Method for rapidly determining iodixanol content in iodixanol injection by ultra-high performance liquid chromatography

Abstract

The invention belongs to the technical field of content determination, and particularly relates to a method for rapidly determining the content of iodixanol in an iodixanol injection by using an ultra-high performance liquid chromatography. The method comprises the following steps: preparing a detection reagent by using iodixanol; detecting the detection reagent by using the ultra-high performance liquid chromatography; wherein the detection conditions are as follows: the chromatographic column is Waters BEH HILLIC (2.1 * 100 mm, 1.7 m); a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; isocratic elution: carrying out isocratic elution for 0-5 minutes by using 30% of water and 70% of acetonitrile; the flow velocity is 0.3 mL / min. The detection method disclosed by the invention is high in sensitivity, rapid in acquisition and low in detection cost.

Description

Technical Field

[0001] The invention relates to the technical field of content determination, and in particular to a method for quickly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography. Background Art

[0002] Iodine-containing contrast agents have been widely used in clinical practice for many years. With the development of modern medicine, clinical studies have found that most contrast agents have varying degrees of nephrotoxicity. At present, ionic iodine-containing contrast agents have been basically eliminated, and non-ionic iodine-containing contrast agents are widely used. Low-osmotic non-ionic iodine-containing contrast agents are recognized by everyone because of their safety, effectiveness and good tolerance. Iodixanol, as a new type of soluble non-ionic isotonic dimer hexaiodine X-ray contrast agent, is injected into the body. The organically bound iodine will absorb radiation in blood vessels and tissues, thereby producing artificial contrast and playing a contrast examination function. Therefore, it is very necessary to accurately determine the effective content in its injection solution.

[0003] In addition, iodixanol injection is currently an X-ray contrast agent, clinically used for cardiovascular angiography, cerebral angiography, peripheral arterial angiography, abdominal angiography, urography, venography and CT enhancement examination in adults; it is also used for cardiovascular angiography, urography and CT enhancement examination in children. Due to its high viscosity, the determination of its content in the preparation has always been a difficult problem that has troubled technical personnel. The existing determination methods include titration and high-performance liquid chromatography. However, due to its long determination time and complex operation, it is very limited by the experience level of the operator. Therefore, it is very urgent to develop a simple and efficient method for determining the content of iodixanol in iodixanol injection. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, in order to solve the problems, the present invention provides a method for quickly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography. The detection method has high sensitivity and fast detection speed, and data collection can be completed within 5 minutes.

[0005] The present invention provides the following technical solutions: A method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography comprises the following steps: Preparation of detection reagent using iodixanol: detection of detection reagent using ultra-high performance liquid chromatography; The testing conditions include: Chromatographic column: Waters BEH HILLIC (2.1×100 mm, 1.7µm); mobile phase A: ultrapure water, mobile phase B: acetonitrile; isocratic elution: 0~5min, 30% water-70% acetonitrile; flow rate: 0.3 mL / min.

[0006] Furthermore, the detection conditions also include: injection volume: 5 μL; injection port plate temperature control: 5° C.; column temperature 30° C.; detector DAD, detection wavelength 254 nm.

[0007] Preferably, the iodixanol content of the detection reagent is detected by ultra-high performance liquid chromatography.

[0008] Preferably, the detection reagent is prepared using a diluent and iodixanol.

[0009] Preferably, the diluent includes water or acetonitrile.

[0010] Preferably, the volume ratio of water to acetonitrile is 30:70.

[0011] Preferably, iodixanol and a diluent are mixed to prepare an iodixanol reference substance stock solution.

[0012] Preferably, the iodixanol reference substance stock solution and the diluent are mixed to prepare the iodixanol reference substance solution.

[0013] Preferably, the iodixanol reference solution and the diluent are mixed to prepare the iodixanol sensitivity solution.

[0014] Preferably, the iodixanol injection and the diluent are mixed to prepare the iodixanol injection test solution.

[0015] Different from the prior art, the beneficial effects of the present invention are: In the prior art, the cost of ultra-high performance liquid chromatography is high, and the particle size of the chromatographic column is usually small, which will lead to a high column pressure. For example, the Waters BEH HILLIC (2.1×100 mm, 1.7µm) selected in the present invention, therefore, it is difficult to balance the column efficiency and the analysis speed. It is even more difficult to increase the analysis speed while maintaining a high accuracy. In addition, the viscosity of the iodixanol injection itself is relatively high, which further increases the difficulty of detection. Therefore, the present invention overcomes many technical difficulties, achieves a balance in various aspects, and successfully explores a suitable detection method, which is as follows: 1. The present application can complete data collection in 5 minutes, reduce reagent waste, and save costs. It should be noted that the 5 minutes of the present invention includes the time for isocratic elution, which is difficult to achieve with the prior art.

[0016] 2. The standard curve of this application has a high degree of fit and can be accurately quantified in the range of 0.18 mg / mL~1.2 mg / mL. The linear correlation coefficient is r = 1.000, the accuracy recovery rate is 98.4%~101.3%, and the RSD (n=9) is 1.1%.

[0017] In summary, the detection method of the present invention has high sensitivity, rapid collection and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Other features, purposes and advantages of the present invention will become more obvious by reading the detailed description of the non-limiting embodiments with reference to the following drawings.

[0019] Figure 1 This is the specificity spectrum 1 of the present invention; Figure 2 This is the second specificity graph of the present invention; Figure 3 A spectrum for repeatability and accuracy detection of the present invention; Figure 4 It is the standard curve diagram of the present invention. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods for which specific conditions are not specified in the preferred embodiments are usually carried out under conventional conditions. The examples are provided to better illustrate the contents of the present invention, but the contents of the present invention are not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned invention contents, which still fall within the scope of protection of the present invention. All chemical reagents are analytically pure unless otherwise specified, and all reagents are commercial conventional reagents unless otherwise specified, which can be purchased or prepared according to the instructions.

