Method for measuring concentration of inorganic copper by liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for measuring the concentration of inorganic copper through liquid chromatography-tandem mass spectrometry, and relates to the technical field of detection. The method comprises the following steps: extracting inorganic copper in a sample by adopting acetic acid to obtain an extracting solution; adding a DDDC-Na solution into the extracting solution, mixing, standing, and deriving inorganic copper in the sample into copper diethyl dithiocarbamate to obtain a derived solution; and purifying the derivatized liquid through liquid-liquid extraction and / or solid-phase extraction, and injecting the purified liquid into a liquid chromatography tandem mass spectrometer for detection. Interference of other ions can be reduced, and complex samples can be analyzed. Strong acid, strong alkali and toxic compounds are not used in the pretreatment process, the steps are simple, and operation is easy. The method is high in sensitivity and analysis speed, the detection lower limit LOD is 0.00001 mg / L, and the quantification lower limit LOQ is 0.0005 mg / L. And the analysis speed can reach 2-3 minutes for each sample.

Description

Cross-reference to related applications

[0001] This disclosure claims priority to Chinese Patent Application No. 2025106893380, filed on May 27, 2025, entitled "A Method for Determining Inorganic Copper Concentration by Liquid Chromatography-Tandem Mass Spectrometry", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of detection technology, and more specifically, to a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry. Background Technology

[0003] Copper ions (Cu) 2+ Copper ions show no significant absorption in the visible light region and require a colorimetric reagent (such as dithizone, sodium diethyldithiocarbamate (DDTC), or neocubic copper reagent) to form a colored complex. Currently, there are methods for detecting copper ion concentration using a UV spectrophotometer based on this colorimetric reaction. The steps are as follows: (1) Sample pretreatment: Digestion: If the sample contains organic matter or solids (such as soil or biological tissue), it needs to be digested by microwave or wet digestion with nitric acid (HNO3) and hydrogen peroxide (H2O2) to convert it into a clear solution.

[0004] Filtration: Removes insoluble impurities and avoids interference.

[0005] (2) Adjust the pH value: Adjust the solution to a weakly alkaline state (pH 8-9) by adding ammonia (NH3·H2O) and confirm with a pH meter or precision test paper.

[0006] Add a buffer solution (such as NH3-NH4Cl buffer) to maintain the stability of the reaction system.

[0007] (3) Color reaction: Add the colorimetric reagent DDTC solution (0.1% aqueous solution), shake well, and let stand for 10-15 minutes to allow the complex to fully form.

[0008] (4) Eliminate interference: Add masking agents (such as EDTA, ammonium citrate) to mask Fe 3+ Pb 2+ Interfering ions.

[0009] If the solution is turbid, centrifugation or filtration is required to remove the precipitate.

[0010] (5) Measure absorbance: The absorbance was measured at a wavelength of 440 nm using a blank solution (a reagent solution without copper) as a reference.

[0011] Standard curve method: Prepare a series of copper standard solutions with different concentrations (e.g., 0, 0.2, 0.5, 1.0, 2.0 mg / L), measure the absorbance after color development, and plot the concentration-absorbance standard curve.

[0012] However, the above-mentioned method for detecting copper ion concentration using an ultraviolet spectrophotometer is susceptible to Fe. 3+ Al 3+ Plasma interference and inability to analyze complex samples are common problems. Traditional methods, which use ultraviolet light to detect standards, have difficulty analyzing samples below 0.5 mg / L. Furthermore, their pretreatment processes involve strong acids, strong bases, and toxic compounds, making the procedures complex and unsafe.

[0013] In view of this, the present invention is proposed. Summary of the Invention

[0014] The purpose of this invention is to provide a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry.

[0015] This invention is implemented as follows: In a first aspect, the present invention provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, comprising: The inorganic copper in the sample was extracted using acetic acid to obtain the extract. Add DDTC-Na solution to the extract, mix, let stand, and derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain the derivatized solution; The derivatized liquid was purified by liquid-liquid extraction and / or solid-phase extraction, and then injected into a liquid chromatography-tandem mass spectrometer for detection.

