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

Technical Field

[0001] The present invention relates to the field of detection technology, and more particularly, to a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry. Background Art

[0002] Copper ions (Cu 2 +) have no obvious absorption in the visible light region and need to react with color reagents (such as dithizone, sodium diethyldithiocarbamate (DDTC), neocuproine, etc.) to form colored complexes. Currently, there is a method for detecting the concentration of copper ions using a UV spectrophotometer by means of a color reaction. The steps are as follows:

[0003] (1) Sample pretreatment:

[0004] Digestion: If the sample contains organic matter or solids (such as soil, biological tissue), it needs to be digested by microwave digestion or wet digestion with nitric acid (HNO) and hydrogen peroxide (HO) to be converted into a clear solution.

[0005] Filtration: Remove insoluble impurities to avoid interference.

[0006] (2) Adjust the pH value:

[0007] Add ammonia water (NH·HO) to adjust the solution to weakly alkaline (pH 8-9), and confirm with a pH meter or precision test paper.

[0008] Add a buffer solution (such as NH-NH Cl buffer solution) to maintain the stability of the reaction system.

[0009] (3) Color reaction:

[0010] Add DDTC solution (0.1% aqueous solution) as the color reagent, shake well and let stand for 10-15 minutes to fully form the complex.

[0011] (4) Eliminate interference:

[0012] Add masking agents (such as EDTA, ammonium citrate) to mask interfering ions such as Fe 3 + and Pb 2 +.

[0013] If the solution is turbid, centrifuge or filter to remove the precipitate.

[0014] (5) Measure the absorbance:

[0015] Using a blank solution (reagent solution without copper) as a reference, measure the absorbance at a wavelength of 440 nm.

[0016] Standard curve method: Prepare a series of copper standard solutions with different concentrations (such as 0, 0.2, 0.5, 1.0, 2.0 mg / L), measure the absorbance after color development, and plot the concentration-absorbance standard curve.

[0017] However, the method for detecting the concentration of copper ions by the above ultraviolet spectrophotometer is susceptible to interference from Fe 3 + and Al 3 + and other ions, and cannot analyze complex samples. It is difficult to analyze samples below 0.5 mg / L by the traditional method of detecting standard products by ultraviolet, and in its pretreatment process, strong acids, strong alkalis and toxic compounds are used, and the steps are complex and the safety is poor.

[0018] In view of this, the present invention is specifically proposed. Summary of the Invention

[0019] The purpose of the present invention is to provide a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry.

[0020] The present invention is implemented as follows:

[0021] In the first aspect, the present invention provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes:

[0022] Using acetic acid to extract inorganic copper in the sample to obtain an extract;

[0023] Adding a DDTC-Na solution to the extract, mixing and standing to derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain a derivatized solution;

[0024] After purifying the derivatized solution by liquid-liquid extraction and / or solid-phase extraction, injecting it into a liquid chromatography-tandem mass spectrometer for detection.

[0025] In an optional embodiment, the concentration of the acetic acid added to the sample is 10%-30%;

[0026] and / or, the extraction time is 5-15 min;

[0027] and / or, the sample includes a water sample, a soil sample or a biological sample; when the sample is a water sample, directly add an acetic acid solution to the water sample; when the sample is a soil sample or a biological sample, add an acetic acid solution according to a ratio of 1 g:4-6 mL;

[0028] In an optional embodiment, the volume ratio of the extract to the DDTC-Na solution is 1 mL:1-3 mL;

[0029] and / or, the solvent of the DDTC-Na solution is methanol;

[0030] and / or, the concentration of the DDTC-Na solution is 0.5-2 g / L;

[0031] And / or, the standing time is 10 - 30 min.

[0032] In an optional embodiment, the liquid-liquid extraction includes extracting the post-derivatization solution with an extraction reagent, taking the organic phase, drying it, and then re-dissolving it with a re-dissolution reagent.

[0033] In an optional embodiment, the extraction reagent includes at least one of cyclohexane, n-hexane, and toluene;

[0034] And / or, the drying includes drying with a nitrogen blower;

[0035] And / or, the re-dissolution reagent includes at least one of acetonitrile and methanol.

[0036] In an optional embodiment, the solid-phase extraction includes passing the post-derivatization solution through a solid-phase extraction column for adsorption, and then eluting it with an elution reagent.

[0037] In an optional embodiment, the elution reagent includes at least one of acetonitrile and methanol.

