Measurement of Magnesium, Zinc, and Copper in Human Serum by Using Isotope Dilution Inductively Coupled Plasma Mass Spectrometry (ID ICP-MS)

In order to evaluate the reliability of the ID ICP-MS method for the measurement of magnesium, zinc, and copper in human serum, we investigated the traceability, precision, trueness, and uncertainty of the method. This method traces the contents of magnesium, zinc, and copper in human serum to the standard materials NIST SRM3131a, SRM3168a, and SRM3114 respectively, thus completing the traceability to SI unit. The repeatability of this method for measuring magnesium, zinc, and copper in the human serum reference material GBW09152 was found to be 0.2%, 0.7%, and 0.6% (n = 9), respectively. The measurement, when employed to measure the magnesium, zinc, and copper in standard materials, had caused a maximum deviation of less than 0.88%, 1.35%, and 1.15%, respectively. The measurement results are within the stated uncertainty range of standard materials. The expanded uncertainties were 0.2 mg·kg−1, 0.04 mg·kg−1, and 0.08 mg·kg−1 (K = 2) for magnesium, zinc, and copper, respectively. Therefore, this method has high trueness, good reproducibility, and simple operation and is suitable for tracing the values of magnesium, zinc, and copper in human serum.


Introduction
Isotope dilution mass spectrometry, which has many advantages over conventional methods, is the only authoritative method that can directly provide trace and ultratrace values [1].In common analysis methods, the accuracy of the results may be afected by the loss of the elements to be tested in the process of sample pretreatment, matrix efects, and instrument signal drift [2][3][4][5].Isotope dilution mass spectrometry is the method which measures only the isotope abundance ratio in the sample.Te abundance ratio becomes a constant value when the concentration of the isotopic spike is added to the sample and reaches the equilibrium with the absence of external contamination, and the sample loss in the process of sample separation and concentration does not afect the abundance ratio [6].Isotope dilution method can greatly eliminate interference and errors caused by sample pretreatment and has thus been confrmed by the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) as one of the fve methods with absolute measurement properties [7,8].Tis isotope dilution mass spectrometry originated from the UK Government Chemist Laboratory (GCL) [9,10].We have established isotope dilution mass spectrometry for the detection of potassium and selenium in human serum using this principle [11].Compared with conventional isotope dilution mass spectrometry, it has the advantage of not having to calibrate the concentration of the isotopic spike during the standardization process.It can determine the chemical composition in serum only by means of measuring the selected pair of isotope abundance ratios, thus reducing the requirements on the performance of the mass spectrometry instrument [12][13][14][15][16][17].
Trace elements of magnesium, zinc, and copper in the human body are closely related to people's health [18][19][20][21][22]. Currently, there is no reference method for measuring zinc and copper in serum by using isotope dilution mass spectrometry in the Joint Committee on Traceability on Laboratory Medicine (JCTLM) list.Only the measurement of the concentration of magnesium in serum by using traditional isotope dilution mass spectrometry is available [23][24][25].A precise method for analyzing magnesium, zinc, and copper in human serum by using two-step isotope dilution mass spectrometry was established in this laboratory.Te repeatability of this method for measuring magnesium, zinc, and copper in the human serum reference material GBW09152 was found to be 0.2%, 0.7%, and 0.6% (n � 9), respectively.Te expanded uncertainties were 0.2 mg•kg −1 , 0.04 mg•kg −1 , and 0.08 mg•kg −1 (K � 2) for magnesium, zinc, and copper, respectively.With the quality of easy operation, high trueness, and good reproducibility, this method is suitable as a reference method for measuring magnesium, zinc, and copper elements in human serum.

Materials and Reagents.
Te laboratory is a class 100,000 cleanroom.Te experimental water was provided by a water purifcation system: Milli-Q Advantage (Millipore, USA).Te nitric acid (Ultrapure-BVIII) and hydrochloric acid (MOS grade) used in the experiment were produced by the Beijing Institute of Chemical Reagents, China.Te microanalytical balance used for sample weighing was Mettler Toledo XS205 (Switzerland), and the sample analysis was performed on an ICP mass spectrometer: ELAN DRC-e (PerkinElmer, USA).Te standard materials used for method tracing were magnesium (Mg) standard solution SRM3131a (NIST, USA), copper (Cu) standard solution SRM3114 (NIST, USA), and zinc (Zn) standard solution SRM3168a (NIST, USA).Te isotope spikes used in the method were 25 Mg (assay: 97%), 65 Cu (assay: 99%), and 67 Zn (assay: 94%), concentrated isotopes from the Oak Ridge National Laboratory, USA.Trueness verifcation was performed by using inorganic components in frozen human serum GBW09152 (National Institute of Metrology, China), ERM-DA120a (LGC, UK) and electrolytes in frozen human serum SRM956d (NIST, USA).

