CN113984943A - Method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum - Google Patents
Method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a method for simultaneously detecting a gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum, and belongs to the technical field of clinical detection of gadolinium contrast agents. In order to solve the problems that a gadolinium contrast agent and free gadolinium ions cannot be effectively separated and the detection accuracy and sensitivity are low in the existing clinical detection, the invention provides a method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in blood plasma or blood serum, which comprises the steps of determining detection conditions, preparing a mixed standard sample group containing the gadolinium contrast agent and gadolinium elements, drawing a standard curve, pre-treating clinical samples, detecting the samples and processing data. The high-efficiency molecular exclusion chromatographic column has smaller particle size and higher separation efficiency, can effectively separate gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum, and has higher detection sensitivity and more accurate detection result. According to the invention, the plasma or serum sample is treated by acetonitrile or hydrogen peroxide during pretreatment, the extraction effect is good, the gadolinium contrast agent structure is not influenced, and the accuracy of the detection result is ensured.
Description
Technical Field
The invention belongs to the technical field of clinical detection of gadolinium contrast agents, and particularly relates to a method for simultaneously detecting gadolinium contrast agents and free gadolinium ions in blood plasma or blood serum by high-efficiency molecular exclusion chromatography combined with inductively coupled plasma mass spectrometry (HPSEC-ICP-MS).
Background
Magnetic resonance imaging is an important imaging examination method, has higher resolution, but the resolution cannot meet the clinical requirement in some cases, and the enhancement of magnetic resonance can provide more diagnostic information, and has important value for early diagnosis and differential diagnosis of various systemic diseases of the whole body. Enhancing magnetic resonance examination requires the use of magnetic resonance contrast agents for contrast enhancement purposes. The magnetic resonance contrast agent generally enters a human body through intravenous injection, and the contrast between a focus and a normal tissue is improved by changing the relaxation time of protons in the surrounding tissue, so that the detection rate of a lesion is increased, and the diagnosis accuracy is improved.
Gadolinium-based contrast agents (GBCAs) are a type of magnetic resonance contrast agent commonly used in clinical practice, and are mainly complexes of Gadolinium, Gadolinium ion (Gd)3+) Is a metal ion with strong paramagnetism and can show larger magnetic momentum, but free Gd3+Has high toxicity and is easy to deposit in vivo. GBCAs release free Gd3+Formation of polyaminocarboxylic acid chelates, thereby circumventing free Gd3+While maintaining its excellent paramagnetism, has been recognized as a contrast agent with higher safety. However, with the popularization and promotion of enhanced magnetic resonance, the use of GBCA has increased year by year, and its safety has received more and more attention. GBCAs are mainly filtered through glomeruli in an original shape, 98% of GBCAs can be removed within 24 hours after injection for patients with normal renal functions, and the complete removal needs about 24-48 hours. However, it has been shown that GBCAs release free Gd after entry into the body3+Free Gd3+Can be rapidly distributed in tissues and organs such as skin, bone, liver and brain, and has small amount of deposition. On the other hand, free Gd3+With Ca2+Similar in size, can compete for Ca2+Voltage-dependent channels and enzymes, inhibition of Ca2+Influx and a series of related enzyme activities. Free Gd3+Prolonged residence in the body may also inhibit reticuloendothelial system function. More notably, the use of GBCAs in patients with severe renal injury may also cause systemic nephrogenic activityFibrosis, characterized by symmetrically distributed thickening, stiffening and pigmentation of the skin, can also affect other tissues and organs of the body such as joints, skeletal muscles, cardiac muscles, kidneys, nerves, etc. Patients often have diseases hidden and symptoms are gradually aggravated, no effective treatment method is available at present, and the death rate is extremely high. Pathogenesis may be associated with impaired GBCAs excretion, Gd3+Accumulation in vivo has been implicated and studies have shown that nephrogenic systemic fibrosis is associated with GBCAs injection doses. The determination of the content of GBCAs in vivo is helpful for exploring the occurrence mechanism of diseases and reducing the occurrence of adverse reactions.
To evaluate the in vivo kinetic Process of GBCAs and to liberate free Gd3+The dynamic characteristics of the composition, researches on the correlation between the deposition amount of gadolinium and the toxicity of gadolinium, reduces the adverse reaction of patients, and is necessary to treat in vivo GBCAs and free Gd3+The content is measured. The GBCAs in biological samples such as plasma, urine, bile and the like are measured by using detection means such as high performance liquid chromatography, liquid chromatography-mass spectrometry, inductively coupled plasma emission spectrometry and the like, and the detection of GBCAs and free Gd in hair by using molecular exclusion chromatography (SEC) combined with inductively coupled plasma mass spectrometry is reported in foreign countries3+However, the degree of separation is poor, and the two cannot be completely separated; the pretreatment method uses tetramethylammonium hydroxide for digestion, but the tetramethylammonium hydroxide induces partial GBCAs to be dissociated into free Gd3+Resulting in inaccurate determination of GBCAs and free Gd in the sample3+. Furthermore, this method failed to detect free Gd in urine3+This may be related to insufficient sensitivity of the method.