[0016] In an optional embodiment, the concentration of the acetic acid added to the sample is 10%-30%; And / or, the extraction time is 5-15 min; And / or, the sample includes a water sample, a soil sample, or a biological sample; when the sample is a water sample, acetic acid solution is added directly to the water sample; when the sample is a soil sample or a biological sample, acetic acid solution is added at a ratio of 1g:4-6mL; In an optional embodiment, the volume ratio of the extract to the DDTC-Na solution is 1 mL: 1-3 mL; And / or, the solvent of the DDTC-Na solution is methanol; And / or, the concentration of the DDTC-Na solution is 0.5-2 g / L; And / or, the settling time is 10-30 minutes.

[0017] In an optional embodiment, the liquid-liquid extraction includes extracting the derivatized liquid with an extraction reagent, taking the organic phase, drying it, and then resolving it with a redissolving reagent.

[0018] In an optional embodiment, the extraction reagent includes at least one of cyclohexane, n-hexane, and toluene; And / or, the drying includes drying using a nitrogen blower; And / or, the redissolving agent includes at least one of acetonitrile and methanol.

[0019] In an optional embodiment, the solid-phase extraction includes passing the derivatized liquid through a solid-phase extraction column for adsorption, followed by elution with an elution reagent.

[0020] In an optional embodiment, the elution agent includes at least one of acetonitrile and methanol.

[0021] In an optional embodiment, the conditions for determination by the liquid chromatography-tandem mass spectrometry are as follows: (1) Chromatographic conditions: Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50 mm, 1.8-Micron; column temperature: 30-40℃; flow rate: 0.5-0.7 mL / min; injection volume: 1 µL; external standard method for quantification; Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; The gradient elution procedure is as follows: 0.00-0.80 min: The concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%; 0.80-0.90 min: The concentration of mobile phase A changes from 65%-75% to 15%-25%, and the concentration of mobile phase B changes from 25%-35% to 75%-85%; 0.90-2.20 min: The concentration of mobile phase A changes from 15%-25% to 3%-8%, and the concentration of mobile phase B changes from 75%-85% to 92%-97%; 2.20-2.30 min: The concentration of mobile phase A changes from 3%-8% to 65%-75%, and the concentration of mobile phase B changes from 92%-97% to 25%-35%; 2.30-3.0 min: The concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%; (2) Mass spectrometry conditions: Ion source: Electrospray ionization (ESI); Scan mode: MRM mode; Mass analyzer: tandem quadrupole; Drying gas temperature: 300℃; Drying gas flow rate: 5 L / min; Nebulizer gas pressure: 45 psi; Sheath gas temperature: 250 ℃; Sheath gas flow rate: 11 L / min; Capillary voltage: 3500 V; Nozzle voltage: 500 V; MRM mode parameters: Copper Diethyldithiocarbamate (Positive); *361>116.2 (Fragmentation voltage: 100 V; Collision energy: 30 V; Acceleration voltage: 5 V); 361>88.1 (Fragmentation voltage: 100 V; Collision energy: 40 V; Acceleration voltage: 5 V); Scan time (msec): 50.

[0022] In an optional embodiment, before performing the liquid chromatography-tandem mass spectrometry (LC-MS / MS) determination, the method further includes plotting a quantitative curve: weighing an inorganic copper standard substance, making up to volume with acetic acid solution to prepare a standard stock solution; diluting the stock solution with acetic acid solution to prepare the required standard working solution; derivatizing and purifying the standard working solution in the same manner as the sample; and then detecting it using liquid chromatography-tandem mass spectrometry (LC-MS / MS) and establishing a quantitative curve.

[0023] In an optional embodiment, the limit of detection (LOD) of the liquid chromatography-tandem mass spectrometry method for determining the concentration of inorganic copper is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L.

[0024] The present invention has the following beneficial effects: The method for determining inorganic copper concentration using liquid chromatography-tandem mass spectrometry (LC-MS / MS) provided by this invention reduces interference from other ions through sample extraction, derivatization, and purification pretreatment. Subsequent separation and ion pair qualitative analysis using the LC-MS / MS column allows for the analysis of complex samples. The pretreatment process avoids the use of strong acids, strong bases, and toxic compounds, is simple, requires minimal operator skill, and utilizes readily available instruments in analytical laboratories. Traditional methods using UV detection standards struggle to reach concentrations below 0.5 mg / L, while this method, using LC-MS / MS, can detect inorganic copper standards down to 0.0005 mg / L. Sample concentration can be further enhanced through pretreatment, achieving a detection limit of 0.00001 mg / L. Furthermore, LC-MS / MS offers rapid analysis, with each sample requiring only 2-3 minutes of analysis. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Standard curves for copper hydroxide (0.002-0.15 mg / L) (copper ions (0.0013-0.098 mg / L) were prepared. Figure 2 It is a standard of 0.05 mg / L copper hydroxide (copper ions 0.0325). Figure 3 The recovered sample was added to fishpond water containing 0.0001 mg / L copper hydroxide (0.000065 copper ions). Figure 4 Chromatogram of inorganic copper in soil sample; Figure 5 This is a chromatogram of inorganic copper in a plasma sample. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] This invention provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, comprising the following steps: S1. Extraction solution was obtained by extracting inorganic copper from the sample using acetic acid.

