Method for determining impurity calcium in gadolinium-based contrast agent

Abstract

The invention discloses a method for determining impurity calcium in a gadolinium-based contrast agent, which comprises the following steps of: diluting a sample with nitric acid, directly determining by adopting ICP-MS / MS (Inductively Coupled Plasma Mass Spectrometry / Mass Spectrometry), adopting Sc as an internal standard solution, inhibiting ionization of plasma gas Ar through low temperature under a cold plasma condition, reducing formation of 40Ar < + >, adopting N2O as reaction gas in an MS / MS mode, generating 40Ca16O < + > based on O atom transfer reaction between 40Ca < + > and N2O, and determining the impurity calcium in the gadolinium-based contrast agent. Other interfering ions do not have mass transfer reaction with N2O, and mass spectrum interference is eliminated by using a mass transfer method. The accuracy, reliability and precision of the method are evaluated by analyzing a standard reference substance and performing standard recovery. The result shows that the detection limit of Ca is as low as 0.11 ng / L, and the measured value and the certified value of Ca in NIST SRM 1640a and NIST SRM 1643f are basically consistent. The work represents a systematic method for accurately quantifying the challenging element Ca in the gadolinium-based contrast agent through ICP-MS / MS, can be expanded and applied to other fields, and has great reference value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for determining impurity calcium in a gadolinium-based contrast agent. Background Art

[0002] Gadolinium-based contrast agent (GBCA) injection is sealed in a pharmaceutical glass bottle, and calcium oxide is usually added to pharmaceutical glass to lower the melting temperature of the glass and improve the tolerance of the inner surface of the glass. Due to the interaction between GBCA and the inner surface of the glass, the Ca element in the glass may migrate into the GBCA, affecting the quality and safety of GBCA. According to the "Technical Guidelines for Compatibility Studies of Chemical Drug Injections and Pharmaceutical Glass Packaging Containers", the Ca content in GBCA must be accurately determined to fully understand the migration characteristics of Ca in glass and correctly evaluate the safety of GBCA.

[0003] Among various atomic spectroscopy techniques used to determine Ca, inductively coupled plasma mass spectrometry (ICP-MS) has been widely used. Ca has 6 stable isotopes, namely 40 Ca, 42 Ca, 43 Ca, 44 Ca, 46 Ca and 48 Ca, among which 40 Ca and 44 The abundances of Ca were 96.94% and 3.06%, respectively, and the abundances of the remaining four isotopes were too low to be used for ICP-MS analysis. 40 Ca + The main mass spectrometric interferences are from the isobaric isotopes formed by the plasma gas Ar. 40 Ar + , even with high-resolution ICP-MS (SF-ICP-MS), it is impossible to separate them. Existing ICP-MS analysis can only select low-abundance 44 Ca is an analytical isotope, which results in low analytical sensitivity. Cold plasma technology avoids a large amount of 40 Ar + formation, but it is still possible to form a small amount 40 Ar + interference 40 Ca + At the same time, due to the metastable ion NO + The ionization potential of Ar is close to that of Ar, even without 40 Ar + Formation, NO + It also undergoes a charge transfer reaction with Ar (NO + +Ar→NO+Ar + ),generate 40Ar + Still interfering 40 Ca + The collision / reaction cell (CRC) technology provides a general method for eliminating mass spectrometry interferences, among which the H2 reaction mode can quickly eliminate Ar ion interferences, providing a general method for ICP-MS to eliminate mass spectrometry interferences, but it is still extremely challenging for the determination of Ca. Under cold plasma conditions combined with the H2 reaction mode, the Ar-based ion interference can be completely eliminated, but all ions from the plasma enter the CRC. Due to unpredictable side reactions, there is still a possibility that the interference cannot be completely eliminated. The emergence of inductively coupled plasma tandem mass spectrometry (ICP-MS / MS) technology has made it possible to select high-abundance 40 In order to make the analysis of Ca isotopes possible, the reaction process in CRC can be controlled by controlling the m / z of the primary quadrupole mass filter (Q1). The main method at present is to block some ions outside CRC through Q1, use H2 reaction gas under cold plasma conditions, and use in-situ mass method to eliminate interference. However, for some polyatomic ions (such as 24 Mg 16 O + ) is not completely eliminated, and high-sensitivity and accurate determination of Ca cannot be achieved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a method for determining the impurity calcium in gadolinium-based contrast agents. N2O is used as a reaction gas to eliminate mass spectrometry interference, and ICP-MS / MS is used in cold plasma / reaction mode to determine the content of Ca in GBCA.

