Method for rapidly measuring pH value of trace PET probe through spectrometry and application of method

Abstract

The invention discloses a method for rapidly measuring the pH value of a trace PET probe through a spectrum method and application of the method. The method has the advantages that the using amount of a sample is extremely small, a standard spectrum curve is drawn after experimental conditions are optimized, the measuring steps are simple, the measuring result is high in accuracy and sensitivity, and meanwhile microfluidic automatic online measuring can be achieved. The PET tracer agent has the characteristics of small synthesis amount, high price, radioactivity and the like, so that the PET tracer agent has huge application value in the aspects of testing the use amount of samples, simplifying detection, realizing automatic testing and the like.

Description

Technical Field

[0001] The invention relates to the field of pH value measurement, and in particular to a method for quickly measuring the pH value of a trace PET probe using a spectral method and an application thereof. Background Art

[0002] Radiopharmaceutical therapy (RPT) is becoming a safe and effective targeted method for treating many types of cancer. Radiopharmaceuticals usually refer to a special class of drugs labeled with radionuclides for medical diagnosis and treatment. Radioactive labeling diagnoses electromagnetic emitter rays (gamma rays or X-rays). Radionuclide imaging includes positron emission tomography (PET) and single photon emission tomography (SPECT). Radioactive probes are injected to evaluate and quantify the anabolism and gene expression of organisms. This imaging method is widely used in clinical diagnosis and treatment, as well as the development and evaluation of new therapies in clinical research, by injecting radioactive drugs labeled with positron-emitting or gamma-emitting isotopes into patients.

[0003] Radiopharmaceuticals must undergo strict quality control (QC) testing to ensure product purity, effectiveness and safety. According to the nature of the test, the test indicators are divided into physical testing, chemical testing and biological testing, as shown in Table 1 for PET tracers. 18 F-FDG quality control system detection indicators and commonly applicable detection methods for microfluidics. After QC inspection, the physiological compatibility of the final preparation and the absence of microbial, pyrogen, chemical or particulate contamination are ensured. In addition, radioactivity testing is required before it can be used clinically. pH is a scale used to indicate the acidity and alkalinity of aqueous solutions. It is an important physical and chemical indicator of reaction solution systems. The determination of the pH value of radioactive drugs can measure the stability of the drug and ensure that it will not degrade or fail during manufacturing and injection. At the same time, as an important indicator of in vivo release, radioactive drugs are used as intravenous drugs. The pH value of human blood ranges from 7.35 to 7.45. The compatibility of pH with the organism and its stability in the body make the quality control of pH very important.

[0004] Table 1: PET tracers 18 F-FDG quality control system detection indicators and commonly used microfluidic detection methods

[0005]

[0006]

[0007] a: The residual amount of chemical solvents in radioactive drugs is extremely small. The boiling points of acetonitrile, acetone and ethanol are all below 100°C and are very volatile. The existing synthesis process can ensure their removal.

[0008] Common methods for testing pH include pH test paper and glass electrode. pH test paper can only test the pH range and cannot accurately measure pH. Glass electrode is one of the earliest systems for measuring pH and is currently the most accurate measurement method. Its first model was proposed by F. Haber and Z. Klemansiewicz in 1909. Based on the results obtained by M.Cremer, he noticed the potential difference of the thin glass membrane in 1906 and separated two solutions with different pH values. The error range of pH test paper is relatively large. Glass electrodes have defects such as fragility, frequent calibration, need to be stored in protective solution, need to rinse the electrode before testing, and large demand for test solution. At the same time, the test methods are limited by manual operation, which greatly promoted the development of devices with trace samples, accurate testing, high sensitivity, and online automation. Therefore, researchers began to explore other methods such as optical, electrochemical, transistor-based sensor research for measuring pH, and combined with microfluidic technology in pH determination. Electrochemistry usually uses the test solution as a component of a chemical cell, and conducts measurements based on a certain relationship between a certain electrical parameter of the cell (such as resistance, conductivity, potential, current, charge or current-voltage curve, etc.) and the concentration of the substance being measured. The pH value is a scale for the acidity and alkalinity of aqueous solutions, and the pH value is usually expressed as the negative logarithm of the H ion concentration. In addition to acid and base ions, there are also a large number of metal ions and negative ions in the solution, which have a great influence on the electrical parameters. The electrochemical method is interfered by various ions in the test solution, and the error is large.

