Method for rapidly determining K.2. 2.2. 2 content in FDG card sleeve kit leacheate

Abstract

The invention relates to a method for rapidly determining the content of K.2. 2.2. 2 in an FDG card sleeve kit leacheate, and belongs to the technical field of radiopharmaceutical analysis. The method comprises the following steps: selecting a 1110 + / -5 cm <-1 > peak as a quantitative characteristic peak of K.2.2. 2, wherein the peak corresponds to symmetric stretching vibration of a C-O-C ether bond in a K.2.2. 2 molecule; selecting a 2253 + / -3cm <-1 > peak as an internal standard peak, wherein the peak corresponds to C = N triple bond stretching vibration of acetonitrile in the leacheate; preparing a K.2. 2.2. 2 gradient solution with the concentration of 0.05 to 5 mg / mL, and fixing the concentration of K2CO3 to be 0.5 to 2 mg / mL; the method comprises the following steps: acquiring a spectrum by adopting an ATR technology, setting the resolution ratio to be 4cm <-1 > and the scanning frequency to be 32, and establishing a K.2. 2.2. 2 linear model according to the ratio of A1110 / A2253; eliminating baseline drift by adopting a second derivative spectrum; and a PLS algorithm is adopted to correct trace interference of K2CO3.

Description

A rapid method for determining the K.2.2.2 content in the eluent of an FDG cassette kit. Technical Field

[0001] This invention belongs to the technical field of radiopharmaceutical analysis, and more specifically, relates to a method for rapidly determining the K.2.2.2 content in the eluent of an FDG cartridge kit. Background Technology

[0002] [18F]Fluorodeoxyglucose (FDG) is the most commonly used radiotracer in PET imaging. Its synthesis typically uses K.2.2.2 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) as a phase transfer catalyst. However, K.2.2.2 has potential neurotoxicity, and pharmacopoeias strictly stipulate that the residual K.2.2.2 in the final product must be below 50 μg / mL, while the concentration of K.2.2.2 in the eluent needs to be precisely controlled within the range of 0.8-1.2 mg / mL to ensure synthesis efficiency and purification effect. Therefore, K.2.2.2 content determination is a core quality control step to ensure the safety, effectiveness, and reliability of FDG as a clinical diagnostic tool.

[0003] Currently, industry standard methods for K.2.2.2 content detection include ultraviolet spectrophotometry, HPLC, and thin-layer chromatography. However, ultraviolet spectrophotometry for K.2.2.2 detection requires reaction with metal ions (such as Pb) in a specific buffer system. 2+ The formation of stable complexes and quantification by measuring the characteristic absorption peaks of the complexes in the ultraviolet region are methods, but baseline interference may affect sensitivity. HPLC detection of K.2.2.2 requires large amounts of solvent, complex pretreatment, and is time-consuming, making it unsuitable for rapid quality control. Thin-layer chromatography is semi-quantitative and has poor reproducibility. Therefore, existing technologies suffer from drawbacks such as long analysis times, insufficient specificity, and complex operation, failing to meet the requirements for rapid release detection under GMP conditions.

[0004] While Fourier transform infrared spectroscopy (FTIR) has applications in pharmaceutical analysis, it has not yet been used for the determination of K.2.2.2 content, especially in complex matrices such as FDG eluent, where the infrared absorption peaks of K.2.2.2 and K2CO3 in acetonitrile overlap (e.g., the COC peak of K.2.2.2 and the CO3 peak of K2CO3). 2- Peaks cannot be directly quantified by traditional FTIR. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid method for determining the K.2.2.2 content in the eluent of an FDG card sleeve reagent kit. By optimizing the selection of characteristic peaks and chemometric algorithms, this method overcomes the shortcomings of existing technologies, such as long processing time, complex operation, and the need to use hazardous reagents, and achieves highly selective detection.

[0006] The objective of this invention can be achieved through the following technical solution: A method for rapidly determining the K.2.2.2 content in the eluent of an FDG card sleeve reagent kit, comprising the following steps: S1, characteristic peak selection: selecting 1110±5cm -1 The peak, as a quantitative characteristic peak of K.2.2.2, corresponds to the symmetric stretching vibration of the COC ether bond in the K.2.2.2 molecule; the peak value is 2253±3 cm⁻¹. -1 The peak serves as an internal standard, corresponding to the C≡N triple bond stretching vibration of acetonitrile in the eluent; S2, Standard curve establishment: Prepare 0.05-5 mg / mL K.2.2.2 gradient solutions, fixing the K2CO3 concentration at 0.5-2 mg / mL; Acquire spectra using ATR technology, setting the acquisition parameters to a resolution of 4-8 cm⁻¹. -1 The number of scans is 16-32, with A 1110 / A 2253 The ratio establishes a linear model for K.2.2.2; S3, interference correction: second derivative spectroscopy is used to eliminate baseline drift; and the PLS algorithm is used to correct the trace interference of K2CO3.