[0005] To achieve the above object, the present invention adopts the following technical solution: a method for determining impurity calcium in a gadolinium-based contrast agent, characterized in that it comprises the following steps:

[0006] (1) Preparation of calibration solution: dilute the Ca standard solution with nitric acid solution to prepare a series of concentration calibration solutions;

[0007] (2) Preparation of internal standard solution: Take 10 mg / L of Li, Sc, Ge, Y, In, Tb, Bi internal standard mixed solution and dilute it with nitric acid solution to prepare internal standard solution;

[0008] (3) sample treatment, diluting the sample with a nitric acid solution to obtain a sample solution;

[0009] (3) On-machine testing: use a standard T-type internal standard mixing joint to mix the internal standard solution into the sample solution, calibration solution, and blank solution online; in MS / MS mode, select N2O as the reaction gas and eliminate mass spectrometry interference through O atom transfer; according to CaO +With internal standard ion ScO + The relative signal intensity of the calibration solution corresponding to the concentration was linearly regressed to establish a calibration curve and calculate the Ca content in the sample solution; the isotope selection was analyzed 40 Ca + .

[0010] The ICP-QQQ operating parameters were:

[0011] Scan typeMS / MS

[0012] RF power 600W

[0013] Carrier gas flow rate 0.70L / min,

[0014] Compensation gas flow rate 0.8L / min,

[0015] Sampling depth 18mm

[0016] Atomization chamber temperature 2°C

[0017] Reaction gas N2O

[0018] Reaction gas flow rate 0.5mL / min

[0019] Octopole bias voltage -16V

[0020] Kinetic energy discrimination voltage -5V,

[0021] The mass-to-charge ratio (m / z) of the first-stage quadrupole mass filter (Q1) was set to 40 and 44,

[0022] The mass-to-charge ratio (m / z) of the second-stage quadrupole mass filter (Q2) was set to 56 and 60, respectively.

[0023] In the above scheme: the mass concentration of the nitric acid solution is 2%.

[0024] In the above scheme: the concentrations of the series of concentration calibration solutions are Ca series calibration solutions with concentrations of 0, 1.0, 5.0, 20, 100, and 500 μg / L, respectively.

[0025] In the above scheme: the concentration of the internal standard solution is 1 mg / L.

[0026] In the above scheme: the sample is diluted 100 times with nitric acid having a mass concentration of 2% to obtain a sample solution.

[0027] The molecular-ion interaction process of using N2O as a reaction gas to eliminate mass spectrum interference includes: O atom transfer reaction, N atom transfer reaction, charge transfer reaction and addition reaction, which provides multiple possibilities for eliminating mass spectrum interference. The present invention uses ICP-MS / MS to determine the Ca content in GBCA in cold plasma / reaction mode, selects N2O as a reaction gas in MS / MS mode, and uses N2O and Ca for the first time based on the fact that N2O is a more effective O atom transfer gas than O2. + The efficient O atom transfer reaction eliminates interference and can achieve high sensitivity and interference-free accurate determination of Ca content in GBCA. It has the characteristics of simplicity, rapidity, safety, reliability and high analytical accuracy.