[0009] Acid-base indicators are a class of organic weak acids or organic weak bases with relatively complex structures. They can partially ionize into indicator ions and hydrogen ions (or hydroxide ions) in solutions, and due to structural changes, their molecules and ions have different colors, and thus show different colors in solutions with different pH values. Commonly used in pH test paper, pH titration, etc., a single indicator has a narrow pH value indication range, but can effectively indicate changes in pH value through color changes within a specified range, that is, the spectral value changes, and the pH value can be determined by spectroscopic determination of changes in the solution spectrum. Bromothymol Blue (BTB), also known as bromothymol blue, is an acid-base indicator and adsorption indicator with a chemical formula of C 27 H 28 O5SBr2 is easily soluble in ethanol, ether, methanol and dilute alkali hydroxide solutions. Summary of the invention

[0010] The present invention aims to address the deficiencies of the prior art and to provide a method for rapidly determining the pH value of a trace PET probe using a spectral method.

[0011] The objective of the present invention is achieved through the following technical scheme: a method for quickly determining the pH value of a trace PET tracer by spectroscopy, preparing a 0.02% (m:V=g:mL) bromothymol blue BTB solution (mg / μL), drawing a standard spectral curve of a standard solution with a pH value of 4.00-8.45 by spectroscopy, taking a PET tracer sample of 50 μL, adding 1 μL of BTB solution, determining the absorbance value with a micro-colorimeter, and calculating the pH value of the PET tracer by the pH standard spectral curve.

[0012] On the other hand, the present invention also provides an application of bromothymol blue spectroscopy in the rapid determination of the pH value of a trace PET probe, wherein the PET probe sample is added to a bromothymol blue BTB solution, the spectral absorption curve at 300 to 980 nm is measured, the absorbance at the wavelength of the maximum absorbance value of the characteristic wavelength is taken, and the pH value is calculated using a standard curve.

[0013] Furthermore, the standard curve preparation process is: taking pH standard mother solutions with different pH values ​​and adding bromothymol blue BTB solution, measuring the spectral absorption curve of 300-980nm, and taking the absorbance at the wavelength of the maximum absorbance value of the characteristic wavelength, and drawing the standard curve with pH value as the horizontal axis and absorbance as the vertical axis.

[0014] Furthermore, a Britton-Robinson wide-range buffer solution (pH 1.8-11.9) was prepared by mixing phosphoric acid, boric acid and acetic acid system (H3PO4-HAc-H3BO3) and adding NaCl to prepare a 0.9% saline pH standard stock solution.

[0015] Beneficial effects of the present invention: The pH value QC of the PET probe provided by the present invention can achieve trace sample (within 50ul or even less) through spectroscopy, without calibration, rinsing, non-destructive, high sensitivity, high precision, and it is easier to achieve the advantages of online automated measurement through the combination of microfluidic chip technology and application. The development of methods and test processes can realize the development of microfluidic disposable chips, making the test more convenient, and by broadening the test range through different indicators, it is completely possible to achieve accurate measurement of the full range of pH values ​​from 1 to 14. This method uses very little sample for measurement, the test method is convenient, the operation steps are simple, the measurement results are accurate, and it is easy to achieve fully automated online operation. It is of great significance for the quality control of medical imaging probes, radioactive drugs, and other radioactive drugs with low production and high prices. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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.

[0017] Figure 1 is the spectral value of BTB solution in physiological saline solution with pH value of 4.00-8.45;

[0018] Figure 2 It is the absorbance value of BTB solution at 615nm in physiological saline solution with a pH value of 4.00 to 8.45;

[0019] Figure 3 It is the standard curve of pH5.00~6.00 spectrum;

[0020] Figure 4 Absorbance value at 430nm (406nm wavelength at pH 6) DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific implementation examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The invention provides a method for quickly determining the pH value of a trace PET probe by spectroscopy. The invention finds through experiments that the indicator can characterize a pH value between 5 and 8 by spectroscopy, meets the pH value limit requirement of the PET probe, can be developed as a spectroscopy reagent to test the pH value of the PET probe, and can accurately identify abnormal samples. The PET probe pH value is accurately determined by accurately pipetting a trace PET probe sample, adding a bromothymol blue (Bromothymol Blue, BTB) solution, and using a spectrometer to determine the change in the spectral value of the PET probe sample solution.