In summary, the prior art relates to GBCAs and free Gd in biological samples3+The main difficulty of simultaneous detection is as follows: 1. GBCAs and free Gd3+The separation degree is poor, and the effective separation cannot be realized; 2. the GBCAs are damaged in the pretreatment process, and the detection accuracy is influenced; 3. free Gd in biological samples3+The content is low, and the sensitivity of the method needs to be improved to meet the detection aim.
Disclosure of Invention
For solving the existing clinical biological sampleGBCAs and free Gd in this assay3+The invention provides a method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum, and solves the problems that effective separation cannot be realized, the detection accuracy is influenced by sample pretreatment, and the sensitivity of the detection method is low.
The technical scheme of the invention is as follows:
a method for simultaneously detecting a gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum comprises the following steps:
step one, determining HPSEC-ICP-MS detection conditions;
step two, preparing mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements:
pretreatment of blank blood: centrifuging the collected blank blood, collecting supernatant to obtain blank plasma or blank serum, adding acetonitrile into the blank plasma or blank serum, mixing uniformly, centrifuging, and collecting the centrifuged supernatant as a blank plasma sample or a blank serum sample;
(II) preparing a mixed standard sample: adding gadolinium contrast agent standard solution and gadolinium element standard solution with corresponding concentrations into the obtained blank plasma sample or blank serum sample to obtain mixed standard sample groups containing gadolinium contrast agent and gadolinium element at different concentrations;
step three, drawing a gadolinium contrast agent standard curve and a gadolinium element standard curve:
sequentially injecting and detecting the mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements according to the HPSEC-ICP-MS detection conditions determined in the step one; respectively taking the concentration of the gadolinium contrast agent or gadolinium element as a horizontal coordinate and taking the corresponding peak area as a vertical coordinate to perform linear regression so as to respectively obtain a gadolinium contrast agent standard curve equation and a gadolinium element standard curve equation;
step four, pretreatment of clinical biological samples:
centrifuging the collected blood sample to be detected, collecting supernatant to obtain blood plasma or blood serum to be detected, adding acetonitrile into the blood plasma or blood serum to be detected, mixing uniformly, centrifuging, and taking the centrifuged supernatant as a sample to be detected;
step five, sample detection and data processing:
and step four, the sample to be detected obtained in the step one is subjected to sample injection detection according to the HPSEC-ICP-MS detection condition determined in the step one, the retention time is used for qualitative detection, the peak area is used for quantitative detection, the standard curve equation obtained in the step three is used for quantitative detection through an external standard method, and the concentration of the gadolinium contrast agent and the concentration of gadolinium elements in the sample to be detected are obtained through calculation.
Further, the detection conditions of the HPSEC-ICP-MS in the first step are as follows:
conditions for high performance size exclusion chromatography: size exclusion chromatography column: TSK gel G2000SWXL, 7.8 mm. times.300 mm, 5 μm; mobile phase: 20mM Tris-HCl solution at pH 7.4 at a flow rate of 1.0 mL/min; the sample volume is 10 mu L;
ICP-MS analysis conditions: radio frequency power: 1500W, atomizing gas flow rate: 0.8L/min, cooling gas flow rate: 15.0L/min, assist gas flow rate: 1.0L/min, dwell time 100ms, detection mode: a bump gas mode.
Further, the blank blood centrifugation condition in the second step is centrifugation for 10min at 3000 rpm; the blank plasma or blank serum and acetonitrile are mixed according to the volume ratio of 1:4, and the centrifugation condition after mixing is 13000rpm centrifugation for 15min at 4 ℃; the volume ratio of the gadolinium contrast agent standard solution with the corresponding concentration to the gadolinium element standard solution to the blank plasma sample or the blank serum sample is 10:10: 80.
Further, in the second step, the concentrations of the gadolinium contrast agent and the gadolinium element in the mixed standard sample group containing both the gadolinium contrast agent and the gadolinium element are sequentially as follows: gadolinium contrast agent 0.005 μ M + gadolinium element 1ng/mL, gadolinium contrast agent 0.025 μ M + gadolinium element 5ng/mL, gadolinium contrast agent 0.05 μ M + gadolinium element 10ng/mL, gadolinium contrast agent 0.1 μ M + gadolinium element 25ng/mL, gadolinium contrast agent 0.15 μ M + gadolinium element 50ng/mL, gadolinium contrast agent 0.25 μ M + gadolinium element 75ng/mL, and gadolinium contrast agent 0.5 μ M + gadolinium element 100 ng/mL.