[0021] Example 1 A method for determining the content of iodixanol in iodixanol injection, the specific steps are as follows: The specific conditions for detection by UPLC method refer to Table 1.

[0022] Table 1

[0023] Example 2 Prepare detection reagents: 1) Diluent / blank solution: water and acetonitrile, the volume ratio of water to acetonitrile is 30:70.

[0024] 2) Preparation of reference solution: a. Weigh 240 mg of iodixanol reference substance into a 10 mL volumetric flask, dilute to scale with purified water, shake for 30 seconds, and let stand for 5 minutes to obtain a 24 mg / mL iodixanol reference substance stock solution.

[0025] b. Pipette 0.25 mL of iodixanol reference stock solution into a 10 mL volumetric flask, dilute to scale with diluent, and shake well to obtain a 0.6 mg / mL iodixanol reference solution.

[0026] c. Pipette 3 mL of iodixanol reference solution into a 10 mL volumetric flask, dilute to scale with diluent, and shake well to obtain a 0.18 mg / mL iodixanol sensitivity solution.

[0027] 3) Preparation of test solution: Prepare the iodixanol injection test solution: dilute the injection 100-fold using 70% acetonitrile as the diluent.

[0028] Example 3 The specificity test was performed using the method of Example 1. The specificity verification solutions were the blank solution, reference solution, and test solution prepared in Example 2. The samples were injected and analyzed according to the chromatographic conditions of Example 1. The UV chromatograms of the two solutions were superimposed on the same horizontal axis. Figure 1 and Figure 2 As shown, the blank solution has no interference at the target peak, and the other peaks in the test sample spiked solution have no obvious interference at the target peak. The peak shape is symmetrical and the baseline is good, indicating that the method has good specificity. For detailed results, see Figure 1 and Figure 2 .

[0029] Example 4 Prepare linear detection reagents: Take 3 mL of the iodixanol reference solution prepared in Example 2, and 0.2 mL, 0.25 mL, 0.3 mL and 0.5 mL of the iodixanol reference stock solution are placed in a 10 mL volumetric flask, and diluted to volume with 70% acetonitrile to obtain linear solutions at different concentration levels, which are marked as L-30%, L-80%, L-100%, L-120%, and L-200%, respectively.

[0030] In this example, a linear test was performed, and a linear test solution was taken and tested according to the method of Example 1. The peak area was used as the ordinate and the concentration was used as the abscissa to draw a standard curve and obtain a regression equation. Figure 4 .

[0031] Table 2

[0032] Example 5 Preparation of repeatability and accuracy test reagents: Take 0.125 mL, 0.25 mL, and 0.375 mL of the iodixanol reference substance stock solution prepared in Example 2 and add them to a 10 mL volumetric flask together with 0.1 mL of iodixanol injection, dilute with 70% acetonitrile, and obtain test sample spiked solutions of different concentration levels, which are named RE-80%-1, RE-80%-2, RE-80%-3, RE-100%-1, RE-100%-2, RE-100%-3, RE-120%-1, RE-120%-2, and RE-120%-3. It should be noted that 3 copies of each concentration level are prepared in parallel. For example, use the suffixes "-1", "-2", and "-3" to distinguish them.

[0033] The prepared repeatability and accuracy detection reagent was taken and tested according to the method of Example 1 and calculated according to the single-point control method. The results are shown in Table 3 and Figure 3 shown.

[0034] Table 3

[0035] Reference Figure 1-Figure 2 Based on Tables 1 to 3 above, it can be seen that the method of the present application can measure the iodixanol content in 5 minutes, and can be accurately quantified to 0.18 mg / mL. The standard curve of the present application has a high degree of consistency, a recovery rate of 98.4% to 101.3%, and an RSD (n=9) of 1.1%, which saves costs.

[0036] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the spirit and scope of the present invention to implement and invent the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the detection conditions. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography, characterized in that: The steps include: Preparation of detection reagent using iodixanol: detection of detection reagent using ultra-high performance liquid chromatography; The testing conditions include: Chromatographic column: Waters BEH HILLIC, 2.1×100 mm, 1.7µm; mobile phase A: ultrapure water, mobile phase B: acetonitrile; isocratic elution: 0~5min, 30% water-70% acetonitrile; flow rate: 0.3 mL / min.

2. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 1, characterized in that: The detection conditions also include: injection volume: 5 μL; injection port plate temperature control: 5°C; column temperature 30°C; detector DAD, detection wavelength 254 nm.

3. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 1, characterized in that: The iodixanol content of the detection reagent was detected by ultra-high performance liquid chromatography.

4. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 1, characterized in that: Prepare the assay reagent using diluent and iodixanol.

5. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 4, characterized in that: Diluents include water and acetonitrile.

6. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 4, characterized in that: The iodixanol and diluent were mixed to prepare the iodixanol reference substance stock solution.

7. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 4, characterized in that: The iodixanol reference solution was prepared by mixing the iodixanol reference stock solution and the diluent.

8. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 4, characterized in that: The iodixanol reference solution and the diluent were mixed to prepare the iodixanol sensitivity solution.

9. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 4, characterized in that: The iodixanol injection solution and the diluent were mixed to prepare the iodixanol injection test solution.

10. The method for rapidly determining the content of iodixanol in iodixanol injection by ultra-high performance liquid chromatography according to claim 5, characterized in that: The volume ratio of water to acetonitrile was 30:70.

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