[0029] In this invention, the samples include water samples, soil samples, or biological samples. When the sample is a water sample, an acetic acid solution is added directly to the water sample to make the acetic acid concentration reach 10%-30%. The source of the water sample includes, but is not limited to, river water, tap water, and fishpond water. When the sample is a soil sample or a biological sample, an acetic acid solution with a concentration of 10%-30% is added at a ratio of 1g:4-6mL. The source of the soil sample includes, but is not limited to, paddy soil and black soil. The source of the biological sample includes, but is not limited to, whole blood and plasma.

[0030] After adding acetic acid solution, the inorganic copper (such as Cu) in the sample is removed using acetic acid. 2+ Cu + Extraction was performed using (etc.) for 5-15 minutes; acetic acid can dissolve the sample, generating soluble copper acetate. Furthermore, acetate ions (CH3COO)- It can be used with Cu 2+ A complex is formed ([Cu(CH3COO))). + This process promotes the dissolution and stabilization of copper. During extraction, some cuprous ions are unstable in aqueous solution and can undergo a spontaneous disproportionation reaction (2Cu₂O₃ + acetic acid) under acetic acid conditions. + →Cu 0 +Cu 2+ ).

[0031] S2. Add DDTC-Na solution to the extract, mix and let stand to derivatize the inorganic copper in the sample into copper diethyldithiocarbamate, and obtain the derivatized solution.

[0032] DDTC-Na (sodium diethyldithiocarbamate) for Cu 2+ It has high selectivity and can react with Cu 2+ The complex Cu(DDTC)₂ is formed. The solvent for the DDTC-Na solution is methanol, and the concentration of the DDTC-Na solution is 0.5-2 g / L. The volume ratio of the extract to the DDTC-Na solution is 1 mL: 1-3 mL. In this invention, by limiting the concentration and amount of DDTC-Na, it is possible to ensure that it fully reacts with the Cu in the extract. 2+ Complexation can be achieved by mixing and then allowing the mixture to stand for 10-30 minutes to ensure a more complete reaction.

[0033] S3. The derivatized liquid is purified by liquid-liquid extraction and / or solid-phase extraction.

[0034] Due to the use of acetic acid to extract copper ions and the use of DDTC-Na with Cu 2+ Both of these can be affected by other metal elements (such as Fe) when forming complexes. Directly injecting and detecting the derivatized liquid will result in a lot of interference and make accurate detection impossible.

[0035] Therefore, the present invention also purifies the above-mentioned derivatized liquid. Specifically, liquid-liquid extraction includes extracting the derivatized liquid with an extraction reagent, taking the organic phase, drying it, and then redissolving it with a redissolving reagent.

[0036] The extraction reagent includes, but is not limited to, at least one of cyclohexane, n-hexane, and toluene; and / or, drying includes drying with a nitrogen evaporator; and / or, the redissolving reagent includes, but is not limited to, at least one of acetonitrile and methanol.

[0037] Solid-phase extraction involves passing the derivatized liquid through a solid-phase extraction column for adsorption, followed by elution with an eluent. The eluent includes, but is not limited to, at least one of acetonitrile and methanol.

[0038] The present invention uses liquid-liquid extraction and / or solid-phase extraction to effectively remove interfering factors from the derivatized liquid. Therefore, the present invention can analyze complex samples and expand the sample range.

[0039] S4. Preparation of standard stock solution and standard working solution.

[0040] Weigh out the inorganic copper standard substance, dilute it to volume with acetic acid solution, and prepare a standard stock solution; dilute the stock solution with acetic acid solution to prepare the required standard working solution; derivatize and purify the standard working solution in the same way as the sample, and then detect it using liquid chromatography-tandem mass spectrometry and establish a quantitative curve.