Further, the standard curve equation of the gadolinium contrast agent in the third step is that Y is 5184.12X +6954.52, r20.9981, linear range of 0.005-0.5 μ M; the standard curve equation of gadolinium element is that Y is 8746.77X +1114.80, r20.9986, the linear range is 1-100 ng/mL.
Further, the volume ratio of the blood plasma or blood serum to be detected to acetonitrile in the step four is 1: 4; after mixing, the centrifugation condition is that the centrifugation speed is 13000rpm for 15min at 4 ℃.
Further, acetonitrile used for the pretreatment of the leucocyte in the step two (first) and acetonitrile used for the pretreatment of the clinical biological sample in the step four are all replaced by hydrogen peroxide with the mass concentration of 30%.
Further, when blank blood is pretreated by hydrogen peroxide with the mass concentration of 30%, the volume ratio of white blood plasma or blank blood serum to the hydrogen peroxide with the mass concentration of 30% is 2:1, and after uniform mixing, the blank blood is centrifuged for 15min at the centrifugal speed of 13000rpm under the centrifugal condition of 4 ℃.
Further, only acetonitrile used in blank blood pretreatment is replaced by hydrogen peroxide with the mass concentration of 30%, a mixed standard sample group containing gadolinium contrast agent and gadolinium element with the same concentration as that in acetonitrile pretreatment is prepared, a standard curve is drawn according to the same method, and the standard curve equation of the gadolinium contrast agent is obtained respectively, namely Y is 4.23e4X+7510.94，r20.994, linear range is 0.005-0.5 mu M; the standard curve equation of gadolinium element is Y-5.51 e4X+940.47，r2The linear range is 1-100 ng/mL, which is 0.991.
Further, when the blood to be detected is pretreated by hydrogen peroxide with the mass concentration of 30%, the volume ratio of the blood plasma or blood serum to be detected to the hydrogen peroxide with the mass concentration of 30% is 2:1, and after the blood plasma or blood serum to be detected is uniformly mixed, the blood plasma or blood serum to be detected is centrifuged for 15min at the centrifugation speed of 13000rpm at 4 ℃.
The invention has the beneficial effects that:
the method adopts high-efficiency molecular exclusion chromatography combined with inductively coupled plasma mass spectrometry to simultaneously detect the gadolinium contrast agent and free gadolinium ions in the blood plasma or the blood serum, and has the advantages of high specificity, high sensitivity, high accuracy, small sample volume, simple sample pretreatment and the like. The high-efficiency molecular exclusion chromatographic column has smaller particle size and higher separation efficiency, and can effectively separate GBCAs and free Gd in blood plasma or blood serum3+The method has the advantages of higher detection sensitivity, more accurate detection result, strong universality and easy popularization.
The method adopts acetonitrile or hydrogen peroxide when a plasma or serum sample is pretreated, the structure of GBCAs is not influenced when the acetonitrile precipitates proteins, and the hydrogen peroxide can effectively separate the gadolinium combined with the proteins, so that the extraction effect is good, the structure of the GBCAs is not influenced, and the accuracy of a detection result is ensured. And the dilution factor is smaller than that of acetonitrile pretreatment during hydrogen peroxide pretreatment, and the detection sensitivity of hydrogen peroxide pretreatment is higher. The pretreatment method is simple, the sample size is small, the operability is strong, and the clinical popularization is convenient.
The method is used for detecting gadolinium deposition, gadolinium clearance and gadolinium release degree in a patient body which is examined through magnetic resonance, can provide accurate basis for evaluating the kinetic characteristics of the gadolinium contrast agent in the patient body, is beneficial to researching the correlation between the gadolinium deposition amount in the body and gadolinium toxicity, provides early warning for the use of the gadolinium contrast agent, and reduces the occurrence of adverse reactions of the patient.
Drawings
FIG. 1 is a total chromatogram of a mixed standard sample of gadolinium butol and gadolinium element plasma as gadolinium contrast agents in example 1;
FIG. 2 is a chromatogram of a gadolinium contrast agent gadobutrol plasma standard sample;
FIG. 3 is a chromatogram of a gadolinium element plasma standard sample;
FIG. 4 is a total chromatogram of a mixed standard sample of gadolinium butol as gadolinium contrast agent and gadolinium element plasma of example 2.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in plasma by HPSEC-ICP-MS, which comprises the following steps:
step one, determining HPSEC-ICP-MS detection conditions;
conditions for high performance size exclusion chromatography in this example: size exclusion chromatography column: TSK gel G2000SWXL, 7.8 mm. times.300 mm, 5 μm; mobile phase: 20mM Tris-HCl solution at pH 7.4 at a flow rate of 1.0 mL/min; the sample volume is 10 mu L;
ICP-MS analysis conditions: radio frequency power: 1500W, atomizing gas flow rate: 0.8L/min, cooling gas flow rate: 15.0L/min, assist gas flow rate: 1.0L/min, dwell time 100ms, detection mode: a bump gas mode.