[0038] In an optional embodiment, the conditions for determination by the liquid chromatography-tandem mass spectrometer are as follows:

[0039] (1) Chromatographic conditions:

[0040] Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50 mm, 1.8 - Micron; Column temperature: 30 - 40 °C; Flow rate: 0.5 - 0.7 mL / min; Injection volume: 1 μL; Quantitative analysis by external standard method;

[0041] Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: acetonitrile;

[0042] The gradient elution program is as follows:

[0043] 0.00 - 0.80 min: The concentration of the mobile phase A is maintained at 65% - 75%, and the concentration of the mobile phase B is maintained at 25% - 35%;

[0044] 0.80 - 0.90 min: The concentration of the mobile phase A changes from 65% - 75% to 15% - 25%, and the concentration of the mobile phase B changes from 25% - 35% to 75% - 85%;

[0045] 0.90 - 2.20 min: The concentration of the mobile phase A changes from 15% - 25% to 3% - 8%, and the concentration of the mobile phase B changes from 75% - 85% to 92% - 97%;

[0046] 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%.

[0047] 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%.

[0048] (2) Mass spectrometry conditions:

[0049] Ion source: Electrospray ionization source (ESI); Scanning mode: MRM mode; Mass analyzer: Tandem quadrupole; Dry gas temperature: 300 °C; Dry gas flow rate: 5 L / min; Nebulizing gas pressure: 45 psi; Sheath gas temperature: 250 °C; 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); Scanning time (msec): 50.

[0050] In an alternative embodiment, before performing the liquid chromatography - tandem mass spectrometry determination, it further includes plotting a calibration curve: Weigh an inorganic copper standard substance, make up the volume with acetic acid solution to prepare a standard stock solution; Dilute the stock solution with acetic acid solution to prepare the required standard working solutions, derivatize and purify the standard working solutions in the same manner as the samples, and then detect using liquid chromatography - tandem mass spectrometry and establish a calibration curve.

[0051] In an alternative embodiment, the lower limit of detection LOD of the method for determining the concentration of inorganic copper by liquid chromatography - tandem mass spectrometry is 0.00001 mg / L, and the lower limit of quantification LOQ is 0.0005 mg / L.

[0052] The present invention has the following beneficial effects:

[0053] The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry provided by the present invention can reduce the interference of other ions through pretreatment operations such as extraction, derivatization, and purification of samples. Subsequently, through the separation of the chromatographic column and the qualitative analysis of ion pairs in liquid chromatography-tandem mass spectrometry, complex samples can be analyzed. Strong acids, strong bases, and toxic compounds are not used in the pretreatment process. The steps are simple and the requirements for personnel operation are not high, and the instruments used are commonly available in analytical laboratories. It is difficult to analyze below 0.5 mg / L by the traditional method through ultraviolet detection of standards. This method can detect inorganic copper standards up to 0.0005 mg / L by liquid chromatography-tandem mass spectrometry, and the detection limit of samples can reach 0.00001 mg / L through concentration in the pretreatment. In addition, using liquid chromatography-tandem mass spectrometry for detection, the analysis speed is fast, up to 2 - 3 minutes for each sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0055] Figure 1 is the standard curve of copper hydroxide at 0.002 - 0.15 mg / L (copper ion 0.0013 - 0.098);

[0056] Figure 2 is the standard of copper hydroxide at 0.05 mg / L (copper ion 0.0325);

[0057] Figure 3 is the recovered sample added to pond water with copper hydroxide at 0.0001 mg / L (copper ion 0.000065);

[0058] Figure 4 is the chromatogram of inorganic copper in soil samples;

[0059] Figure 5 is the chromatogram of inorganic copper in plasma samples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0060] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0061] The present invention provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which comprises the following steps:

[0062] S1. Extract inorganic copper in the sample with acetic acid to obtain an extract.

[0063] In the present invention, the sample includes a water sample, a soil sample or a biological sample. When the sample is a water sample, an acetic acid solution is directly added to the water sample to make the concentration of acetic acid reach 10%-30%. The sources of the water sample include, but are not limited to, river water, tap water, and fish pond water. When the sample is a soil sample or a biological sample, an acetic acid solution with a concentration of 10%-30% is added in a ratio of 1 g: 4-6 mL. Among them, the sources of the soil sample include, but are not limited to, paddy soil and black loam soil. The sources of the biological sample include, but are not limited to, whole blood and plasma.

