous solution is 8-25mmol / L, the volume flow rate is 0.8-1.2 mL / min, the column temperature is 15-25 DEG C, the sample size is 8-12mu L, and the pH value of the mobile phase A is adjusted to 3-4 by formic acid; and the temperature of the CAD detector is 30-40 DEG C. According to the method, taurosulfonamide and taurine in taurolidine can be detected at the same time through an HPLC-CAD method, the method does not need a pretreatment process, direct determination is achieved, operation is easy, accuracy is high, stability and repeatability are good, and a new control means is provided for quality research of taurolidine.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis, and in particular to a method for simultaneously detecting tauramide and taurine in taurolidine using an HPLC-CAD method. Background Art

[0002] Taurolidine, whose chemical name is 4,4'-methylenebis[tetrahydro-2H-1,2,4-thiadiazine]1,1,1',1'-tetraoxide, has a molecular formula of C7H 16 N4O4S2 is in the form of white or off-white crystalline powder, and its chemical structure is as follows:

[0003]

[0004] Taurolidine has antibacterial, antifungal, antiendotoxin and anti-adhesion properties. In Europe, taurolidine has been used as an active ingredient in the lock solution for central venous catheters and intravenous indwelling needles to prevent catheter-related bloodstream infections (CRBSI) and maintain catheter patency.

[0005] The literature (Taurolidine Antiadhesive Properties on Interaction with E.coli; Its Transformation in Biological Environment and Interaction with Bacteria Cell Wall, PLoS One. 2010; 5(1): e8927) discloses that the anti-infection mechanism of taurolidine is related to its hydrolysis mechanism in aqueous solution; the research results of the literature (Electron and light microscopic observations of bacterial cell surface effects due to taurolidine treatment, Lett Appl Microbiol, 1987, 4(5): 103-109) show that the hydroxymethyl-containing substances produced during the hydrolysis of taurolidine can interact with the bacterial cell wall, thereby preventing the colonization of microorganisms and causing microbial apoptosis.

[0006] Taurolidine will have tauramide and taurine impurities generated during the synthesis and preparation process, which need to be strictly controlled. Its structural formula is shown below. However, tauramide and taurine impurities have no obvious ultraviolet characteristic absorption, and their quality control methods have great difficulties. At present, there are few reports on the analysis methods of tauramide and taurine in taurolidine at home and abroad. The methods reported in the existing literature mainly include thin layer chromatography, HPLC-UV method after pre-column derivatization, etc. However, there are still many shortcomings in the reported methods, such as low sensitivity of thin layer chromatography, complex pre-column derivatization operation, poor specificity, and inability to accurately measure.

[0007]

[0008] As a new type of high-sensitivity, universal, mass-spectrum detector, electrospray ionization detector (CAD) has the advantages of good repeatability, wide detection range, and high sensitivity compared with traditional evaporative light scattering and ultraviolet diode array detectors. Its detection signal does not depend on the chemical structure of the substance being detected, which provides a better choice for the quantitative detection of such low-content impurities.

[0009] Therefore, it is particularly important to establish a detection method that is simple to operate, highly accurate, stable and reproducible, and can simultaneously detect tauramide and taurine in taurolidine. Summary of the invention

[0010] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for simultaneously detecting tauramide and taurine in taurolidine by HPLC-CAD method. The method provided by the present invention does not require additional pretreatment process, directly determines, is simple to operate, has high accuracy, good stability and repeatability, and provides a new control means for the quality research of taurolidine.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] The invention provides a method for simultaneously detecting tauramide and taurine in taurolidine by HPLC-CAD method, which is characterized by adopting a SIELC Primesep 500 chromatographic column, taking an ammonium formate aqueous solution (A)-acetonitrile (B) as a mobile phase, gradient elution, a volume flow rate of 0.8-1.2 mL / min, a column temperature of 15-25 DEG C, an injection volume of 8-12 μL; a CAD detector temperature of 30-40 DEG C; and a procedure of the gradient elution as follows:

[0013]

[0014] 10≤t1≤18; 20≤t2≤30;

[0015] 10≤V A1 ≤20; 25≤VA2 ≤35.