[0023] The method of the present invention comprises the following steps:

[0024] Preparation of pH standard stock solution: PET probe is a radioactive drug dissolved in saline, which is used clinically by intravenous injection. This method uses Britton-Robinson wide-range buffer solution (pH 1.8-11.9), which is mixed with phosphoric acid, boric acid and acetic acid system (H3PO4-HAc-H3BO3), and NaCl is added to prepare 0.9% saline pH standard stock solution. Take a certain volume of stock solution, add 0.2 mol / L NaOH solution to adjust the pH, and measure other standard pH solutions with a desktop pH meter. Prepare 0.02% BTB solution, accurately weigh 2 mg BTB, and fully dissolve it in 10.0 mL ethanol solution for use.

[0025] Accurately pipette 50 μL of pH 4.0-8.45 standard pH solution into a 48 μL micro cuvette, accurately add 1 μL BTB solution, measure the spectral absorption curve from 300 to 980 nm, and take the absorbance at the wavelength of the maximum absorbance value of the characteristic wavelength. Draw the standard absorption curve with pH as the horizontal axis and absorbance as the vertical axis. PET probe pH value determination: Accurately pipette 1 μL BTB solution into a 48 μL micro cuvette, accurately add 50 μL PET probe sample, measure the spectral absorption curve from 300 to 980 nm, and take the absorbance at the wavelength of the maximum absorbance value of the characteristic wavelength. Calculate the pH value through the standard curve.

[0026] (1) Optimization of spectral curves at different pH values

[0027] Prepare pH 4.00, 5.00, 6.00, 7.00, and 8.45 solutions with different pH values ​​using pH standard mother solution, pipette 1 μl of BTB into a micro-cuvette, accurately pipette 50 μl of the above solution into 48 μl of micro-cuvettes, measure the spectral value, and draw a standard spectral curve, such as Figure 1 As shown in the figure, there are characteristic absorption peaks at 430nm and 615nm. As the pH value increases, there is an obvious and stable increase in the spectrum value at 615nm. The pH value can be determined by drawing a standard curve at 615nm. Figure 2 As shown in the figure, there is no obvious change in the spectral value between pH 4 and 5, and a smooth curve is presented in the range of pH 5 to 8.45.

[0028] Figure 1 The results show that there is no obvious change between pH 4 and 5, and the spectral value between pH 5 and 6 has a significant jump, so it is impossible to accurately measure these two ranges. The experimental method accurately measures the different spectral values ​​of pH 5.00, 5.20, 5.40, 5.60, 5.80, and 6.00. Figure 3, where 5 to 6 produce a sudden change at 615nm, but show a stable change at 430nm. The standard curve of the maximum absorbance value at 430nm is drawn as follows Figure 4 The figure shows that it is basically linear in the range of 5 to 6. At pH 6, there is a sudden change and the characteristic peak is slightly shifted. A more precise pH standard spectrum can be drawn to obtain an accurate standard curve.

[0029] The PET probe presents different characteristic peaks in different pH ranges. Through the experimental method design, the pH value of the PET probe sample is calculated based on the 615nm characteristic peak. If the pH value is in the range of 6 to 8.45, it is the pH measurement result. If the pH value of the PET probe sample calculated based on the 615nm characteristic peak is lower than 6, the pH value is recalculated based on the 430nm characteristic peak as the measurement result. This method has high sensitivity to PET tracer measurement and can still accurately measure pH value under trace conditions.

[0030] (2) PET probe and micro-sample method validation

[0031] The traditional test method for PET probe pH determination is the pH determination method 0631 in the 2020 edition of the Chinese Pharmacopoeia. This method has the problem that the sample volume is larger than 10mL, and the test process usually requires pH standard solution calibration, glass electrode rinsing and other operations. At the same time, the instrument needs to be operated manually, the laboratory cannot be automated, and the glass electrode is fragile and easy to damage. The development of micro-glass electrodes only reduces the sample volume, and the electrode diameter is reduced to 3mm. The required sample volume is about 100 microliters, but in actual use, if accurate results are to be measured, it still needs to far exceed this volume.

[0032] The spectral method for measuring pH value greatly reduces the amount of PET samples used and improves the accuracy and sensitivity of the test. The spectral method uses a light source and a spectrometer system to non-contactly measure the spectral value of sample solutions or solids. It can be realized by developing a spectral microfluidic chip to achieve automated online testing of pH value and other quality control indicators. 18 For F-FDG samples 1#, 2#, and 3#, the pH values ​​of the samples were measured 6 times in parallel by spectral method and glass electrode method as shown in Table 2 and Table 3. The results show that the results of multiple measurements by spectral method are accurate and have a smaller error than those by glass electrode method.