[0007] The order of steps S1, S2, and S3 is not important. In step S1, select 1110±5cm. -1 The peak is used as the quantitative characteristic peak of K.2.2.2 to avoid the CO3 peak of K2CO3. 2- Peak (1450cm) -1 Interference. Acetonitrile is a constant component in the eluent of FDG synthesis. In existing techniques, the internal standard method often uses an external standard substance, introducing an additional variable. This technical solution uses the acetonitrile C≡N peak (2253±3cm). -1 As an internal standard, it not only simplifies the operation but also eliminates the influence of sample thickness, ATR crystal contact differences, and optical path fluctuations.

[0008] In step S2, ATR technology, also known as attenuated total reflection sampling technology, is used in FTIR analysis with a 4cm depth. -1 -8cm -1 This high resolution ensures quantitative accuracy while reducing acquisition time. Furthermore, this technical solution uses A... 1110 / A 2253 A K.2.2.2 concentration standard curve was established based on the peak area ratio to simulate the composition of the real eluent and improve accuracy.

[0009] In step S3, the PLS algorithm, also known as the partial least squares algorithm, combines the second derivative with PLS to effectively handle complex matrix interference.

[0010] Furthermore, the ATR crystal is a diamond crystal with a surface temperature of 22-28°C, more preferably 24-26°C.

[0011] Further, in step S2, the K2CO3 concentration is fixed at 0.8-1.2 mg / mL, more preferably 1 mg / mL, and the acetonitrile concentration is fixed at 8-12 vol%, more preferably 10 vol%.

[0012] Furthermore, in step S2, the resolution is set to 4cm. -1 The number of scans was 16, and the wavenumber range of the acquired spectra was 4000-600 cm⁻¹. -1 .

[0013] Furthermore, in step S2, before the first analysis each day, the suitability of the system is verified by measuring the quality control sample; and / or, a quality control sample is inserted every 5 samples for intermediate precision monitoring.

[0014] Further, in step S3, the second derivative spectrum refers to the Savitzky-Golay second derivative method with a window size of 13-17 points and a polynomial order of 2; the use of the PLS algorithm to correct the trace interference of K2CO3 refers to the use of the PLS algorithm to construct a correction model to correct the trace interference of K2CO3, where the concentration of K2CO3 in the eluent is in the range of 0.5-2 mg / mL.

[0015] Furthermore, the principal component number of the PLS algorithm is 5-7.

[0016] Furthermore, step S3 also includes the following step: using principal component analysis to identify samples that are outside the calibration range.

[0017] Furthermore, after step S3, the following steps are also included: S4, quantitative analysis: substituting the processed spectral data into the pre-established calibration model to calculate the K.2.2.2 content.

[0018] The beneficial effects of the present invention are as follows: The K.2.2.2 content determination method of the present invention adopts ATR sampling technology, which eliminates the complicated sample preparation process of the traditional transmission method, and has fast detection speed and high accuracy. It does not require the use of toxic and dangerous reagents such as copper sulfate-pyridine, thus reducing operational risks. Moreover, through the multivariate correction algorithm, it can eliminate the influence of common interfering substances such as K2CO3, water and ethanol, and has strong anti-interference ability. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0020] Example 1 Characteristic Peak Confirmation: Preparation of Simulated Eluent: Dissolve 50 mg K.2.2.2 in methanol and dilute to 10 mL to obtain a K.2.2.2 methanol solution with a concentration of 5 mg / mL; dissolve 50 mg K2CO3 in water and dilute to 10 mL to obtain a K2CO3 aqueous solution with a concentration of 5 mg / mL; accurately transfer 2 mL of the 5 mg / mL K.2.2.2 methanol solution, 2 mL of the 5 mg / mL K2CO3 aqueous solution, and 1 mL of pure acetonitrile into a 10 mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain a simulated eluent containing 1 mg / mL K.2.2.2, 1 mg / mL K2CO3 fixed solution, and 10 vol% acetonitrile.