[0028] In ICP-MS / MS, N2O was used as the reaction gas under cold plasma conditions. + O atom transfer reaction with N2O to generate CaO + (Ca + +N2O→CaO + +N2), while other interfering ions do not undergo mass transfer reaction with N2O. The mass transfer method is used to eliminate mass spectrometry interference, and Ca + →CaO + The mass spectrometry interference was completely eliminated for the ion pair determination of Ca content in GBCA. Compared with the common H2 on-site mass method under cold plasma conditions, the sensitivity of the N2O mass shift method is slightly lower, but the established analytical method has lower BEC and LOD, and achieves accurate determination of two Ca isotopes at the ng / L level, providing technical support for the determination of Ca content in GBCA, and it can be expected that this method can be easily expanded and applied to other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 2 shows the mass spectra of Ca in different reaction modes under cold plasma conditions. 500 μg / L Mg and 500 μg / L Si standard solutions were intentionally added to 1 μg / L Ca standard solution as interfering elements. (a) N2O mass transfer method was used to measure Ca. 40 Ca + ; (b) N2O mass transfer method was used to measure 44 Ca + .

[0030] Figure 2 To obtain the 40 Ca 16 O + and 44 Ca 16 O + The BEC and the octopole bias voltage (V oct )relation.

[0031] Figure 3 Effect of N2O flow rate on target product ion CaO in cold plasma / N2O reaction mode + The impact of signal strength.

[0032] Figure 4 The stability of Ca analysis signal within 2 h in cold plasma / N2O reaction mode. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below through specific embodiments:

[0034] Example 1

[0035] 1. Instruments and Equipment

[0036] Inductively coupled plasma tandem mass spectrometer (ICP-MS / MS 8800, Agilent, USA) was equipped with a quartz torch with a central inner diameter of 2.5 mm, a MicroMist glass concentric nebulizer, a Peltier-cooled quartz double-pass Scott-type nebulizer, and an octopole collision reaction system (ORS). The ICP-MS / MS operating parameters are shown in Table 1

[0037] Table 1 ICP-MS / MS operating parameters

[0038]

[0039] 2 Reagents and Materials

[0040] 1000 mg / L single element standard solutions of Ca, Mg, and Si were from the National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials; 10 mg / L internal standard mixed solutions of Li, Sc, Ge, Y, In, Tb, and Bi were from Agilent, USA; 65% nitric acid solution was purchased from Merck, Germany; standard reference materials NIST SRM 1640a (trace elements in natural water) and NIST SRM1643f (trace elements in water) were from the National Institute of Standards and Technology of the United States; GBCA samples were gadoterate meglumine injection (specification: 15 mL) and gadopentetate meglumine injection (specification: 12 mL) produced by four different domestic manufacturers.

[0041] 3 Experimental methods

[0042] The GBCA sample was diluted 100 times with 2% nitric acid to obtain a sample solution (if the concentration exceeded the linear range, it was further diluted to within the linear range). The Ca standard solution was diluted with 2% nitric acid to prepare a series of Ca calibration solutions of 0, 1.0, 5.0, 20, 100, and 500 μg / L. Under the same mass spectrometry conditions, the calibration solution, blank solution (nitric acid with a mass concentration of 2%), and sample solution were measured by ICP-MS / MS, and 1 mg / L of the internal standard mixed solution was added online through a standard "T" type internal standard mixing joint. In the MS / MS mode, N2O was selected as the reaction gas, and mass spectrometry interference was eliminated by O atom transfer. According to CaO + With internal standard ion ScO + The relative signal intensity was linearly regressed with the concentration of the calibration solution corresponding to the sample to establish a calibration curve and calculate the Ca content in the sample solution.

[0043] 4 Results and discussion

[0044] 4.1 Mass spectrometric behavior of Ca in MS / MS reaction mode

[0045] The most abundant isotope of Ca 40 Ca + Influenced by 40 Ar + and 24 Mg 16 O + potential mass spectral interferences, while low-abundance isotopes 44 Ca + Affected by polyatomic ions from C, N, O, and Si 12 C 16 O2 + , 14 N2 16 O + , 28 Si 16 O + He collision mode provides a general technique for eliminating polyatomic ion interferences. 44 Ca + The mass spectrum is affected by the kinetic energy discrimination of the collision mode and the limitation of low abundance. 44 Ca + The sensitivity is very low. 40 Ca isotope can greatly improve the analytical sensitivity, but He collision mode cannot be eliminated 40 Ar + right 40 Ca +The ability of reaction mode to eliminate interference is significantly better than that of collision mode, but the reaction process between ions and gases in ORS and the types of product ions must be known in advance to prevent new interference. In MS / MS mode, O2 as a reaction gas cannot eliminate 40 Ar 40 In order to prevent the interference of Ca, this paper uses N2O as the reaction gas and compares it with H2, which is the most commonly used reaction gas at present. 40 Ca and 44 The analytical sensitivity and background equivalent concentration (BEC) of Ca are shown in Table 2.

