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An Evaluation of Different Digestion Methods for the Quantitation of Inorganic Elements in Human Hair Using ICP-MS

An Evaluation of Different Digestion Methods for the Quantitation of Inorganic Elements in Human... Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 5742468, 9 pages https://doi.org/10.1155/2022/5742468 Research Article An Evaluation of Different Digestion Methods for the Quantitation of Inorganic Elements in Human Hair Using ICP-MS 1,2 3,4 2 5 6 Yue Liu , Yang Yang, Yin-Yin Xia, Jamie V. de Seymour , De-Zhang Zhao, 7 3,4 1,4 Yang-Mei Li, Hua Zhang , and Ting-Li Han Department of Obstetrics andGynaecology, TeSecond Afliated Hospital of Chongqing Medical University, Chongqing, China Department of Occupational and Environmental Hygiene, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, China Department of Obstetrics and Gynaecology, Te First Afliated Hospital of Chongqing Medical University, Chongqing, China Mass Spectrometry Center of Maternal-Fetal Medicine, Chongqing Medical University, Chongqing, China College of Health, Massey University, Auckland, New Zealand College of Pharmacy, Chongqing Medical University, Chongqing, China School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, China Correspondence should be addressed to Hua Zhang; zh2844@gmail.com and Ting-Li Han; tinglihan@cqmu.edu.cn Received 23 August 2022; Revised 28 October 2022; Accepted 21 November 2022; Published 1 December 2022 Academic Editor: Radosław Kowalski Copyright © 2022 Yue Liu et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te inorganic elements have unique properties in biochemical processes in humans. An increasing number of pathologies have been associated with essential element ions, such as lead, mercury, and cadmium. Hair has become an attractive clinical specimen for studying the longitudinal exposure to elements from the external environment. Inductively coupled plasma-mass spectrometry (ICP- MS) coupled with nitric acid (HNO ) digestion is the most common approach for determining inorganic elements from human hair. Tis study aims to optimize the digestion method for the absolute quantitation of 52 elements using ICP-MS, for a large cohort study in human hair. Five diferent HNO (65%) digestion methods were investigated and evaluated for their internal standard solution stability, reproducibility, element coverage, and standard solution recovery efciency, namely, room temperature for 24 h (RT), 90 C for 4h (T90), ultrasonic-assisted digestion (UltraS), programmed digestion of microwave digestion (MicroD), and ordinary mi- crowave oven digestion (O-MicroD). Our results demonstrated that O-MicroD, MicroD, and RTwere the best performing digestion methods for coefcient of variation (CV) scores, coverage, and recovery efciency, respectively. In particular, the O-MicroD method detected multiple elements in a small quantity of hair (3 mg), with minimum nitric acid usage (200μl) and a short digestion time (30 min). Te O-MicroD method had excellent reproducibility, as demonstrated by a continuous thousand injections of hair samples 103 115 209 with three internal standards (CV: Rh � 3.59%, In � 3.61%, and Bi � 6.31%). Future studies of the elemental content of hair should carefully select their digestion method to meet the primary purpose of their study. sodium maintain the electrochemical gradient of the cell 1. Introduction membranes [1]. Tere are many metal ions that are im- Inorganic elements have unique properties that cannot be portant for cellular metabolism and mitochondrial function, + 2+ 2+ performed by organic compounds. Some elements are in- such as K , Mg , and Zn [2]. Nevertheless, growing dispensable for humans and essential in biochemical pro- evidence suggests that excessive exposure to some elements cesses. For example, iron (Fe), copper (Cu), and manganese could cause adverse health outcomes. Te role of cadmium (Mn) are all positioned at the metal-catalytic site of enzymes (Cd), mercury (Hg), and copper (Cu) in type 2 diabetes, involved in redox reactions [1]. Calcium, magnesium, and renal dysfunction, cardiovascular diseases, osteoporosis, and 2 Journal of Analytical Methods in Chemistry Tis study aims to