[0016] Further, the procedure of the gradient elution is:

[0017]

[0018] 10≤t1≤18; 20≤t2≤30; 33≤t3≤37; t4=t3+n; 50≤t5≤60; n=0.5~2;

[0019] 10≤V A1 ≤20; 25≤V A2 ≤35;10≤V A3 ≤20.

[0020] Furthermore, the concentration of the ammonium formate aqueous solution is 8 to 25 mmol / L.

[0021] Furthermore, the concentration of the ammonium formate aqueous solution is 10 to 20 mmol / L.

[0022] Furthermore, the volume flow rate is 1.0 mL / min.

[0023] Furthermore, the CAD atomizer temperature is 35°C.

[0024] Furthermore, the method uses formic acid to adjust the pH value of the mobile phase A to 3-4.

[0025] Furthermore, the method uses formic acid to adjust the pH value of mobile phase A to 3.4.

[0026] Furthermore, the column temperature is 20°C.

[0027] Furthermore, the injection volume is 10 μL.

[0028] The method described is used for the quality control of taurolidine.

[0029] The most preferred technical solution is: using SIELC Primesep500 (4.6mm×150mm, 5μm) as a chromatographic column, using 10mmol / L ammonium formate aqueous solution (adjusting the pH value to 3.4 with formic acid) (A)-acetonitrile (B) as the mobile phase, gradient elution, flow rate of 1.0mL / min, column temperature of 20°C, CAD detector temperature of 35°C, injection volume of 10μL; the gradient elution program is:

[0030]

[0031] That is, t1 = 15; t2 = 25; t3 = 35; t4 = 36; t5 = 55; V A1 =15; VA2 =30; V A3 =15.

[0032] Taurine and taurine were both baseline separated and had good linear relationships.

[0033] The present invention has the following beneficial effects:

[0034] The HPLC-CAD method established by the present invention can simultaneously detect tauramide and taurine in taurolidine, and a specific gradient elution program is selected to significantly improve the accuracy of the detection. At the same time, the use of a CAD detector can achieve accurate determination at extremely low concentrations, and strict methodological verification is performed to ensure the scientific rigor of the method, meeting the needs of research and development and production. The method provided by the present invention does not require an additional pre-treatment process, is directly measured, is simple to operate, has high accuracy, and has good stability and repeatability, and provides a new control means for the quality research of taurolidine.

[0035] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the chromatogram of the system suitability test under the conditions of Example 1.

[0037] Figure 2 This is a chromatogram of the test solution determined using 10 mmol / L ammonium formate aqueous solution-acetonitrile as the mobile phase.

[0038] Figure 3 This is a chromatogram of the test solution determined using 20 mmol / L ammonium formate aqueous solution-acetonitrile as the mobile phase.

[0039] Figure 4 This is a chromatogram of the test solution measured with 10 mmol / L ammonium acetate aqueous solution-acetonitrile as the mobile phase. DETAILED DESCRIPTION

[0040] The following examples are intended to further illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the raw materials and reagents used are all commercially available products, and the methods and operating modes adopted are all conventional methods in the art.

[0041] Example 1

[0042] 1. Instruments and test drugs

[0043] Thermo Ultimate 3000 high performance liquid chromatograph-electrospray detector (Thermo Fisher, USA); Sielc Primesep 500 chromatographic column (4.6 mm × 150 mm, 5 μm; Sielc, USA); KQ-400DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XS205Du one-hundred-thousandth balance, XP56 one-millionth balance (METTLER TOLEDO, Switzerland).

[0044] Taurine amide reference substance (Jiangsu Aosaikang Pharmaceutical Co., Ltd., content 99.5%, batch number S20090901).

[0045] Taurine reference substance (China Food and Drug Inspection Institute, content 100.0%, batch number 111616-201605).