[0041] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer for detection.

[0042] The conditions for determination using liquid chromatography-tandem mass spectrometry are as follows: (1) Chromatographic conditions: Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50 mm, 1.8-Micron; column temperature: 30-40℃; flow rate: 0.5-0.7 mL / min; injection volume: 1 µL; external standard method for quantification; Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; The gradient elution procedure is as follows: 0.00-0.80 min: The concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%; 0.80-0.90 min: The concentration of mobile phase A changes from 65%-75% to 15%-25%, and the concentration of mobile phase B changes from 25%-35% to 75%-85%; 0.90-2.20 min: The concentration of mobile phase A changes from 15%-25% to 3%-8%, and the concentration of mobile phase B changes from 75%-85% to 92%-97%; 2.20-2.30 min: The concentration of mobile phase A changes from 3%-8% to 65%-75%, and the concentration of mobile phase B changes from 92%-97% to 25%-35%; 2.30-3.0 min: The concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%; (2) Mass spectrometry conditions: Ion source: Electrospray ionization (ESI); Scan mode: MRM mode; Mass analyzer: tandem quadrupole; Drying gas temperature: 300℃; Drying gas flow rate: 5 L / min; Nebulizer gas pressure: 45 psi; Sheath gas temperature: 250 ℃; Sheath gas flow rate: 11 L / min; Capillary voltage: 3500 V; Nozzle voltage: 500 V; MRM mode parameters: Copper Diethyldithiocarbamate (Positive); *361>116.2 (Fragmentation voltage: 100 V; Collision energy: 30 V; Acceleration voltage: 5 V); 361>88.1 (Fragmentation voltage: 100 V; Collision energy: 40 V; Acceleration voltage: 5 V); Scan time (msec): 50.

[0043] The limit of detection (LOD) for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1 Please combine Figure 1 , Figure 2 and Figure 3 This embodiment provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes the following steps: S1. Add acetic acid solution to the water sample (fishpond water) to make the acetic acid concentration reach 20%, vortex mix for 10 min to obtain the extract.

[0046] S2. Take 1 ml of extract, add 2.0 mL of 1 g / L DDTC-Na methanol solution, mix and let stand for 20 min, and derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain the derivatized solution.

[0047] S3. Take the derivatized solution, add cyclohexane, and extract copper diethyldithiocarbamate into the organic solvent. Use a nitrogen blower to dry the extraction solvent, redissolve it with acetonitrile, and detect the redissolved solution using liquid chromatography-tandem mass spectrometry.

[0048] S4. Weigh an appropriate amount of inorganic copper standard substance and dilute it to volume with 20% acetic acid solution to prepare a standard stock solution. Dilute the stock solution with 20% acetic acid solution to prepare the required standard working solution. Derivatize and purify the standard working solution according to steps S2 and S3, and then detect it using liquid chromatography-tandem mass spectrometry to establish a quantitative curve.

[0049] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity 11-6470) for detection.

[0050] The conditions for determination using liquid chromatography-tandem mass spectrometry are as follows:

[0051] *: indicates a quantitative ion pair.

[0052] S6. In this embodiment, the limit of detection (LOD) is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L. The inorganic copper content in the sample determined through the above steps is less than 0.00001 mg / L.

[0053] Example 2 Please combine Figure 1 , Figure 2 and Figure 4 This embodiment provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes the following steps: S1. Add 30% acetic acid solution to the soil sample (paddy soil) at a ratio of 1g:10mL, vortex mix for 10 min, and then centrifuge for 5 min to obtain the extract.

[0054] S2. Take 1 ml of extract, add 1.0 mL of 2 g / L DDTC-Na methanol solution, mix and let stand for 15 min, and derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain the derivatized solution.

[0055] S3. Take the derivatized solution, add n-hexane, and extract copper diethyldithiocarbamate into the organic solvent. Use a nitrogen blower to dry the extraction solvent, redissolve it with acetonitrile, and detect the redissolved solution using liquid chromatography-tandem mass spectrometry.

[0056] S4. Weigh an appropriate amount of inorganic copper standard substance and dilute it to volume with 20% acetic acid solution to prepare a standard stock solution. Dilute the stock solution with 20% acetic acid solution to prepare the required standard working solution. Derivatize and purify the standard working solution according to steps S2 and S3, and then detect it using liquid chromatography-tandem mass spectrometry to establish a quantitative curve.