[0064] After adding the acetic acid solution, acetic acid is used to extract inorganic copper (such as Cu 2+ 、Cu + etc.) in the sample, and the extraction time is 5-15 min. Acetic acid can dissolve the sample to generate soluble copper acetate. In addition, acetate (CH3COO-) can form a complex ([Cu(CH3COO)]+) with Cu 2 +, promoting the dissolution and stability of copper. During the extraction process, some cuprous ions are unstable in aqueous solution and can also undergo a spontaneous disproportionation reaction (2Cu + →Cu 0 +Cu 2+ ) under the condition of acetic acid.

[0065] S2. Add a DDTC-Na solution to the extract, mix and let stand to derivatize the inorganic copper in the sample into copper diethyldithiocarbamate to obtain a derivatized solution.

[0066] DDTC-Na (sodium diethyldithiocarbamate) has high selectivity for Cu 2+ and can form a complex Cu(DDTC)2 with Cu 2+ . The solvent of 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 the present invention, by limiting the concentration and dosage of DDTC-Na, it can be ensured that it fully complexes with Cu 2+ in the extract. By mixing and letting stand, the reaction can be ensured to be more sufficient, and the standing time is 10-30 min.

[0067] S3. Purify the derivatized solution by liquid-liquid extraction and / or solid-phase extraction.

[0068] Since acetic acid was used to extract copper ions and DDTC-Na and Cu 2+ To form a complex, both will be affected by some other metal elements (such as Fe). Directly injecting and detecting the derivatized liquid will cause more interference and cannot be accurately detected.

[0069] Therefore, the present invention also purifies the above-mentioned derivatized liquid. Specifically, the liquid-liquid extraction includes extracting the derivatized liquid with an extraction reagent, taking the organic phase, drying it, and then re-dissolving it with a re-dissolving reagent.

[0070] Wherein, the extraction reagent includes but is not limited to at least one of cyclohexane, n-hexane and toluene; and / or, the drying includes drying with a nitrogen blower; and / or, the re-dissolution reagent includes but is not limited to at least one of acetonitrile and methanol.

[0071] Solid phase extraction includes passing the derivatized liquid into a solid phase extraction column for adsorption, followed by elution with an elution reagent, which includes but is not limited to at least one of acetonitrile and methanol.

[0072] In the present invention, the interference factors in the derivatized liquid can be removed well by liquid-liquid extraction and / or solid phase extraction. Therefore, the present invention can analyze complex samples and expand the range of samples.

[0073] S4. Preparation of standard stock solution and standard working solution.

[0074] Weigh the inorganic copper standard substance, make up 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 manner as the sample, and then use liquid chromatography tandem mass spectrometry to detect and establish a quantitative curve.

[0075] S5. Inject the sample into a liquid chromatography tandem mass spectrometer for detection.

[0076] The conditions for the determination by liquid chromatography tandem mass spectrometry are:

[0077] (1) Chromatographic conditions:

[0078] Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50mm, 1.8-Micron; column temperature: 30-40℃; flow rate: 0.5-0.7mL / min; injection volume: 1μL; external standard method for quantification;

[0079] Mobile phase: Mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile;

[0080] The gradient elution program was:

[0081] 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%.

[0082] 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%.

[0083] 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%.

[0084] 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%.

[0085] 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%.

[0086] (2) Mass spectrometry conditions:

[0087] Ion source: Electrospray ionization source (ESI); Scanning mode: MRM mode; Mass analyzer: Triple quadrupole; Drying gas temperature: 300 °C; Drying gas flow rate: 5 L / min; Nebulizing gas pressure: 45 psi; Sheath gas temperature: 250 °C; 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); Scanning time (msec): 50.

[0088] The detection limit LOD of the method for determining the concentration of inorganic copper by liquid chromatography - tandem mass spectrometry is 0.00001 mg / L, and the quantification limit LOQ is 0.0005 mg / L.

[0089] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0090] Example 1

[0091] Please refer to Figure 1 、 Figure 2 and Figure 3 , this example provides a method for determining the concentration of inorganic copper by liquid chromatography - tandem mass spectrometry, which includes the following steps:

[0092] S1. Add acetic acid solution to the water sample (pond water) to make the concentration of acetic acid reach 20%, and vortex for 10 min to obtain the extract.

[0093] S2. Take 1 mL of the 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 post-derivatization solution.