[0046] Table 2 In MS / MS mode 40 Ca and 44 Ca Sensitivity and Background Equivalent Concentration (BEC)

[0047]

[0048] In MS / MS mode, set the m / z of Q1 to 40, allowing only 40 Ca + and its potential interfering ions 40 Ar + , 24 Mg 16 O + Enter ORS; set the m / z of Q1 to 44 and only allow 44 Ca + and its potential interfering ions 12 C 16 O2 + , 14 N2 16 O + , 28 Si 16 O + Entering the ORS, preventing a large number of ions with m / z other than 40 and 44 from the plasma from entering the ORS, thereby precisely controlling the reaction pathway and product ions between the ions in the ORS and the reaction gas. 40 Ar + The formation of N2O as the reaction gas, Ca + With N2O, an efficient O atom transfer reaction (Ca + +N2O→CaO + +N2), while interfering ions 24 Mg 16 O + , 12 C 16 O2 + , 14 N2 16 O+ and 28 Si 16 O + There is no O atom transfer reaction with N2O, and these interfering ions are eliminated, even if there is a small amount of 40 Ar + Since the ionization energy of Ar (15.6 eV) is greater than that of N2O (12.9 eV), Ar + It will react with N2O with high efficiency charge transfer reaction (Ar + +N2O→Ar+N2O + , reaction enthalpy (ΔH r )<0), thereby eliminating Ar + Ca + Therefore, the N2O mass transfer method can be used to eliminate the interference of Ca + The interference suffered.

[0049] In MS / MS mode, H2 was used as the reaction gas, Ca + Does not react with H2, even if a small amount is generated under cold plasma conditions 40 Ar + , and can also quickly react with H2 to eliminate Ca + At the same time, other interfering ions ( 24 Mg 16 O + , 12 C 16 O2 + , 14 N2 16 O + , 28 Si 16 O + etc.) can also undergo mass transfer reactions with H2, so Ca can be eliminated by H2 in-situ mass method. + However, since H2 can only react relatively quickly with argon-based ions and reacts at a lower rate with other interfering ions, the ability to eliminate interference using H2 on-site mass method is very limited. Although the sensitivity obtained in H2 reaction gas mode is higher than that in N2O reaction mode, the background equivalent concentration (BEC) is higher than that in N2O reaction mode, indicating that the interference is not completely eliminated in H2 reaction mode.

[0050] To further evaluate the ability of N2O mass-shift method to eliminate interference, 500 μg / L Mg and 500 μg / L Si were deliberately added to the 1 μg / L Ca standard solution as interfering elements. 40 Ca + and 44 Ca +The mass spectrometric behavior in N2O reaction mode and H2 reaction mode is shown in the results. Figure 1 It can be seen that when N2O is used as the reaction gas, 40 Ca + and 44 Ca + The interference can be completely eliminated by mass-shift method ( Figure 1 (a) and Figure 1 (b)) confirms that the interference can be completely eliminated by using the N2O mass-shift method under cold plasma conditions.

[0051] 4.2 Optimization of ORS parameters

[0052] Ca + The progress of the reaction with N2O is mainly controlled by the octopole bias potential (V oct ), by setting V oct is a negative voltage, Ca + is accelerated by increasing Ca before the reaction + The kinetic energy accelerates Ca + The mass transfer reaction with N2O increases the CaO + The yield of Ca is improved, but the negative V oct Will cause the BEC of Ca to increase. 40 Ca and 44 Ca in different V oct BEC( Figure 2 ),Depend on Figure 2 It can be seen that as V oct The more negative, 40 Ca and 44 The BEC of Ca gradually decreases. oct When it is -16V, 40 Ca and 44 The BEC of Ca reaches the minimum value. Therefore, V oct -16V, ensuring that the BEC of Ca is still at a minimum value while providing a large amount of additional energy, thus accelerating Ca + The purpose of the reaction process with N2O.