[0046] 2. Methods and Results

[0047] 2.1 Chromatographic conditions

[0048] Chromatographic column: Sielc Primesep 500 (150 mm × 4.6 mm, 5 μm)

[0049] Mobile phase: 10 mmol / L ammonium formate aqueous solution (adjust pH to 3.4 with formic acid) (A) - acetonitrile (B)

[0050] Flow rate: 1.0mL / min

[0051] Column temperature: 20℃

[0052] CAD atomizer temperature: 35℃

[0053] Injection volume: 10 μL

[0054] Elution method: gradient elution

[0055]

[0056]

[0057] 2.2 Solution preparation

[0058] 2.2.1 Preparation of test solution: Take an appropriate amount of taurolidine, dissolve and dilute it with acetonitrile-water mixed solution (80:20, v / v) to prepare a solution with a concentration of 0.8-1.1 mg taurolidine per 1 mL as the test solution;

[0059] 2.2.2 Preparation of reference solution: Accurately measure appropriate amount of taurine and taurine reference substances, dissolve in acetonitrile-water mixed solution (80:20, v / v) and dilute to prepare mixed reference stock solution containing 15.080 μg / mL and 14.789 μg / mL of taurine and 15.080 μg / mL of taurine, respectively; then dilute with acetonitrile-water mixed solution (80:20, v / v) to prepare a solution containing 1.5080 μg and 1.4789 μg of taurine per 1 mL, respectively, as reference solution;

[0060] 2.2.3 Preparation of system suitability test solution: Take an appropriate amount of taurolidine, add an appropriate amount of mixed reference substance stock solution, add acetonitrile-water mixed solution (80:20, v / v) to dissolve and dilute to make a solution containing 1 mg of taurolidine, 1.5080 μg of tauramide and 1.4789 μg of taurine per 1 mL as the system suitability test solution.

[0061] 2.3 Methodological validation

[0062] 2.3.1 System suitability test

[0063] Accurately measure 10 μL of blank solvent and system suitability solution, inject them into the HPLC, and measure according to the chromatographic conditions under "2.1". Record the chromatogram. The results show that the separation of taurolidine, tauramide and taurine peaks is not less than 1.5, and the theoretical plate number is not less than 8000. The blank solvent has no interference with the determination of the above three components. The results are shown in Figure 1 .

[0064] 2.3.2 Linear range test

[0065] Accurately measure an appropriate amount of the reference stock solution prepared under "2.2.2", dilute it with an acetonitrile-water mixed solution (80:20, v / v) to prepare a linear series of solutions with 5 different concentrations, and inject the samples according to the chromatographic conditions under "2.1" to determine the peak area. Use the injection concentration c (μg / mL) as the abscissa and the peak area A as the ordinate to perform linear regression to obtain the regression equation and linear range of each component. The results show that the components have a good linear relationship within the injection volume range. The results are shown in Table 1.

[0066] Table 1 Linearity and range test results

[0067]

[0068] 2.3.3 Determination of limit of quantification and limit of detection

[0069] Accurately measure an appropriate amount of the reference stock solution prepared under "2.2.2", dilute it step by step with an acetonitrile-water mixed solution (80:20, v / v), inject and measure according to the chromatographic conditions under "2.1", and record the peak area of ​​each chromatographic peak. Calculate the limit of quantification and the limit of detection according to the signal-to-noise ratio (S / N) of 10 and 3, respectively. The results showed that the limits of quantification of taurine and taurine were 0.2029μg / mL and 0.1991μg / mL, respectively, and the limits of detection of taurine and taurine were 0.0609μg / mL and 0.0597μg / mL, respectively.

[0070] 2.3.4 Precision test

[0071] Accurately measure the mixed reference solution prepared under "2.2.2" and inject 6 times continuously. Record the peak area of ​​each chromatographic peak. The RSDs of the peak areas of tauramide and taurine in the sample are 2.0% and 1.7%, respectively, indicating good injection precision.

[0072] 2.3.5 Stability test

[0073] The mixed reference solution and the test solution were placed at room temperature for 0, 2, 4, 6, 12, 24 and 48 hours, and then the samples were analyzed. The peak area at each time point was recorded. The RSD of the peak area of ​​taurine amide and taurine in the mixed reference solution was 2.2% and 3.6%, respectively; taurine amide and taurine were not detected in the test solution. The results showed that the reference solution and the test solution were stable at 25°C for 48 hours.