establish a method for analysing the cancers, has been reported in experimental and epidemio- logical studies [3–5]. Excessive aluminium (Al), lead (Pb), inorganic elements in human hair samples from a large cohort, using low hair mass. We analysed fve diferent and arsenic (As) exposures have also been associated with oxidative stress, intestinal diseases, dyslipidemia, and met- sample preparation methods for their reproducibility, ele- abolic diseases [3, 6, 7]. Exposure to metals can be mediated ment coverage, and extraction efciency. Te results from through food, dermal contact, air pollution, and drinking our fndings have informed the methodology for a large water [7]. Elements absorbed by the pulmonary tract and the cohort study of hair from pregnant women to investigate the digestive system can be distributed via the bloodstream to a link between exposure to inorganic elements and pregnancy/ range of diferent organs and also to the hair [8]. Fur- fetal outcomes. thermore, some elements might be deposited on the hair through external contamination. Hence, measurements of 2. Experimental the composition of the hair can refect both endogenous and exogenous exposures to various inorganic elements. 2.1. Hair Sample Collection. Hair samples were collected Hair is a proteinaceous fbre predominantly consisting of from pregnant women in the Complex Lipids in Mothers keratin proteins [9]. Most inorganic elements have a high and Babies (CLIMB) cohort (Chinese Clinical Trial Register afnity for the sulfhydryl group of amino acids in keratin. number: ChiCTR-IOR-16007700) [22]. Te 3–6 hair strands Terefore, inorganic elements are easily incorporated and were taken from the occipital area, 0.5 cm away from the retained in human hair [9]. Te accumulation of inorganic scalp. Te hair samples were cut into pieces and stored in a elements in hair refects long-term exposure. Since metal self-sealing bag at −20 C. Te collection and segmentation of concentrations are reduced in urine and blood after days and hair were done with scissors made of polytetrafuoroethylene weeks, respectively, human hair appears to be a more robust (PTFE) to avoid elemental contamination. A thousand hair specimen for estimating past and ongoing exposure to inor- samples collected from CLIMB were used to test the re- ganic elements. Hair also has less background matrix than producibility of the chosen method. Tese hair samples were urine and blood, and the inorganic elements are easier to detect collected in accordance with the method published by analytically because they are usually present at higher levels in Delplancke et al. [23] and in full agreement with the the hair. Moreover, hair samples are collected noninvasively principles of the International Conference on Harmo- and can be transported and stored without refrigeration or nisation Good Clinical Practice E6 (ICH-GCP) and the prior processing. Hence, hair has become an attractive clinical Declaration of Helsinki. Te study was approved by the specimen for studying the longitudinal exposure of inorganic Ethics Committee of Chongqing Medical University elements from the external environment. (No.2014034), and written informed consent was obtained Numerous instruments have been employed for element from all participants. detection and quantitation, including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), instru- 2.2. Reagents and Calibration Standard Solutions. mental neutron activation analysis (INAA), electrothermal- Analytical and internal standards were purchased from atomic absorption spectrometry (ET-AAS), and fame Agilent Technologies. ICP-MS-grade nitric acid (65%) and atomic absorption spectrometry (FAAS) [10–14]. For acetone were obtained from ANPEL Laboratory Technol- qualitative and quantitative measurement of hair elements, ogies (Shanghai, China). Ultrapure deionized water (18 mΩ) ICP-MS is our selected platform because it has the most was obtained from a water purifcation system (Aoside, superior sensitivity, resolving power, and improved limits of China). detection (LODs) to measure elements [8]. A variety of hair A working standard solution was prepared by diluting digestion techniques for element detection via ICP-MS 10μg/mL of mixed-element