[0021] Spectral acquisition: Infrared spectra were acquired using an ATR-FTIR spectrometer; FTIR parameter settings: resolution 4cm. -1 The number of scans was 32, and the wavenumber range covered 4000-600cm. -1 Each sample was measured three times.

[0022] Data processing: Second derivative processing was used with parameters of 15-point window and 2nd order; calculations were performed on each component at 1110±20cm. -1 and 2253±15cm -1 The contribution of the region to light absorption.

[0023] The results showed that pure K.2.2.2 at 1110 cm⁻¹ -1 A strong absorption peak is observed at 2253 cm⁻¹ (absorbance 0.782±0.015), while K₂CO₃ and acetonitrile show no significant absorption in this region (absorbance <0.02); pure acetonitrile shows a strong absorption peak at 2253 cm⁻¹. -1 A sharp absorption peak is observed at 1110 cm⁻¹ (absorbance 0.854±0.018), while K₂.₂.₂ and K₂CO₃ show no significant absorption in this region (absorbance <0.03); in the simulated eluent, 1110 cm⁻¹... -1 96.3% of the peak originated from K.2.2.2, and 3.7% from other components; 2253 cm⁻¹ -1 98.5% of the peak comes from acetonitrile, and 1.5% comes from other components.

[0024] Therefore, we can conclude that: 1110cm -1 The peak is a specific quantitative peak for K.2.2.2, at 2253 cm⁻¹. -1 The peak is a specific internal standard peak for acetonitrile, and the two do not significantly interfere with each other, making them suitable for simultaneous detection.

[0025] Example 2: Standard Curve Establishment: Standard Series Preparation: Referring to the preparation method of the simulated eluent in Example 1, a series of standard solutions with K.2.2.2 concentration gradients of 0.05, 0.1, 0.25, 0.5, 1, 2, and 5 mg / mL were prepared. Each solution contained 1 mg / mL K2CO3 fixative and 10 vol% acetonitrile. Each concentration was replicated in triplicate, for a total of 21 samples.

[0026] Spectral acquisition and processing: Referring to the spectral acquisition and data processing method in Example 1, calculate 1110±5cm. -1 Absorbance of characteristic peak at K.2.2.2 (A) 1110 ) and 2253±2cm -1 Absorbance of acetonitrile internal standard peak (A) 2253 The ratio R=A 1110 / A 2253 Plotting R-values ​​on the ordinate and K.2.2.2 concentration on the abscissa, a weighted least squares method was used to fit a linear regression equation: R = k × C + b, where C is the K.2.2.2 concentration (mg / mL), k is the slope, and b is the intercept. The linear range was determined by analyzing the residual plot and R-values. 2 Values ​​and RSDs at each concentration point were confirmed. Limits of detection (LOD) and limits of quantitation (LOQ) were calculated according to ICH Q2(R1) guidelines: LOD = 3.3σ / S, LOQ = 10σ / S, where σ is the standard deviation of the calibration curve residuals and S is the regression slope. All statistical analyses were performed using R4.1.2 software, with a significance level set at α = 0.05.

[0027] The results showed that the calibration curve exhibited good linearity in the range of 0.12-5 mg / mL, R 2 =0.9987, slope k = 0.852 ± 0.008 AU·mL / mg, intercept b = 0.005 ± 0.003 AU. Residual analysis showed that the model assumptions were satisfied (normality p = 0.821, homoscedasticity p = 0.125). The method detection limit (LOD) was 0.12 mg / mL, the quantitation limit (LOQ) was 0.3 mg / mL, the precision RSD ≤ 3%, and the accuracy recovery rate was 95.2-103.8%.

[0028] Therefore, it can be seen that this method has good linearity in the range of 0.05-5 mg / mL, completely covers the actual concentration range of K.2.2.2 in the FDG cassette kit eluent, and has a sufficiently low limit of detection.

[0029] Example 3 Interference correction verification: K2CO3 interference settings: The concentration of K.2.2.2 was fixed at 1 mg / mL, and the concentration of K2CO3 was set to 0.5, 1, 1.5, and 2 mg / mL, respectively, with 3 replicates for each group.

[0030] Water content interference settings: K.2.2.2 concentration was fixed at 1 mg / mL and K2CO3 concentration was fixed at 1 mg / mL, and water content was set at 85%, 90%, and 95% respectively, with 3 replicates for each group.

[0031] Data processing comparison: A blank control group, experimental group 1, and experimental group 2 were set up. The control group contained the original spectra without any processing; experimental group 1 underwent only second derivative processing; experimental group 2 underwent second derivative and PLS correction processing. The relative error was calculated using the HPLC results as reference values. The PLS model used 48 samples with different concentration combinations as the calibration set and 12 independent samples as the validation set for cross-validation to determine the optimal number of principal components.