Instrument Parameters.
Te instrument parameters for this research are listed in Table 1.

Methods
(1) A two-step dilution of NIST SRM3131a was performed by using the weighing method in a ratio of approximately 1 : 20, and a three-step dilution of NIST SRM3168a and NIST SRM3114 in a ratio of approximately 1 : 20.Te fnal dilution must be prepared on the day of the experiment.(2) Te concentrated isotopic metal chips of 25 Mg, 65 C, and 67 Zn were dissolved with BVIII grade nitric acid and diluted to an appropriate concentration with ultrapure water, followed by a two-step dilution on the day of the experiment.(3) A mixed solution of magnesium standard solution and 25 Mg isotope diluent, a mixed solution of zinc standard solution and 67 Zn isotope diluent, and a mixed solution of copper standard solution and 65 Cu isotope diluent were prepared, respectively, by using the weighing method.A mixed solution of the serum sample and 25 Mg isotope diluent, a mixed solution of the serum sample and 67 Zn isotope diluent, and a mixed solution of the serum sample and 65 Cu isotope diluent were also prepared, respectively, by using the weighing method.Te isotopic ratio ( 24 Mg/ 25 Mg or 66 Zn/ 67 Zn or 63 Cu/ 65 Cu) in the mixture solution was close to 1, and at the same time, the cps signal intensities of the corresponding isotopes in the mixture of the standard solution and the isotope diluent as well as in the mixture of the serum sample and the isotope diluent were close.(4) Solutions of magnesium, copper, and zinc with appropriate concentrations were prepared on the day of the experiment so that the signal intensity of 24 Mg, 66 Zn, and 63 Cu in the solution is consistent with that of the corresponding isotopes in the mixed solution in (3).( 5) Solutions of 25 Mg, 67 Zn, and 65 Cu diluents with appropriate concentrations were prepared on the day of the experiment so that the signal intensity of the isotopes in the solution is consistent with that of the corresponding isotopes in the mixed solution in (3).( 6) Mass spectrometric procedures: the concentrations of magnesium, zinc, and copper elements in the serum sample were calculated according to the concentration formula (1) in the isotope dilution mass spectrometry [9]: In this formula, C z is the concentration of the standard solution, m Y is the mass of the enriched isotope added to the serum sample, m X is the mass of the serum sample added to the mixture of the serum sample and the enriched isotope, m Yc is the mass of the enriched isotope added to the standard solution, and m zc is the mass of the standard solution added to the mixture of the standard solution and the enriched isotope.Rz is the isotope ratio of 24 Mg/ 25 Mg or 66 Zn/ 67 Zn or Cu/ 65 Cu) in the mixture of serum sample and the enriched isotope, and R Bc the isotope ratio of 24 Mg/ 25 Mg ( 66 Zn/ 67 Zn or 63 Cu/ 65 Cu) in the mixture of standard solution and the enriched isotope.C B is a blank in the measurement process.
It was found that the fuctuation of the isotope ratio in the standard solution or the enriched isotope had little impact on the fnal results, while the measurement fuctuation of the isotope ratio in the mixed solution would lead to changes in the fnal results.Terefore, the mixed solution of the standard solution and the enriched isotope and the mixed solution of the serum sample and the enriched isotope must be alternately measured six times so as to reduce the error introduced by instrument measurement drift.

Determination of Instrument Measurement Conditions.
In this experiment, the dynamic reaction cell mode of inductively coupled plasma mass spectrometry (ICP-MS) was used to eliminate interference.When analyzing magnesium, interference from NaH was severe, and interference from Ca ++ and LiO may also exist.Te condition optimization was aiming to obtain the best analysis efect when analyzing 24 Mg, and the appropriate fow rate of the reaction gas CH 4 and argon.When analyzing zinc, interference from SO 2 , ClO 2 , and ArP may exist, and interference on 67 Zn was more likely to occur.Te condition optimization was aimed at obtaining the best analysis efect when analyzing 67 Zn and the appropriate fow rate of the reaction gas CH 4 and argon.When analyzing copper, interference from SO 2 and PO 2 may exist, while all these interferences can be ignored in actual analysis.Terefore, this experiment analyzed copper in blood under standard mode.