[0053] The reaction yield of N2O in ORS is mainly controlled by the reaction gas velocity. The greater the N2O flow rate, the greater the density of N2O in ORS. + The reaction yield with N2O is also greater when Ca + After the reaction with N2O reaches equilibrium, excessive N2O will hinder the target product ion CaO + The transmission efficiency of N2O decreased, resulting in a decrease in the analytical signal intensity and affecting the analytical sensitivity. + Transfer to form CaO +The results are shown in Figure 3 It can be seen that 40 Ca + and 44 Ca + The mass spectrometric behaviors of CaO + The signal intensity gradually increases, indicating that a larger N2O flow rate is beneficial to CaO + When the N2O flow rate reaches 0.5mL / min, CaO + The signal intensity is the largest, and then the N2O flow rate is increased, CaO + The signal intensity began to decrease. Therefore, the N2O flow rate was selected to be 0.5 mL / min to ensure that the target product ion CaO + The sensitivity is the greatest.

[0054] 4.3 Stability of the analytical process

[0055] GBCA has many types and complex matrix composition. In this experiment, the internal standard method was used for calibration. A 5 μg / L Ca standard solution was added to the GBCA sample. 45 Sc + , 89 Y + , 103 R + and its oxygen atom transfer product ions ( 45 Sc 16 O + , 89 Y 16 O + , 103 R 16 O + ) was used as the internal standard ion, and the determination was repeated 6 times to investigate the effect of different internal standard ions on the recovery rate of Ca spike addition. The results are shown in Table 3. It can be seen that the recovery rates of all internal standard elements are between 93.8% and 105.2%, among which the recovery rates of Ca are 96.0% and 102.4% respectively when Sc is used as the internal standard element, which is better than using Y and Rh as the internal standard elements; using mass transfer products as internal standard ions is significantly better than in-situ mass product ions. Therefore, the experiment was selected 45 Sc 16 O + as the internal standard ion.

[0056] Table 3 Effect of internal standard elements on Ca recovery in GBCA

[0057]

[0058] Compared with thermal plasma, low-temperature cold plasma provides low energy, low matrix decomposition efficiency, and poor tolerance to high-matrix samples. As the analysis time goes by, deposits may form on the interface cone and ion lens, affecting the stability of the analysis signal. In this experiment, a 100-fold dilution was performed during sample processing to maintain the Gd concentration in the GBCA sample solution at <800 mg / L, and the stability of the analysis process was confirmed by monitoring the signal stability over a long period of time. A 5 μg / L Ca standard solution was added to the GBCA sample solution for long-term stability testing, and the signal was measured every 10 minutes. 40 Ca + and 44 Ca + The signal intensity was measured 6 times each time to investigate the stability of Ca in the spiked sample solution for 2 h under cold plasma conditions. Figure 4 It can be seen that the normalized ion counts of Ca varied between 1 ± 0.05, indicating good stability of the analysis process.

[0059] 4.4 Evaluation of analytical performance of the method

[0060] The Ca series calibration solutions were measured according to the operating conditions of ICP-MS / MS, and the calibration curve was established and linear regression was performed. The detection limit (LOD) and quantification limit (LOQ) of Ca were obtained by measuring the blank solution 10 times and 3 times the standard deviation. The results showed that 40 Ca and 44 The linear relationship of Ca was good (the linear correlation coefficients were 1.0000 and 0.9999, respectively). 40 Ca and 44 The LOD of Ca was 0.11ng / L and 0.16ng / L, and the LOQ was 3.7ng / L and 5.2ng / L, respectively. Compared with the ICP-MS reported in the literature, the LOD of this method was at the lowest level.