[0094] S3. Take the post-derivatization solution, add cyclohexane, extract copper diethyldithiocarbamate into the organic solvent, dry the extraction solvent with a nitrogen evaporator, re-dissolve with acetonitrile, and detect the re-dissolved solution by liquid chromatography-tandem mass spectrometry.

[0095] S4. Weigh an appropriate amount of inorganic copper reference material, make up the volume with 20% acetic acid solution to prepare the 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 by liquid chromatography-tandem mass spectrometry and establish a quantitative curve.

[0096] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity ll-6470) for detection.

[0097] The determination conditions of the liquid chromatography-tandem mass spectrometer are as follows:

[0098]

[0099]

[0100] *: Quantitative ion pair.

[0101] S6. The detection limit LOD of this example is 0.00001 mg / L, and the lower limit of quantification LOQ is 0.0005 mg / L. The content of inorganic copper in the sample measured by the above steps is lower than 0.00001 mg / L.

[0102] Example 2

[0103] Please combine Figure 1 , Figure 2 and Figure 4 , this example provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes the following steps:

[0104] S1. Add acetic acid solution with a concentration of 30% to the soil sample (paddy soil) at a ratio of 1 g:10 mL, vortex for 10 min, and then centrifuge for 5 min to obtain the extract.

[0105] S2. Take 1 mL of the extract, add 1.0 mL of a 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 post-derivatization solution.

[0106] S3. Take the post-derivatization solution, add n-hexane, extract copper diethyldithiocarbamate into the organic solvent, dry the extraction solvent with a nitrogen evaporator, re-dissolve with acetonitrile, and detect the re-dissolved solution by liquid chromatography-tandem mass spectrometry.

[0107] S4. Weigh an appropriate amount of the inorganic copper reference material, make up the 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 by liquid chromatography-tandem mass spectrometry and establish a quantitative curve.

[0108] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity ll-6470) for detection. The detection conditions of the liquid chromatography-tandem mass spectrometer are the same as those in Example 1.

[0109] S6. The limit of detection LOD of this example is 0.00001 mg / L, and the limit of quantification LOQ is 0.0005 mg / L. The content of inorganic copper in the sample measured by the above steps is lower than 0.0001 mg / kg.

[0110] Example 3

[0111] Please combine Figure 1 、 Figure 2 and Figure 5 This example provides a method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry, which includes the following steps:

[0112] S1. Add a 10% acetic acid solution to the biological sample (plasma) at a ratio of 1 mL:5 mL, vortex for 10 min, and then centrifuge for 5 min to obtain the extract.

[0113] S2. Take 1 mL of the extract, add 3.0 mL of a 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 post-derivatization solution.

[0114] S3. Take the post-derivatization solution, pass it through a solid phase extraction column (AgelaCleanert SC18) for adsorption, then elute with acetonitrile, and detect the eluate by liquid chromatography-tandem mass spectrometry.

[0115] S4. Weigh an appropriate amount of inorganic copper reference material, dilute it to a constant 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 solutions. Derive and purify the standard working solutions according to steps S2 and S3, and then detect them using liquid chromatography-tandem mass spectrometry and establish a quantitative curve.

[0116] S5. Inject the sample into a liquid chromatography-tandem mass spectrometer (Agilent 1290 Infinity ll-6470) for detection.

[0117] The determination conditions of the liquid chromatography-tandem mass spectrometer are the same as those in Example 1.

[0118] S6. The detection limit LOD of this example is 0.00001 mg / L, and the quantification limit LOQ is 0.0005 mg / L. The content of inorganic copper in the sample determined by the above steps is lower than 0.0001 mg / L.

[0119] Example 4

[0120] This example is basically the same as Example 1, except that in this example, the derivation is allowed to stand for 10 min. The result of derivation at room temperature for 10 min is the same as that in Example 1, indicating that the derivation at room temperature for 10 min reaches stability.

[0121] Example 5

[0122] This example is basically the same as Example 1, except that in this comparative example, n-hexane and toluene are selected as extraction reagents.

[0123] The results of this example and Example 1 are the same, indicating that the type of extraction reagent selected in this application does not affect the detection results.

[0124] Comparative Example 1

[0125] This comparative example is basically the same as Example 3, except that in this comparative example, 30% acetic acid solution is selected as the extraction reagent.