[0074] 2.3.6 Repeatability test

[0075] Take an appropriate amount of taurolidine (batch number: S20111917), add 1 mL of the mixed reference stock solution prepared under "2.2.2", add acetonitrile-water mixed solution (80:20, v / v) to dissolve and dilute to make a reproducible solution containing 1 mg, 1.508 μg and 1.4789 μg of taurolidine, tauramide and taurine per 1 mL, respectively.

[0076] Accurately measure 10 μL of blank solvent, reference solution and repeatability solution, respectively, and inject and measure according to the chromatographic conditions under "2.1", and record the chromatogram. Calculate the content and RSD of taurine amide and taurine. The results are shown in Table 2, indicating that the repeatability of this method is good.

[0077] Table 2 Repeatability test results

[0078]

[0079] 2.3.7 Accuracy test

[0080] Low concentration recovery solution (0.05%): Take about 100 mg of taurolidine (batch number: S20111917), add 3.5 mL of the mixed reference stock solution prepared under "2.2.2", place in a 100 mL volumetric flask, add acetonitrile-water mixed solution (80:20, v / v) to dissolve and dilute to the scale, shake well, and obtain.

[0081] Medium concentration recovery solution (0.15%): Take about 100 mg of taurolidine (batch number: S20111917), add 10 mL of the mixed reference stock solution prepared under "2.2.2", place in a 100 mL volumetric flask, add acetonitrile-water mixed solution (80:20, v / v) to dissolve and dilute to the scale, shake well, and obtain.

[0082] High concentration recovery solution (0.3%): Take about 100 mg of taurolidine (batch number: S20111917), add 20 mL of the mixed reference stock solution prepared under "2.2.2", place in a 100 mL volumetric flask, add acetonitrile-water mixed solution (80:20, v / v) to dissolve ice and dilute to the scale, shake well, and obtain.

[0083] Three solutions were prepared in parallel for each concentration, for a total of 9 solutions.

[0084] Accurately measure 10 μL of blank solvent, reference solution, test solution and recovery solution, respectively, and inject and measure according to the chromatographic conditions under "2.1", and record the chromatogram. Calculate the average recovery and RSD of taurine and taurine, and the results are shown in Table 3.

[0085] Table 3 Accuracy test results

[0086]

[0087] The results showed that the average recoveries of taurine amide and taurine (n=9) were 102.8% and 101.8%, respectively, and the RSDs were 3.4% and 2.2%, respectively. The accuracy of the method was good.

[0088] 2.4 Sample determination

[0089] Take appropriate amount of taurolidine from different batches and prepare test solutions according to the method under "2.2.1". Accurately measure 10 μL of blank solvent, reference solution and test solution, respectively, and inject them into HPLC instrument, and measure according to the chromatographic conditions under "2.1". Record the chromatogram and calculate the content of tauramide and taurine in the sample by external standard method. The results are shown in Table 4.

[0090] Table 4 Sample measurement results

[0091]

[0092] 2.5 Chromatographic Condition Optimization Process

[0093] 2.5.1 Using 10mmol / L ammonium formate aqueous solution-acetonitrile as the mobile phase, the test solution was determined using the gradient elution conditions in Table 5. Other determination conditions were the same as those in Item "2.1". The determination results are shown in Tables 6 and Figure 2 .

[0094] Table 5 Gradient elution conditions with 10 mmol / L ammonium formate aqueous solution-acetonitrile as mobile phase

[0095]

[0096] Table 6 Results of gradient elution conditions with 10 mmol / L ammonium formate aqueous solution-acetonitrile as mobile phase

[0097]

[0098] 2.5.2 The sample solution was determined using 20 mmol / L ammonium formate aqueous solution-acetonitrile as the mobile phase and the gradient elution conditions in Table 7. Other determination conditions were the same as those in Item "2.1". The determination results are shown in Tables 8 and Figure 3 .