standard solutions (Multiele- analysis have been published, including using a microwave ment Calibration Standard 2A: Ag, Al, As, Ba, Be, Ca, Cd, digestion instrument, leaving the sample at room temper- Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, ature for 24 h, using a heat block, and using an ultrasonic Tl, U, V, and Zn; Multielement Calibration Standard 4: B, water bath [15–19]. Tese approaches are generally coupled Ge, Mo, Nb, P, Re, S, Si, Ta, Ti, W, and Zr; Multielement with efcient acid decomposition procedures such as di- Calibration Standard 1: Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, gestion in concentrated nitric acid (65–68%), and only a few Pr, Sc, Sm, Tb, T, Tm, Y, and Yb) or a single-element studies use other solutions [8, 20, 21]. However, these standard solution (Multielement Calibration Standard 2A- methods often use a large amount of hair biomass HG: Hg). Two sets of seven standard solutions for cali- (30–150 mg), excessive acid solution (3–10 ml per sample), bration curves were prepared. Te frst set contained all the and extensive digestion time (1 hr–2 days) [15–19]. Tese elements (except Hg) diluted in 5% HNO in 15 ml cen- factors are not conducive to the efcient analysis of a large trifuge tubes (concentration gradient: 0.0μg/L, 0.1μg/L, cohort of samples, and not all participants are willing to 0.5μg/L, 2.5μg/L, 25μg/L, 250μg/L, and 2500μg/L). Te donate a large volume of hair. It is clear that the method- second set contained the low concentration Hg (<5μg/L) ology for analysing inorganic elements in hair needs to be diluted in 5% HNO in 15 ml centrifuge tubes (concentra- improved in order to efectively and efciently analyse hair tion gradient: 0.0μg/L, 0.02μg/L, 0.1μg/L, 0.5μg/L, and element concentrations in large cohorts. 2.5μg/L). An internal standard (IS) solution was also Journal of Analytical Methods in Chemistry 3 prepared by diluting 100μg/mL ICP-MS Internal Standard reach more than 3000, 10000, and 6000, respectively; oxide Mix in 5% HNO (Bi, Ge, In, Li, Lu, Rh, Sc, and Tb) (ICP-MS <2%; doubly charged <3%; peak width -10%: 0.65–0.80), Internal Standard Mix: 5% HNO �1 : 200). using a 1μg/L tuning solution (Ce, Co, Li, Mg, Tl, and Y; Agilent Technologies). Te main ICP-MS detection pa- rameters were as follows: radio-frequency (RF) power: 2.3. Hair Digestion and Element Extraction Methods. Five 1550.0 w, RF matching: 1.80 V, sampling depth: 10.0 mm, diferent extraction methods were chosen based on previ- nebulizer gas fow: 1.05 L/min, peristaltic pump speed: ously reported studies analysing human hair acid decom- 0.1 rps, atomization chamber temperature: 2 C, extract 2: position. Te extraction methods were as follows: (1) HNO −230.0 V, omega bias: −105.0 V, omega lens: 8.8 V, collision digestion at room temperature for 24 h (RT), (2) HNO cell entrance: −50.0 V, collision cell exit: −60.0 V, Q1 en- digestion at 90 C for 4 h in a constant temperature drying trance: −50.0 V, Q1 exit: 1.0 V, Q1 bias: −1.0 V, fow rate of oven (T90) (Termo Scientifc), (3) ultrasonic-assisted acid argon cell for collision cell: 5.5 mL/min, OctP RF: 150.0 V, digestion with HNO (UltraS) (SCIENTZ, SB-5200DT), (4) and OctP bias: −18.0 V. An internal standard solution was programmed digestion using microwave digestion with online to join by peristaltic pump. HNO (MicroD, Table S1) (PreeKem, WX-6000), and (5) ordinary microwave oven digestion with HNO (O-MicroD, 100% power, 30 min) (Galanz, G70D20CN1P-D2(S0)). 2.6. Elemental Quantitation, Normalization, and Statistical Analysis. Inorganic element identifcation and absolute quantitation were performed using MassHunter 4.6 2.4. Sample Preparation. Te hair strands were washed two Workstation Software 8900 ICP-QQQ Data Analysis times with acetone and then once with deionized water, (C.01.06). Te calibration standard’s regression equation prior to drying in an oven at 37 C. Hair segments were mixed and correlation coefcient (r) for individual elements were together in 2 mL eppendorf tubes with PTFE beads to break determined (Table S2). Only elements not in the calibration the hairs into powder using tissue lysis U (QIAGEN), to 103 115 209 standard solution were selected as ISs ( Rh, In, Bi). ensure homogeneity of the