[0126] The 30% acetic acid solution in this comparative example has a high solubility for proteins, resulting in clogging of the extraction column during solid-phase extraction, reducing the efficiency. When using the instrument for detection, it is found that the sample has high noise and the peaks of the noise and the analyte cannot be completely separated.

[0127] Comparative Example 2

[0128] This comparative example is basically the same as Example 1, except that the derivation is allowed to stand for 5 min.

[0129] The derivation in this comparative example is not complete after standing for 5 min at room temperature.

[0130] Comparative Example 3

[0131] This comparative example is basically the same as Example 1, except that in this comparative example, the purification step in step S3 is omitted, and the derivatized solution is directly injected and detected.

[0132] There is no response in the mass spectrometry of this comparative example, and the parent ion of copper diethyldithiocarbamate cannot be found even by changing the instrument conditions, indicating that without the purification step of the sample, the response of copper diethyldithiocarbamate will be completely inhibited.

[0133] Comparative Example 4

[0134] This comparative example is basically the same as Example 1, except that in this comparative example, the mobile phase is acetonitrile and pure water.

[0135] The lower limit of detection LOD of this comparative example is 0.001 mg / L, and the lower limit of quantification LOQ is 0.002 mg / L. The content of inorganic copper in the sample measured by the above steps is lower than 0.001 mg / kg, and the peak shape has serious tailing.

[0136] In summary, the method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry provided by the present invention can reduce the interference of other ions through pretreatment operations such as extraction, derivatization, and purification of the sample. Subsequently, through the separation of the chromatographic column and the qualitative analysis of the ion pair in liquid chromatography-tandem mass spectrometry, complex samples can be analyzed. Strong acids, strong bases, and toxic compounds are not used in the pretreatment process, the steps are simple, the requirements for personnel operation are not high, and the instruments used are commonly available in analytical laboratories. It is difficult for traditional methods to analyze below 0.5 mg / L by ultraviolet detection of standards. This method can detect inorganic copper standards up to 0.0005 mg / L by liquid chromatography-tandem mass spectrometry, and the detection limit of the sample can reach 0.00001 mg / L through the concentration of the pretreatment. In addition, using liquid chromatography-tandem mass spectrometry for detection, the analysis speed is fast, up to 2 - 3 minutes for each sample.

[0137] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope 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: Extracting inorganic copper in the sample with acetic acid to obtain an extract; Adding a DDTC-Na solution to the extract, mixing, standing, and derivatizing the inorganic copper in the sample into copper diethyldithiocarbamate to obtain a derivatized solution; After purifying the derivatized solution by liquid-liquid extraction and / or solid-phase extraction, injecting it 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, wherein 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, directly add an acetic acid solution to the water sample; when the sample is a soil sample or a biological sample, add an acetic acid solution according to a ratio of 1 g:4-6 mL.

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 standing time is 10-30 min.

4. The method for determining the concentration of inorganic copper by liquid chromatography-tandem mass spectrometry according to claim 1, wherein The liquid-liquid extraction includes extracting the derivatized solution with an extraction reagent, taking the organic phase, drying, and then redissolving 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 with a nitrogen blower; and / or, the redissolving reagent 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, wherein The solid-phase extraction includes passing the derivatized solution through a solid-phase extraction column for adsorption, and then eluting with an elution reagent.

7. The method for determining the concentration of inorganic copper by liquid chromatography tandem mass spectrometry according to claim 6, wherein 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, wherein The determination conditions of the liquid chromatography-tandem mass spectrometer are: (1) Chromatographic conditions: Chromatographic column: ZORBAX Eclipse Plus C18, 2.1×50 mm, 1.8-Micron; column temperature: 30-40 °C; flow rate: 0.5-0.7 mL / min; injection volume: 1 μL; quantitative analysis by external standard method; Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: acetonitrile; Gradient elution program: 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 source (ESI); Scanning mode: MRM mode; Mass analyzer: Tandem quadrupole; Dry gas temperature: 300 °C; Dry gas flow rate: 5 L / min; Nebulizing gas pressure: 45 psi; Sheath gas temperature: 250 °C; 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); Scanning 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 determination, it further includes plotting a calibration curve: Weigh an inorganic copper reference material, make a constant volume with acetic acid solution to 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 by liquid chromatography-tandem mass spectrometry and establish a calibration 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 detection limit LOD of the method for determining the inorganic copper concentration by liquid chromatography-tandem mass spectrometry is 0.00001 mg / L, and the lower limit of quantification LOQ is 0.0005 mg / L.

Images