[0033] Table 2 PET probe sample spectral value determination pH value

[0034]

[0035] Table 3 pH value of PET probe samples measured by glass electrode method

[0036]

[0037]

[0038] Actual case 1:

[0039] 1. Accurately pipette 1 μL BTB solution into a 48 μL micro cuvette, accurately add 50 μL physiological saline solution, and calibrate the wavelength to zero at 615 nm. Accurately pipette 1 μL BTB into a 48 μL micro cuvette, accurately add 1# 18 The spectrum values ​​of 50 μL of F-FDG sample were measured at 615 nm for 3 times, which were 0.265, 0.262, 0.266, and the average value was 0.264. The calculated pH value was 7.7, and the result of the benchtop pH meter was 7.65, with a relative standard deviation of 3.5%. The relative standard deviation of this example is less than 10%, indicating that the discrete degree of the test results is low. There is no significant difference between the data results detected by the method of the present invention and those by the traditional method.

[0040] Actual case 2:

[0041] 2. Accurately pipette 1 μL BTB solution into a 48 μL micro cuvette, accurately add 50 μL physiological saline solution, and calibrate the wavelength to zero at 615 nm. Accurately pipette 1 μL BTB into a 48 μL micro cuvette, accurately add 2# 18 The spectrum values ​​of 50 μL of F-FDG sample were measured at 615 nm for 3 times, which were 0.245, 0.242, 0.246, and the average value was 0.244. The calculated pH value was 7.2, and the result of the benchtop pH meter was 7.17, with a relative standard deviation of 2.1%. The relative standard deviation of this example is less than 10%, indicating that the discrete degree of the test results is low. There is no significant difference between the data results detected by the method of the present invention and those by the traditional method.

[0042] Actual case three:

[0043] 3. Accurately pipette 1 μL BTB solution into a 48 μL micro cuvette, accurately add 50 μL physiological saline solution, and calibrate the wavelength to zero at 615 nm. Accurately pipette 1 μL BTB into a micro cuvette, accurately add 3# 18 The spectrum values ​​of 50 μL of F-FDG sample were measured at 615 nm for 3 times, which were 0.249, 0.252, and 0.256, with an average value of 0.252. The calculated pH value was 7.5, and the result of the benchtop pH meter was 7.44, with a relative standard deviation of 4.2%. The relative standard deviation of this example is less than 10%, indicating that the discrete degree of the test results is low. There is no significant difference between the data results detected by the method of the present invention and those by the traditional method.

[0044] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for rapidly determining the pH value of a trace PET tracer by spectroscopy, characterized in that: Prepare 0.02% (m:V=g:mL) bromothymol blue BTB solution, and draw a standard spectral curve of the standard solution with a pH value of 4.00-8.45 by spectroscopy. The PET tracer sampling volume is 50 μL. After adding 1 μL BTB solution, the absorbance value is measured with a micro-colorimeter, and the PET tracer pH value is calculated by the pH standard spectral curve.

2. An application of bromothymol blue spectroscopy in the rapid determination of pH value of trace PET probes, characterized in that: The PET probe sample was added to the bromothymol blue BTB solution, and the spectral absorption curve was measured at 300-980 nm. The absorbance at the wavelength with the maximum absorbance value of the characteristic wavelength was taken, and the pH value was calculated using the standard curve.

3. The application of bromothymol blue spectroscopy in the rapid determination of pH value of trace PET probe according to claim 2, characterized in that: The standard curve preparation process is as follows: pH standard mother solutions with different pH values ​​are added with bromothymol blue BTB solution, the spectral absorption curve of 300-980 nm is measured, and the absorbance at the wavelength of the maximum absorbance value of the characteristic wavelength is taken at the same time, and the standard curve is drawn with pH value as the horizontal axis and absorbance as the vertical axis.

4. The application of bromothymol blue spectroscopy in the rapid determination of pH value of trace PET probe according to claim 3, characterized in that: The Britton-Robinson wide-range buffer solution (pH 1.8-11.9) was prepared by mixing phosphoric acid, boric acid and acetic acid system (H3PO4-HAc-H3BO3) and adding NaCl to prepare a 0.9% physiological saline pH standard stock solution.

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