[0032] The interference correction results for K2CO3 and water content are shown in Table 1 below.

[0033] Table 1 As shown in Table 1, the K2CO3 interference correction results indicate that experimental group 2 has the lowest error, proving that the PLS algorithm can effectively identify and correct the chemical interference of K2CO3. The water content interference correction results also show that the errors in experimental groups 1 and 2 are significantly improved compared to the blank control group, demonstrating that second-derivative processing can effectively eliminate physical interference caused by changes in water content. In summary, second-derivative processing effectively eliminates the effects of baseline drift and water content changes. The PLS algorithm further corrects for K2CO3 interference, ensuring that the K2CO3 concentration is within the range of 0.5-2.0 mg / mL, with a result error of <2%.

[0034] Example 4 Method Validation: 1. Accuracy Test: 0.1 mg / mL K₂CO₃ and 10 vol% acetonitrile were added to 18 containers as blank solutions. Then, known amounts of K.2.2.2 were added to the blank solutions to prepare three different concentrations of samples: 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL. Six identical samples were prepared for each concentration. Infrared spectra were acquired using an ATR-FTIR spectrometer; FTIR parameter settings: resolution 4 cm⁻¹. -1 The number of scans was 32, and the wavenumber range covered 4000-600cm. -1 10 μL of standard solution was added dropwise to ATR crystals, and the spectrum was collected after equilibration at room temperature for 15 seconds. A standard curve was established using the method in Example 2 to obtain the concentration of K.2.2.2 in each sample. The recovery rate was calculated as (actual measured concentration / theoretical added concentration) × 100%.

[0035] The accuracy test results are shown in Table 2.

[0036] Table 2 As can be seen from the results in Table 2, the accuracy of the measurement method in this technical solution is good.

[0037] 2. Precision test: 2.1 Intra-day precision: Select a K.2.2.2 sample with a concentration of 1.0 mg / mL, and on the same day, have the same operator use the same instrument to measure the sample 6 times consecutively, and calculate the relative standard deviation (RSD) of the 6 results.

[0038] 2.2 Intraday precision: Select K.2.2.2 samples with a concentration of 1.0 mg / mL, aliquot and store them, and measure them 3 times a day for 3 consecutive days by the same operator, and calculate the RSD of the 9 measurement results.

[0039] Results: The intra-day precision RSD was 2.1%, and the inter-day precision RSD was 2.8%, indicating that the determination method of this technical scheme has good stability within the same day and also has good stability between different days.

[0040] 3. Specificity testing: First, test the characteristic peaks of K.2.2.1 and K.2.2.0 in the FTIR spectrum separately, especially at 1110 cm⁻¹. -1 In the vicinity of the sample, a simulated degradation sample containing 0.5 mg / mL K.2.2.2 and 0.5 mg / mL K.2.2.1 was prepared. The concentration of K.2.2.2 in the mixed sample was measured using the accuracy test method described above, and the measured results were compared with the theoretical value (0.5 mg / mL).

[0041] Results: The specificity test yielded a K.2.2.2 concentration of 0.495 mg / mL, which is very close to the theoretical value. Furthermore, the concentrations of K.2.2.1 and K.2.2.0 were within the range of 1110 cm⁻¹. -1 The fact that there was almost no response indicates that the measurement method of this technical solution has good specificity.

[0042] Example 5: Actual sample analysis: Thirty batches of commercially available FDG cartridge kit eluent (batch numbers 202301-202330) were selected, and the methods of this invention, UV method, and HPLC method were used for determination. The three methods were named Experimental Groups 1-3, respectively. In Experimental Group 1, system suitability testing was performed before the first batch of samples each day; one replicate sample was inserted for every five samples.

[0043] The method for selecting characteristic peaks in experimental group 1 is the same as in Example 1, and the method for establishing the standard curve is the same as in Example 2; the quality control sample for system suitability testing is 1 mg / mL K.2.2.2.

[0044] Spectral acquisition: Preheat the FTIR spectrometer for 30 minutes, wipe it three times with anhydrous ethanol ATR crystals, and dry it with nitrogen; FTIR parameter settings: resolution 4cm. -1The number of scans was 32, and the wavenumber range covered 4000-600cm. -1 ; Take 20 μL of sample and add it to ATR crystal. After equilibration at room temperature for 15 seconds, collect the spectrum. The instrument automatically applies the second derivative and calculates the K.2.2.2 concentration using the PLS model and displays the result.