[0099] Table 7 Gradient elution conditions with 20 mmol / L ammonium formate aqueous solution-acetonitrile as mobile phase

[0100]

[0101]

[0102] Table 8 Results of gradient elution conditions with 20 mmol / L ammonium formate aqueous solution-acetonitrile as mobile phase

[0103]

[0104] 2.5.3 Using 10mmol / L ammonium acetate aqueous solution-acetonitrile as the mobile phase, the test solution was determined using the gradient elution conditions in Table 9. Other determination conditions were the same as those in Item "2.1". The determination results are shown in Tables 10 and Figure 4 .

[0105] Table 9 Gradient elution conditions with 10 mmol / L ammonium acetate aqueous solution-acetonitrile as mobile phase

[0106]

[0107] Table 10 Results of gradient elution conditions with 10 mmol / L ammonium acetate aqueous solution-acetonitrile as mobile phase

[0108]

[0109] The mobile phase has a great influence on the separation degree and peak shape of impurities in the experimental results. In the traditional chromatographic test system, a slight change in the mobile phase ratio or pH value will have a great influence on the test results, and may not even be applicable to the detection of related impurities. The present invention compares several different mobile phase systems such as 10mmol / L ammonium formate aqueous solution-acetonitrile, 20mmol / L ammonium formate aqueous solution-acetonitrile and 10mmol / L ammonium acetate aqueous solution-acetonitrile. The results show that the separation degree of each component is better under the 10mmol / L ammonium formate aqueous solution-acetonitrile system, and the use of formic acid to adjust to different pH values ​​is investigated. The results show that the baseline is stable when the pH value is 3.4, and the symmetry of each peak and the number of theoretical plates are the best. The Sielc Primesep 500 chromatographic column is a chromatographic column with both HILIC and ion modes. Adding ammonium formate aqueous solution to the mobile phase can not only stabilize the pH value, but also prevent compound tailing and improve the peak shape.

[0110] The present invention investigates two elution conditions: gradient elution (0-15 min, 85% B; 15-25 min, 85% B→70% B; 25-35 min, 70% B; 35-36 min, 70% B→85% B; 36-55 min, 85% B) and isocratic elution (0-15 min, 85% B). The results show that under the two elution conditions, the separation of each component is good, and the symmetry of each peak and the number of theoretical plates meet the requirements. Considering that the associated preparation contains buffer salts, which interfere with sample detection, we finally adopted gradient elution.

[0111] The above embodiments are for illustrative purposes only. The present invention is not limited to the above embodiments. A person skilled in the art may make changes and modifications based on the description of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting tauramide and taurine in taurolidine by HPLC-CAD method, characterized in that: A SIELC Primesep 500 chromatographic column was used, with ammonium formate aqueous solution (A)-acetonitrile (B) as the mobile phase, gradient elution, a volume flow rate of 0.8-1.2 mL / min, a column temperature of 15-25° C., an injection volume of 8-12 μL; a CAD detector temperature of 30-40° C.; the gradient elution procedure was: The concentration of the ammonium formate aqueous solution is 8 to 25 mmol / L; 10≤t1≤18; 20≤t2≤30; 10≤V A1 ≤20;25≤V A2 ≤35。 2. The method according to claim 1, characterized in that The procedure of the gradient elution is: 10≤t1≤18; 20≤t2≤30; 33≤t3≤37; t4=t3+n; 50≤t5≤60; n=0.5~2; 10≤V A1 ≤20;25≤V A2 ≤35;10≤V A3 ≤20。 3. The method according to claim 1, characterized in that The concentration of the ammonium formate aqueous solution is 10-20 mmol / L.

4. The method according to claim 1, characterized in that: The volume flow rate is 1.0 mL / min.

5. The method according to claim 1, characterized in that The CAD atomizer temperature is 35°C.

6. The method according to claim 1, characterized in that The pH value of mobile phase A was adjusted to 3-4 with formic acid.

7. The method according to claim 6, characterized in that The pH of mobile phase A was adjusted to 3.4 with formic acid.

8. The method according to claim 1, characterized in that The column temperature was 20°C.

9. The method according to claim 1, characterized in that: The injection volume was 10 μL.

10. The method according to any one of claims 1 to 9, characterized in that This method can be used for the quality control of taurolidine.

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