In the embodiment, the inductively coupled plasma mass spectrometry is combined by high-efficiency molecular exclusion chromatography, wherein the aperture of a high-efficiency molecular exclusion chromatographic column is 0.5-200 kDa, the particle size of the chromatographic column is smaller, the separation efficiency is higher, and the high-efficiency molecular exclusion chromatographic column can effectively separate GBCAs and free Gd from plasma or serum3+The method has the advantages of high specificity, high sensitivity, high accuracy, small sample volume, simple sample pretreatment and the like, and is strong in universality and easy to popularize.
Step two, preparing mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements:
pretreatment of blank blood:
in the embodiment, the blank blood is obtained from mixed blood samples of 7 different negative volunteers, the blood of the volunteers is collected by an anticoagulation tube and mixed, the mixture is centrifuged at 3000rpm for 10min to collect supernatant to obtain blank plasma, 100 mu L of the blank plasma is taken and added with 400 mu L of acetonitrile, the mixture is uniformly mixed by vortex for 15s, the mixture is centrifuged at 13000rpm at 4 ℃ for 15min, protein precipitate is removed, and the centrifuged supernatant is taken as a blank plasma sample;
(II) preparing a mixed standard sample:
pure water is used to prepare stock solutions of a gadolinium contrast agent and a gadolinium element, and the gadolinium contrast agent used in this example is gadolinium bromophenol. Respectively diluting stock solutions of a gadolinium contrast agent and gadolinium elements step by using pure water to obtain a gadolinium contrast agent standard mother solution with the concentration of 1000 mu M and a gadolinium element standard mother solution with the concentration of 20 mg/L; and respectively diluting the gadolinium contrast agent standard mother liquor and the gadolinium element standard mother liquor by using 2% nitric acid aqueous solution to obtain a gadolinium contrast agent standard solution and a gadolinium element standard solution.
In the embodiment, the concentration of the gadolinium contrast agent in the gadolinium contrast agent standard solution is 0.05, 0.25, 0.5, 1, 1.5, 2.5 and 5 μ M in sequence; the concentration of the gadolinium element in the gadolinium element standard solution is 10, 50, 100, 250, 500, 750 and 1000ng/mL in sequence.
Taking 10 mu L of gadolinium contrast agent standard solution and gadolinium element standard solution with corresponding concentrations, and adding 80 mu L of blank plasma sample to obtain a mixed standard sample simultaneously containing gadolinium contrast agent and gadolinium element.
In this example, a total of 7 mixed standard sample sets (STD1-STD7) containing both gadolinium contrast agent and gadolinium element at different concentrations were prepared, wherein the concentrations of gadolinium contrast agent and gadolinium element are shown in table 1.
TABLE 1
As shown in table 1, the concentrations of the gadolinium contrast agent and the gadolinium element in the mixed standard sample group containing both gadolinium contrast agent and gadolinium element are as follows: gadolinium contrast agent 0.005 μ M + gadolinium element 1ng/mL, gadolinium contrast agent 0.025 μ M + gadolinium element 5ng/mL, gadolinium contrast agent 0.05 μ M + gadolinium element 10ng/mL, gadolinium contrast agent 0.1 μ M + gadolinium element 25ng/mL, gadolinium contrast agent 0.15 μ M + gadolinium element 50ng/mL, gadolinium contrast agent 0.25 μ M + gadolinium element 75ng/mL, and gadolinium contrast agent 0.5 μ M + gadolinium element 100 ng/mL.
Step three, drawing a gadolinium contrast agent standard curve and a gadolinium element standard curve:
sequentially feeding and detecting a mixed standard sample group STD1-STD7 containing both gadolinium contrast agent and gadolinium element according to the HPSEC-ICP-MS detection condition determined in the step one; respectively taking the concentration of the gadolinium contrast agent or gadolinium element as a horizontal coordinate, and taking the corresponding peak area as a vertical coordinate to perform linear regression, and respectively obtaining a gadolinium contrast agent standard curve equation and a gadolinium element standard curve equation: the standard curve equation of gadolinium contrast agent is that Y is 5184.12X +6954.52, r20.9981, linear range of 0.005-0.5 μ M; standard curve equation of gadolinium elementIs Y-8746.77X +1114.80, r20.9986, the linear range is 1-100 ng/mL.
Step four, collecting and processing clinical biological samples:
collecting blood to be tested, centrifuging at 3000rpm for 10min, collecting blood to be tested with anticoagulation tube, centrifuging, collecting supernatant to obtain blood plasma to be tested, subpackaging each part with 100 μ L, and storing in-80 deg.C refrigerator;
adding 400 mul acetonitrile into 100 mul blood plasma to be detected, mixing evenly for 15s by vortex, centrifuging for 15min at the centrifugal speed of 13000rpm at 4 ℃, removing protein precipitate, and taking supernatant liquid by a pipettor to obtain a sample to be detected.