Deduction of Signal Background in the Experimental
Method.Solutions of serum magnesium with 6 concentration gradients ranging from 0.5 μg/ml to 9 μg/ml were measured.Te response signals of 24 Mg and 25 Mg are linearly related to the concentration range of magnesium (see Figures 1(a) and 1(b)), with a linear correlation coefcient (r) of 0.99999.However, the ratio of 24 Mg response signal to 25 Mg response signal was not constant but gradually tended to be constant with the increase of concentration (see Figure 1(c)).Tis is because the straight lines in Figures 1(a) and 1(b) have a nonzero intercept, indicating the presence of a blank response signal for 24 Mg and 25 Mg.As the concentration becomes smaller, the relative proportion of the response signal becomes higher, and the impact on the ratio of 24 Mg to 25 Mg response signal is more signifcant.Terefore, this blank response signal cannot be ignored.After subtracting the blank response signal from the measured impact signal, a constant value is obtained for the ratio of the 24 Mg to 25 Mg•g response signals.Te same is true for the determination of 66 Zn/ 67 Zn and 63 Cu/ 65 Cu, so in this experiment, the response signals used are all values after deducting the blank signal.

Efect of Solution Reaction System on Measurement
Precision.It was found in the experiment that using a 0.02% hydrochloric acid system can improve the stability of 66 Zn/ 67 Zn measurement.Terefore, the determination of zinc in serum was indeed carried out in a 0.02% hydrochloric acid system.

Process Blank (LOB) and Detection Limits (LOD).
While preparing the mixture of serum sample and the enriched isotope, an appropriate amount of 25 Mg ( 67 Zn or 65 Cu) solution was taken into the blank sample tube as a process blank, so that the 24 Mg/ 25 Mg ( 66 Zn/ 67 Zn or 63 Cu/ 65 Cu) in the process blank was approximately equal to 2. Te process blank was determined along with the samples in the same batch.Te process blanks for magnesium, zinc, and copper in serum were 0.5 mg/kg, 0.09 mg/kg, and 0.010 mg/kg, respectively.When the confdence interval of 95% was determined, the detection limits of magnesium, zinc, and copper in serum were 0.7 mg/kg, 0.11 mg/kg, and 0.016 mg/kg, respectively.

Method Precision.
Te method precision was the relative standard deviation of the measurement results of 6 bottles of human serum reference materials.Te experiments were carried out in 2 consecutive days, with 3 bottles each day, and 3 parallel for each bottle.Te results are shown in Table 2. Te precision for magnesium, zinc, and copper in diferent concentrations of human serum reference materials was lower than 0.3%, 0.9%, and 0.6%, respectively.
3.6.Method Trueness.Magnesium, zinc, and copper in standard substances NIST956D, ERM-DA120a, and GBW09152 were analyzed by using isotope dilution mass spectrometry.Parallel analysis was conducted three times a day for three consecutive days, and the results were good, as shown in Table 3.

Uncertainty Evaluation.
In this research, the uncertainty caused by factors such as the experimental reagents, the samples, the laboratory environments, the solution preparation, the instrument measurement, and the data processing has been evaluated as the source of uncertainty in the measurement process.It can be concluded that by evaluating the uncertainty of each parameter in formula (1), the uncertainty caused by each factor in the measurement process can be fully included.Each parameter in formula ( 1) is an independent parameter, and the uncertainty u c (y) related to measurement is calculated as follows: Te formula of the sensitivity coefcient (zf/zx i ) of each parameter in formula ( 1) is as follows: International Journal of Analytical Chemistry

Terefore,
Table 4: Sources of uncertainty in the determination.Tis research has evaluated the uncertainty of measurement and Table 4 is the source of uncertainty for measuring the parameters.Te uncertainty of the measurement of Type A is the experimental standard deviation of the 6 repeated instrument measurements, taking the worst result in the experiment as the evaluation data.Te uncertainty of the measurement of Type B is the uncertainty of solution preparation, which is synthesized from the uncertainty resulting from the electronic balance calibration and the uncertainty resulting from weighing.

Conclusions
Tis isotope dilution mass spectrometry established in our laboratory with the quality of easy operation, high trueness, and good reproducibility was used to accurately analyze the contents of magnesium, zinc, and copper in human serum.In the isotope dilution mass spectrometry determination process, there is no need to measure the accurate amount of isotope, the enriched isotope, which reduces the requirements for mass spectrometry instruments [26,27].Tis method is suitable as a reference method for assigning values of magnesium, zinc, and copper in human serum.

Figure 1 :
Figure1: (a) Relationship between the response signal of24 Mg and magnesium concentration; (b) relationship between the response signal of25 Mg and magnesium concentration; (c) relationship between the ratio of24 Mg to25 Mg response signals and sample concentration.

Table 1 :
Instrument parameters.Cu in the standard solution, and R Y is the isotope ratio of24Mg/25Mg ( 66 Zn/ 67 Zn or 63 Cu/ 65 Cu) in the enriched isotope.R B is the isotope ratio of24Mg/ 25 Mg ( 66 Zn/ 67 Zn or 63

Table 2 :
Repeatability of magnesium, zinc, and copper in human serum by ID ICP-MS.