[0045] Results: After 30 days of continuous testing, the quality control sample results ranged from 0.96 to 1.04 mg / mL, with an RSD of 1.8%, indicating that the system suitability test was passed in all cases. In the repeated sample tests, 98.9% passed the repeatability check, with a relative deviation of <3%.

[0046] Experimental group 2 used the ultraviolet method for analysis. The main analytical method was as follows: 1 mL of sample was added to a quartz cuvette, and the absorbance was measured at a wavelength of 280 nm. The concentration of K.2.2.2 was calculated according to the standard curve.

[0047] Experimental group 3 used HPLC analysis. The main analytical method was as follows: the sample was filtered through a 0.45 μm filter, 20 μL was injected, separated by a C18 column, a UV detector at 254 nm, and the peak area of ​​K.2.2.2 was read after 25 minutes. The concentration was calculated according to the external standard method.

[0048] The test results of 30 batches of samples are shown in Table 3 below.

[0049] Table 3 Abnormal sample re-examination results: In batch 16, the initial UV method result (Experiment 2) was abnormally high, while the HPLC result (Experiment 3) was close to the result of the method of this invention (Experiment 1). Re-examination revealed that the sample contained a high concentration of degradation product K.2.2.1, which interfered with UV absorption. The method of this invention (FTIR-PLS) successfully corrected this interference. Similarly, in batch 24, the UV method was interfered with by both K2CO3 and degradation products, resulting in a large deviation in the results. However, the FTIR-PLS model of this invention effectively distinguished between the target analyte and the interfering analyte, resulting in a small deviation in the results.

[0050] As shown in Table 3, the method of the present invention is highly consistent with the HPLC reference method, with a deviation of <2%. The ultraviolet method shows a more serious deviation. Therefore, the method of the present invention has HPLC-level accuracy and has a significant advantage in analysis speed compared with the HPLC method, while maintaining excellent anti-interference ability.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for rapidly determining the K.2.2.2 content in the eluent of an FDG card sleeve reagent kit, characterized in that, Includes the following steps: S1. Characteristic peak selection: Select 1110±5cm. -1 The peak, as a quantitative characteristic peak of K.2.2.2, corresponds to the symmetric stretching vibration of the COC ether bond in the K.2.2.2 molecule; the peak value is 2253±3 cm⁻¹. -1 The peak serves as an internal standard, corresponding to the C≡N triple bond stretching vibration of acetonitrile in the eluent; S2, Standard curve establishment: Prepare 0.05-5 mg / mL K.2.2.2 gradient solutions, fixing the K2CO3 concentration at 0.5-2 mg / mL; Acquire spectra using ATR technology, setting the acquisition parameters to a resolution of 4-8 cm⁻¹. -1 The number of scans is 16-32, with A 1110 / A 2253 The ratio establishes a linear model for K.2.2.2; S3, interference correction: second derivative spectroscopy is used to eliminate baseline drift; and the PLS algorithm is used to correct the trace interference of K2CO3.

2. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, The ATR crystal is a diamond crystal with a surface temperature of 22-28°C, more preferably 24-26°C.

3. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S2, the concentration of K2CO3 is fixed at 0.8-1.2 mg / mL, and the concentration of acetonitrile is fixed at 8-12 vol.

4. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S2, the resolution is set to 4cm. -1 The number of scans was 16, and the wavenumber range of the acquired spectra was 4000-600 cm⁻¹. -1 .

5. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S2, before the first analysis each day, the suitability of the quality control sample for the verification system is determined.

6. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S3, the second derivative spectrum refers to the spectrum obtained by using the Savitzky-Golay second derivative method, with a window size of 13-17 points and a polynomial order of 2.

7. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S3, the use of the PLS algorithm to correct the trace interference of K2CO3 means that a correction model is constructed using the PLS algorithm to correct the trace interference of K2CO3, where the concentration of K2CO3 in the eluent is in the range of 0.5-2 mg / mL.

8. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, The principal component number of the PLS algorithm is 5-7.

9. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, In step S2, A 1110 / A 2253 The ratio is used to establish a linear model K.2.2.2, R. 2 >0.

998.

10. The method for rapidly determining the K.2.2.2 content in the eluent of the FDG card sleeve reagent kit according to claim 1, characterized in that, After step S3, the following steps are also included: S4, quantitative analysis: substituting the processed spectral data into the pre-established calibration model to calculate the K.2.2.2 content.

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