The method adopts acetonitrile to precipitate the protein, does not influence the structure of the gadolinium contrast agent, and is simple, small in sample size, strong in operability and convenient for clinical popularization.
Step five, sample detection and data processing:
and step four, the sample to be detected obtained in the step one is subjected to sample injection detection according to the HPSEC-ICP-MS detection condition determined in the step one, the retention time is used for qualitative detection, the peak area is used for quantitative detection, the standard curve equation obtained in the step three is used for quantitative detection through an external standard method, and the concentration of the gadolinium contrast agent and the concentration of gadolinium elements in the sample to be detected are obtained through calculation.
In this embodiment, when the blank blood is collected by using a common serum tube, the blank serum obtained under the same centrifugal processing conditions is subjected to sample injection processing by the same method of the second step and the third step, and the drawn standard curve is substantially the same as the standard curve obtained by using the blank plasma, so that when a clinical biological sample is processed, data obtained by sample injection detection using a to-be-detected plasma sample or a to-be-detected serum sample can be brought into a standard curve equation to obtain the concentration of a gadolinium contrast agent and the concentration of gadolinium element in the to-be-detected sample, and the detection results of the to-be-detected plasma sample and the to-be-detected serum sample are substantially the same.
Example 2
The embodiment provides a method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in plasma by HPSEC-ICP-MS, which comprises the following steps:
step one, determining HPSEC-ICP-MS detection conditions;
conditions for high performance size exclusion chromatography in this example: size exclusion chromatography column: TSK gel G2000SWXL, 7.8 mm. times.300 mm, 5 μm; mobile phase: 20mM Tris-HCl solution at pH 7.4 at a flow rate of 1.0 mL/min; the sample volume is 10 mu L;
ICP-MS analysis conditions: radio frequency power: 1500W, atomizing gas flow rate: 0.8L/min, cooling gas flow rate: 15.0L/min, assist gas flow rate: 1.0L/min, dwell time 100ms, detection mode: a bump gas mode.
Step two, preparing mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements:
pretreatment of blank blood:
in the embodiment, the blank blood is obtained from mixed blood samples of 7 different negative volunteers, blood of the volunteers is collected by an anticoagulation tube and mixed, the mixture is centrifuged at 3000rpm for 10min to collect supernatant to obtain blank plasma, 100 microliter of the blank plasma is taken, 50 microliter of hydrogen peroxide with the mass concentration of 30% is added, the mixture is uniformly mixed by vortex for 15s, the mixture is centrifuged at 4 ℃ at 13000rpm for 15min, and the centrifuged supernatant is taken as a blank plasma sample;
(II) preparing a mixed standard sample:
pure water is used to prepare stock solutions of a gadolinium contrast agent and a gadolinium element, and the gadolinium contrast agent used in this example is gadolinium bromophenol. Respectively diluting stock solutions of a gadolinium contrast agent and gadolinium elements step by using pure water to obtain a gadolinium contrast agent standard mother solution with the concentration of 1000 mu M and a gadolinium element standard mother solution with the concentration of 20 mg/L; and respectively diluting the gadolinium contrast agent standard mother liquor and the gadolinium element standard mother liquor by using 2% nitric acid aqueous solution to obtain a gadolinium contrast agent standard solution and a gadolinium element standard solution.
In the embodiment, the concentration of the gadolinium contrast agent in the gadolinium contrast agent standard solution is 0.05, 0.25, 0.5, 1, 1.5, 2.5 and 5 μ M in sequence; the concentration of the gadolinium element in the gadolinium element standard solution is 10, 50, 100, 250, 500, 750 and 1000ng/mL in sequence.
Taking 10 mu L of gadolinium contrast agent standard solution and gadolinium element standard solution with corresponding concentrations, and adding 80 mu L of blank plasma sample to obtain a mixed standard sample simultaneously containing gadolinium contrast agent and gadolinium element.
In this example, a total of 7 mixed standard sample sets (STD1-STD7) containing gadolinium as a gadolinium contrast agent and gadolinium element at the same concentrations as those shown in table 1 of example 1 were prepared.
Step three, drawing a gadolinium contrast agent standard curve and a gadolinium element standard curve:
sequentially feeding and detecting a mixed standard sample group STD1-STD7 containing both gadolinium contrast agent and gadolinium element according to the HPSEC-ICP-MS detection condition determined in the step one; respectively taking the concentration of the gadolinium contrast agent or gadolinium element as an abscissa and taking the corresponding peak area as an ordinate to perform linear regression, and respectively obtaining a standard curve equation of the gadolinium contrast agent as Y-4.23 e4X+7510.94，r20.994, linear range is 0.005-0.5 mu M; the standard curve equation of gadolinium element is Y-5.51 e4X+940.47，r2The linear range is 1-100 ng/mL, which is 0.991.