[0057] S5. The sample is injected into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity 11-6470) for detection. The liquid chromatography-tandem mass spectrometry determination conditions are the same as in Example 1.

[0058] S6. In this embodiment, the limit of detection (LOD) is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L. The inorganic copper content in the sample determined through the above steps is less than 0.0001 mg / kg.

[0059] Example 3 Please combine Figure 1 , Figure 2 and Figure 5 This embodiment provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes the following steps: S1. Add 10% acetic acid solution to the biological sample (plasma) at a ratio of 1mL:5mL, vortex mix for 10 min, and then centrifuge for 5 min to obtain the extract.

[0060] S2. Take 1 ml of extract, add 3.0 mL of 1.0 g / L DDTC-Na methanol solution, mix and let stand for 30 min, and derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain the derivatized solution.

[0061] S3. Take the derivatized liquid and pass it through a solid phase extraction column (Agela Cleanert SC18) for adsorption. Then, elute with acetonitrile and detect the eluent using liquid chromatography-tandem mass spectrometry.

[0062] S4. Weigh an appropriate amount of inorganic copper standard substance and dilute it to volume with 20% acetic acid solution to prepare a standard stock solution. Dilute the stock solution with 20% acetic acid solution to prepare the required standard working solution. Derivatize and purify the standard working solution according to steps S2 and S3, and then detect it using liquid chromatography-tandem mass spectrometry to establish a quantitative curve.

[0063] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity 11-6470) for detection.

[0064] The conditions for the determination by liquid chromatography-tandem mass spectrometry were the same as in Example 1.

[0065] S6. In this embodiment, the limit of detection (LOD) is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L. The inorganic copper content in the sample determined through the above steps is less than 0.0001 mg / L.

[0066] Example 4 This embodiment is basically the same as Embodiment 1, except that the derivatization is allowed to stand for 10 minutes in this embodiment. The result of derivatization at room temperature for 10 minutes is consistent with the result of Embodiment 1, indicating that derivatization at room temperature for 10 minutes reaches stability.

[0067] Example 5 This embodiment is basically the same as Example 1, except that hexane and toluene are used as extraction reagents in this embodiment.

[0068] The results of this embodiment are consistent with those of Embodiment 1, indicating that the type of extraction reagent used in this application does not affect the detection results.

[0069] Comparative Example 1 This comparative example is basically the same as Example 3, except that a 30% acetic acid solution is used as the extraction reagent in this comparative example.

[0070] The 30% acetic acid solution in this comparative example has high protein solubility, which causes the extraction column to become clogged during solid-phase extraction, resulting in low efficiency. When the instrument was used for detection, the sample was found to have high noise, and the noise and the peak of the analyte could not be completely separated.

[0071] Comparative Example 2 This comparative example is basically the same as Example 1, except that the derivative was left to stand for 5 minutes.

[0072] The comparative example did not fully degenerate after 5 minutes at room temperature.

[0073] Comparative Example 3 This comparative example is basically the same as Example 1, except that the purification step of step S3 is omitted in this comparative example, and the derivatized liquid is directly injected and detected.

[0074] The comparative sample showed no response in mass spectrometry, and the precursor ion of copper diethyldithiocarbamate was not found even after changing the instrument conditions, indicating that the response of copper diethyldithiocarbamate was completely suppressed without a purification step.

[0075] Comparative Example 4 This comparative example is basically the same as Example 1, except that the mobile phase in this comparative example is acetonitrile and pure water.

[0076] The limit of detection (LOD) for this comparative example was 0.001 mg / L, and the limit of quantitation (LOQ) was 0.002 mg / L. The inorganic copper content in the sample determined using the above steps was less than 0.001 mg / kg, and the peak tailing was severe.