[0061] Standard reference materials (NIST SRM 1640a and NIST SRM 1643f) were used to evaluate the accuracy, reliability and precision of the method. 40 Ca was the analytical isotope, and each sample was measured 6 times and spiked and recovered, and the results are shown in Table 4. It can be seen that the measured values ​​of Ca in the two standard reference materials are basically consistent with the certified values, with relative standard deviations (RSDs) of 0.8% and 1.2%, respectively, and spiked recoveries of 103.3% and 98.8%, respectively, which verifies that the established method has good accuracy, reliability and high precision.

[0062] Table 4 Analysis results of Ca in NIST SRM 1640a and NIST SRM 1643f

[0063]

[0064] 4.5 Sample analysis

[0065] In order to verify the applicability of the established analytical method, four samples of gadoterate dimeglumine injection (sample numbers A and B) and gadopentetate dimeglumine injection (sample numbers C and D) produced by different manufacturers were selected and selected using this method. 40 Ca was analyzed as an isotope, and SF-ICP-MS was used for comparative analysis. Each sample was measured 6 times, and the t test method was used for statistical analysis of the two analysis methods. As can be seen from Table 5, at a confidence level of 95%, the results of this method were not significantly different from those of SF-ICP-MS (p>0.05). There are certain differences in the Ca content in different GBCA samples. According to the FDA's maximum allowable dose of GBCA of 0.3mmol / kg, the maximum daily dose is converted to 42mL (based on 70kg). The maximum possible daily intake of Ca is calculated based on this. According to the Chinese Pharmacopoeia (2020 edition), the PDE limit standard for Ca in injection is 100μg / day. It can be seen that the Ca content in the four GBCAs is less than the limit requirement, indicating that the residual and migration risks of Ca impurities introduced by GBCA during the production process are basically controllable.

[0066] Table 5 Analysis results of Ca in samples

[0067]

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for determining impurity calcium in a gadolinium-based contrast agent, characterized in that: The following steps are involved: (1) Preparation of calibration solution: dilute the Ca standard solution with nitric acid solution to prepare a series of concentration calibration solutions; (2) Preparation of internal standard solution: Take 10 mg / L of Li, Sc, Ge, Y, In, Tb, Bi internal standard mixed solution and dilute it with nitric acid solution to prepare internal standard solution; (3) sample treatment, diluting the sample with a nitric acid solution to obtain a sample solution; (3) On-line testing: using a standard T-type internal standard mixing joint to mix the internal standard solution into the sample solution, calibration solution, and blank solution; In MS / MS mode, N2O was selected as the reaction gas to eliminate mass spectrum interference through O atom transfer; according to CaO + With internal standard ion ScO + The relative signal intensity of the calibration solution corresponding to the concentration was linearly regressed to establish a calibration curve and calculate the Ca content in the sample solution. The ICP-QQQ operating parameters were: Scan typeMS / MS RF power 600W Carrier gas flow rate 0.70L / min, Compensation gas flow rate 0.8L / min, Sampling depth 18mm Atomization chamber temperature 2°C Reaction gas N2O Reaction gas flow rate 0.5mL / min Octopole bias voltage -16V Kinetic energy discrimination voltage -5V, The mass-to-charge ratio (m / z) of the first quadrupole mass filter (Q1) was set to 40.

2. The method for determining the calcium content in a gadolinium-based contrast agent by inductively coupled plasma tandem mass spectrometry according to claim 1, characterized in that: The mass concentration of the nitric acid solution is 2%.

3. The method for determining the calcium content in a gadolinium-based contrast agent by inductively coupled plasma tandem mass spectrometry according to claim 2, characterized in that: The concentrations of the series of concentration calibration solutions are 0, 1.0, 5.0, 20, 100, and 500 μg / L Ca series calibration solutions, respectively.

4. The method for determining the calcium content in a gadolinium-based contrast agent by inductively coupled plasma tandem mass spectrometry according to claim 3, characterized in that: The concentration of the internal standard solution was 1 mg / L.

5. The method for determining the calcium content in a gadolinium-based contrast agent by inductively coupled plasma tandem mass spectrometry according to claim 4, characterized in that: The sample was diluted 100 times with nitric acid having a mass concentration of 2% to obtain a sample solution.

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