Step four, collecting and processing clinical biological samples:
collecting blood to be tested, centrifuging at 3000rpm for 10min, collecting blood to be tested with anticoagulation tube, centrifuging, collecting supernatant to obtain blood plasma to be tested, subpackaging each part with 100 μ L, and storing in-80 deg.C refrigerator;
adding 50 mul of hydrogen peroxide with the mass concentration of 30% into 100 mul of blood plasma to be detected, uniformly mixing the blood plasma for 15s in a vortex manner, centrifuging the blood plasma for 15min at the centrifugal speed of 13000rpm at 4 ℃, and taking supernatant liquid by a pipettor to obtain a sample to be detected.
In the embodiment, hydrogen peroxide is adopted for pretreatment, the hydrogen peroxide can effectively separate gadolinium combined with protein, the extraction effect is good, the structure of a gadolinium contrast agent is not affected, the hydrogen peroxide dilution factor is small, the detection result is accurate, the method is simple, the sample size is small, the operability is high, and the clinical popularization is facilitated.
Step five, sample detection and data processing:
and step four, the sample to be detected obtained in the step one is subjected to sample injection detection according to the HPSEC-ICP-MS detection condition determined in the step one, the retention time is used for qualitative detection, the peak area is used for quantitative detection, the standard curve equation obtained in the step three is used for quantitative detection through an external standard method, and the concentration of the gadolinium contrast agent and the concentration of gadolinium elements in the sample to be detected are obtained through calculation.
In this embodiment, when the blank blood is collected by using a common serum tube, the blank serum obtained under the same centrifugal processing conditions is subjected to sample injection processing by the same method of the second step and the third step, and the drawn standard curve is substantially the same as the standard curve obtained by using the blank plasma, so that when a clinical biological sample is processed, data obtained by sample injection detection using a to-be-detected plasma sample or a to-be-detected serum sample can be brought into a standard curve equation to obtain the concentration of a gadolinium contrast agent and the concentration of gadolinium element in the to-be-detected sample, and the detection results of the to-be-detected plasma sample and the to-be-detected serum sample are substantially the same.
FIG. 1 is a total chromatogram of a standard sample of a mixture of plasma containing gadolinium as contrast agent and gadolinium at a concentration of gadobutrol 0.1. mu.M + gadolinium element 25ng/mL in example 1; FIG. 2 is a chromatogram of a standard sample of acetonitrile-treated plasma containing gadobutrol only 0.1. mu.M; FIG. 3 is a chromatogram of a standard sample of acetonitrile-treated plasma containing only gadolinium element 25 ng/mL; the comparison of fig. 1 to fig. 3 shows that the retention time of the gadolinium contrast agent is 6.13min, the retention time of gadolinium element is 9.28min, and the detection method provided by the invention has the advantages of strong specificity of target analyte, good separation degree and small interference.
FIG. 4 is a total chromatogram of a standard sample of a mixture of plasma containing gadolinium as contrast agent and gadolinium at a concentration of gadobutrol 0.1. mu.M + gadolinium element 25ng/mL in example 2; therefore, the blood sample is pretreated by using the hydrogen peroxide, and the detection result with strong specificity, good separation degree and small interference of the target analyte can be obtained.
Example 3
The present example verifies the accuracy and precision of the method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in plasma provided in example 1 as follows:
respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 0.25 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 50ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 1 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 250ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with gadolinium contrast agent concentration of 2.5 muM and 2.5 muL of gadolinium element standard solution with gadolinium element concentration of 750ng/mL, and adding 95 muL of blank plasma; obtaining a mixed standard product containing gadolinium contrast agent and gadolinium element at three concentrations of low, medium and high.
The measurement was carried out in the same manner as in example 1, and the analysis was repeated for 3 batches, and the recovery rate and the daily precision were as shown in Table 2.
TABLE 2
As can be seen from the data in table 2, the spiked recovery rates of both the gadolinium contrast agent and gadolinium element at different spiked levels are within ± 15% for the method provided in example 1; the precision in the day and the precision in the day are within +/-15%, which shows that the detection method provided by the embodiment 1 has better precision and repeatability and can be used for accurate quantitative analysis.
Example 4
This example demonstrates the stability of the method provided in example 1 for simultaneous detection of gadolinium contrast agent and free gadolinium ions in plasma as follows:
respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 0.25 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 50ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 1 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 250ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with gadolinium contrast agent concentration of 2.5 muM and 2.5 muL of gadolinium element standard solution with gadolinium element concentration of 750ng/mL, and adding 95 muL of blank plasma; obtaining a mixed standard product containing gadolinium contrast agent and gadolinium element at three concentrations of low, medium and high. Three groups are prepared respectively, wherein the group 1 is placed at room temperature for 6h, the group 2 is placed in a refrigerator with the temperature of 80 ℃ below zero and is repeatedly frozen and thawed for three times, and the group 3 is placed in the refrigerator with the temperature of 80 ℃ below zero for 30 days.