[0077] In summary, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for determining inorganic copper concentration provided by this invention reduces interference from other ions through sample extraction, derivatization, and purification pretreatment. Subsequent separation and ion pair qualitative analysis via LC-MS / MS column separation enable the analysis of complex samples. The pretreatment process avoids the use of strong acids, strong bases, and toxic compounds, is simple, requires minimal operator skill, and utilizes readily available instruments in analytical laboratories. Traditional methods using UV detection standards struggle to reach concentrations below 0.5 mg / L, while this method, using LC-MS / MS, can detect inorganic copper standards down to 0.0005 mg / L. Sample concentration can be further enhanced through pretreatment concentration, achieving a detection limit of 0.00001 mg / L. Furthermore, LC-MS / MS offers rapid analysis, with each sample requiring only 2-3 minutes of analysis.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, characterized in that, It includes: The inorganic copper in the sample was extracted using acetic acid to obtain the extract. Add DDTC-Na solution to the extract, mix, let stand, and derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain the derivatized solution; The derivatized liquid was purified by liquid-liquid extraction and / or solid-phase extraction, and then injected into a liquid chromatography-tandem mass spectrometer for detection.

2. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The concentration of the acetic acid added to the sample is 10%-30%; And / or, the extraction time is 5-15 min; And / or, the sample includes a water sample, a soil sample, or a biological sample; when the sample is a water sample, acetic acid solution is added directly to the water sample; when the sample is a soil sample or a biological sample, acetic acid solution is added at a ratio of 1g:4-6mL.

3. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The volume ratio of the extract to the DDTC-Na solution is 1 mL: 1-3 mL; And / or, the solvent of the DDTC-Na solution is methanol; And / or, the concentration of the DDTC-Na solution is 0.5-2 g / L; And / or, the settling time is 10-30 minutes.

4. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The liquid-liquid extraction includes extracting the derivatized liquid with an extraction reagent, taking the organic phase, drying it, and then resolving it with a redissolving reagent.

5. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, The extraction reagent includes at least one of cyclohexane, n-hexane, and toluene; And / or, the drying includes drying using a nitrogen blower; And / or, the redissolving agent includes at least one of acetonitrile and methanol.

6. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The solid-phase extraction includes passing the derivatized liquid into a solid-phase extraction column for adsorption, followed by elution with an elution reagent.

7. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that, The elution reagent includes at least one of acetonitrile and methanol.

8. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The conditions for the determination using the liquid chromatography-tandem mass spectrometry were as follows: (1) Chromatographic conditions: Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50 mm, 1.8-Micron; column temperature: 30-40℃; flow rate: 0.5-0.7 mL / min; injection volume: 1 µL; external standard method for quantification; Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; The gradient elution program is as follows: 0.00-0.80 min: the concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%; 0.80-0.90 min: the concentration of mobile phase A changes from 65%-75% to 15%-25%, and the concentration of mobile phase B changes from 25%-35% to 75%-85%. 0.90-2.20 min: The concentration of mobile phase A changes from 15%-25% to 3%-8%, and the concentration of mobile phase B changes from 75%-85% to 92%-97%; 2.20-2.30 min: The concentration of mobile phase A changes from 3%-8% to 65%-75%, and the concentration of mobile phase B changes from 92%-97% to 25%-35%; 2.30-3.0 min: The concentration of mobile phase A is maintained at 65%-75%, and the concentration of mobile phase B is maintained at 25%-35%. (2) Mass spectrometry conditions: Ion source: Electrospray ionization (ESI); Scan mode: MRM mode; Mass analyzer: tandem quadrupole; Drying gas temperature: 300℃; Drying gas flow rate: 5 L / min; Nebulizer gas pressure: 45 psi; Sheath gas temperature: 250 ℃; Sheath gas flow rate: 11 L / min; Capillary voltage: 3500 V; Nozzle voltage: 500 V; MRM mode parameters: Copper Diethyldithiocarbamate (Positive); *361>116.2 (Fragmentation voltage: 100 V; Collision energy: 30 V; Acceleration voltage: 5 V); 361>88.1 (Fragmentation voltage: 100 V; Collision energy: 40 V; Acceleration voltage: 5 V); Scan time (msec):

50.

9. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, Before performing the liquid chromatography-tandem mass spectrometry (LC-MS / MS) determination, the following steps are included: weighing an inorganic copper standard substance, making up to volume with acetic acid solution to prepare a standard stock solution; diluting the stock solution with acetic acid solution to prepare the required standard working solution; derivatizing and purifying the standard working solution in the same manner as the sample; and then detecting it using liquid chromatography-tandem mass spectrometry (LC-MS / MS) and establishing a quantitative curve.

10. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The limit of detection (LOD) for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry is 0.00001 mg / L, and the limit of quantitation (LOQ) is 0.0005 mg / L.

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