The assay was carried out as described in example 1, and group 1, group 2 and group 3 were analyzed repeatedly for 3 batches, giving room temperature stability, freeze thaw stability, -80 ℃ stability (30 days) as shown in Table 3.
TABLE 3
As can be seen from the data in Table 3, the recovery rates of gadolinium contrast agent and gadolinium element in groups 1, 2 and 3 at different standard addition levels are within + -15%, and the stability of the detection method provided in example 1 meets the standard.
Example 5
The present example verifies the accuracy and precision of the method for simultaneously detecting gadolinium contrast agent and free gadolinium ions in plasma provided in example 2 as follows:
respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 0.25 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 50ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 1 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 250ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with gadolinium contrast agent concentration of 2.5 muM and 2.5 muL of gadolinium element standard solution with gadolinium element concentration of 750ng/mL, and adding 95 muL of blank plasma; obtaining a mixed standard product containing gadolinium contrast agent and gadolinium element at three concentrations of low, medium and high.
The measurement was carried out in the same manner as in example 2, and the analysis was repeated for 3 batches, and the recovery rate and the daily precision are shown in Table 4.
TABLE 4
As can be seen from the data in table 4, the spiked recovery of both the gadolinium contrast agent and gadolinium element at different spiked levels is within ± 15% for the method provided in example 2; the precision in the day and the precision in the day are within +/-15%, which shows that the detection method provided by the embodiment 2 has better precision and repeatability and can be used for accurate quantitative analysis.
Example 6
This example demonstrates the stability of the method provided in example 2 for simultaneous detection of gadolinium contrast agent and free gadolinium ions in plasma as follows:
respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 0.25 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 50ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with the gadolinium contrast agent concentration of 1 muM and 2.5 muL of gadolinium element standard solution with the gadolinium element concentration of 250ng/mL, and adding 95 muL of blank plasma; respectively taking 2.5 muL of gadolinium contrast agent standard solution with gadolinium contrast agent concentration of 2.5 muM and 2.5 muL of gadolinium element standard solution with gadolinium element concentration of 750ng/mL, and adding 95 muL of blank plasma; obtaining a mixed standard product containing gadolinium contrast agent and gadolinium element at three concentrations of low, medium and high. Three groups are prepared respectively, wherein the group 1 is placed at room temperature for 6h, the group 2 is placed in a refrigerator with the temperature of 80 ℃ below zero and is repeatedly frozen and thawed for three times, and the group 3 is placed in the refrigerator with the temperature of 80 ℃ below zero for 30 days.
The assay was performed as described in example 2, and group 1, group 2 and group 3 were analyzed repeatedly for 3 batches, giving room temperature stability, freeze thaw stability, -80 ℃ stability (30 days) as shown in Table 5.
TABLE 5
As can be seen from the data in Table 5, the recovery rates of gadolinium contrast agent and gadolinium element in groups 1, 2 and 3 at different standard addition levels are within + -15%, and the stability of the detection method provided in example 2 meets the standard.
Claims (10)
1. A method for simultaneously detecting a gadolinium contrast agent and free gadolinium ions in blood plasma or blood serum is characterized by comprising the following steps:
step one, determining HPSEC-ICP-MS detection conditions;
step two, preparing mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements:
pretreatment of blank blood: centrifuging the collected blank blood, collecting supernatant to obtain blank plasma or blank serum, adding acetonitrile into the blank plasma or blank serum, mixing uniformly, centrifuging, and collecting the centrifuged supernatant as a blank plasma sample or a blank serum sample;
(II) preparing a mixed standard sample: adding gadolinium contrast agent standard solution and gadolinium element standard solution with corresponding concentrations into the obtained blank plasma sample or blank serum sample to obtain mixed standard sample groups containing gadolinium contrast agent and gadolinium element at different concentrations;
step three, drawing a gadolinium contrast agent standard curve and a gadolinium element standard curve:
sequentially injecting and detecting the mixed standard sample groups with different concentrations and containing gadolinium contrast agents and gadolinium elements according to the HPSEC-ICP-MS detection conditions determined in the step one; respectively taking the concentration of the gadolinium contrast agent or gadolinium element as a horizontal coordinate and taking the corresponding peak area as a vertical coordinate to perform linear regression so as to respectively obtain a gadolinium contrast agent standard curve equation and a gadolinium element standard curve equation;
step four, pretreatment of clinical biological samples:
centrifuging the collected blood sample to be detected, collecting supernatant to obtain blood plasma or blood serum to be detected, adding acetonitrile into the blood plasma or blood serum to be detected, mixing uniformly, centrifuging, and taking the centrifuged supernatant as a sample to be detected;
step five, sample detection and data processing:
and step four, the sample to be detected obtained in the step one is subjected to sample injection detection according to the HPSEC-ICP-MS detection condition determined in the step one, the retention time is used for qualitative detection, the peak area is used for quantitative detection, the standard curve equation obtained in the step three is used for quantitative detection through an external standard method, and the concentration of the gadolinium contrast agent and the concentration of gadolinium elements in the sample to be detected are obtained through calculation.
2. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the blood plasma or the blood serum according to claim 1, wherein the detection conditions of HPSEC-ICP-MS in the step one are as follows:
conditions for high performance size exclusion chromatography: size exclusion chromatography column: TSK gel G2000SWXL, 7.8 mm. times.300 mm, 5 μm; mobile phase: 20mM Tris-HCl solution at pH 7.4 at a flow rate of 1.0 mL/min; the sample volume is 10 mu L;
ICP-MS analysis conditions: radio frequency power: 1500W, atomizing gas flow rate: 0.8L/min, cooling gas flow rate: 15.0L/min, assist gas flow rate: 1.0L/min, dwell time 100ms, detection mode: a bump gas mode.
3. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the plasma or the serum according to claim 1 or 2, wherein the blank blood centrifugation condition in the second step is centrifugation at 3000rpm for 10 min; the blank plasma or blank serum and acetonitrile are mixed according to the volume ratio of 1:4, and the centrifugation condition after mixing is 13000rpm centrifugation for 15min at 4 ℃; the volume ratio of the gadolinium contrast agent standard solution with the corresponding concentration to the gadolinium element standard solution to the blank plasma sample or the blank serum sample is 10:10: 80.
4. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the blood plasma or the blood serum according to claim 3, wherein the concentrations of the gadolinium contrast agent and the gadolinium element in the mixed standard sample group containing the gadolinium contrast agent and the gadolinium element in the step two are sequentially: gadolinium contrast agent 0.005 μ M + gadolinium element 1ng/mL, gadolinium contrast agent 0.025 μ M + gadolinium element 5ng/mL, gadolinium contrast agent 0.05 μ M + gadolinium element 10ng/mL, gadolinium contrast agent 0.1 μ M + gadolinium element 25ng/mL, gadolinium contrast agent 0.15 μ M + gadolinium element 50ng/mL, gadolinium contrast agent 0.25 μ M + gadolinium element 75ng/mL, and gadolinium contrast agent 0.5 μ M + gadolinium element 100 ng/mL.
5. The method of claim 4, wherein the standard curve equation of the gadolinium contrast agent in step three is Y-5184.12X +6954.52, r20.9981, linear range of 0.005-0.5 μ M; the standard curve equation of gadolinium element is that Y is 8746.77X +1114.80, r20.9986, the linear range is 1-100 ng/mL.
6. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the plasma or the serum according to claim 5, wherein the volume ratio of the to-be-detected plasma or the to-be-detected serum to the acetonitrile in the step four is 1: 4; after mixing, the centrifugation condition is that the centrifugation speed is 13000rpm for 15min at 4 ℃.
7. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the blood plasma or the blood serum according to claim 6, wherein acetonitrile used in the pretreatment of the white blood in the step two (first) and acetonitrile used in the pretreatment of the clinical biological sample in the step four are all replaced by hydrogen peroxide with the mass concentration of 30%.
8. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the plasma or the serum according to claim 7, wherein the volume ratio of the white plasma or the blank serum to the hydrogen peroxide with the mass concentration of 30% is 2:1 when the blank blood is pretreated with the hydrogen peroxide with the mass concentration of 30%, and the blank blood is centrifuged for 15min at the centrifugation speed of 13000rpm at 4 ℃ after being uniformly mixed.
9. The method according to claim 8, wherein acetonitrile used in the blank blood pretreatment is replaced with hydrogen peroxide having a mass concentration of 30%, a mixed standard sample set containing gadolinium contrast agent and gadolinium element at the same concentration as that in the acetonitrile pretreatment is prepared, a standard curve is drawn according to the same method, and a standard curve equation of Y4.23 e for gadolinium contrast agent is obtained4X+7510.94，r20.994, linear range is 0.005-0.5 mu M; the standard curve equation of gadolinium element is Y-5.51 e4X+940.47，r2The linear range is 1-100 ng/mL, which is 0.991.
10. The method for simultaneously detecting the gadolinium contrast agent and the free gadolinium ions in the plasma or the serum according to claim 9, wherein the volume ratio of the plasma or the serum to be detected to the hydrogen peroxide with the mass concentration of 30% is 2:1 when the blood to be detected is pretreated by the hydrogen peroxide with the mass concentration of 30%, and the plasma or the serum to be detected is centrifuged for 15min at the centrifugation speed of 13000rpm at 4 ℃ after being uniformly mixed.
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