hairs for all digest methods under Te measuring elements were normalized by the most ap- investigation. Four replicates of 3 mg ± 0.5 mg of mixed hair propriate IS according to the following mass range: Rh for were weighed into 15 mL PTFE digestion tubes. Next, 200μL 115 209 mass 23–107, In for mass 111–169, and Bi for mass of concentrated nitric acid was added, and the tubes were 172–238. Te blank deduction was performed by subtracting capped to carry out the digestion reaction. Importantly, the the blank from the sample results. Calibration curves were MicroD digestion method required adding 3 mg ± 0.5 mg then set up by external calibration, linear ft, and blank ofset. hair sample and 5 ml concentrated nitric acid to the Te y value was obtained by dividing the count of each point matching digestion tank and then concentrated the acid into of the correction curve by the count of the unit concen- 200μl before transfer. After the samples had dissolved, they tration of the internal standard of the same level as follows: were transferred to 15 mL centrifuge tubes and made up to a volume of 3 mL with deionized water. A 100μl aliquot of y � , each of the three mixed standard solutions was transferred (1) y /x􏼁 i i into the PTFE tube, and then 200μl of the concentrated nitric acid solution was added. After digestion, using the fve whenx � 0,y �y ;x is the IS element concentration,y is the i σ i i diferent methods detailed above, the digested solution was IS element count, and y is the sample data count. transferred to 15 ml centrifuge tubes and made up to a Te correlation coefcient of the linear regression cor- volume of 10 mL with deionized water. rection curve was calculated using the following formula: 􏽐 􏼈 x − x􏼁 y − y􏼁 􏼉 2.5.InductivelyCoupledPlasma-MassSpectrometry(ICP-MS) i i i r � , (2) 1/2 2 2 Analysis. Te samples were handled using a SPS 4 auto- 􏽐 x − x y − y 􏽮􏽨 􏼁 􏽩􏽨􏽐 􏼁 􏽩􏽯 i i i i sampler (Agilent Technologies). Te ICP-MS/MS instru- ment was an Agilent 8900 controlled with MassHunter 4.6 where x is the average of x , y is the average of y , x is the i i i Workstation Software 8900 ICP-QQQ Top (C.01.06). Te determination value ofx, and y is the determination value ofy. 8900 ICP-MS/MS uses a tandem mass spectrometer layout, Te relative error (%RE) was calculated according to the with two quadrupole mass spectrometers enabling it to following formula: operate in the MS/MS mode. ICP-QQQ ofers an additional quadrupole mass flter in front of the collision reaction cell x − x i i (3) %RE � × 100, to resolve spectral interferences. Te 8900 has higher sen- sitivity and a specialized fow path with argon gas to provide lower backgrounds (sensitivity: Co ≥ 22 ×106 cps/ppm wherex is the actual value of the calibration standard and x (<10% RSD, relative standard deviation) and background is the concentration of the collected corrected standard. (on mass) mass 78 ≤ 400 cps). Te operating conditions Te relative standard error (%RSE) represented the (lenses, torch position, detector voltage, and gas fows) were ftting index of the correction curve, a value calculated using optimized daily (the count of mass 7, 89, and 205 needs to the following formula: 4 Journal of Analytical Methods in Chemistry Alkali Alkaline earth Transition Post-transition Metalloid Non-metal Actinide Lanthanide metal metal metal metal O-MicroD T90 MicroD UltraS RT Figure 1: Te bar chart represents the recovery efciency (%) of eight diferent element classes across fve diferent digestion methods for the standard solution. -0.3 -0.2 -0.1 0.0 0.1 0.2 0.2 0.3 0.1 0.0 -0.1 -0.2 0.20 0.22 -0.3 0.24 0.26 PC1 (74.7%) Figure 2: Principal component analysis (PCA) of the human hair elements, digested using fve diferent methods. Each color represents a digestion method. An ellipse with dotted lines indicates the 95% confdence interval calculated using Hotelling’s T2 statistics. 􏽳��������������� � Excel version 2019. Te recovery efciency of elements in the n 2 􏽐 􏼂 x − x􏼁 /x 􏼃 i− 1 i i i (4) standard solution was compared and displayed using a %RSE � 100 × , (n − p) Microsoft Excel bar chart. where x is the actual value of the calibration curve leveli, x is i i the collection concentrations of the calibration curve level i, p 3. Results and Discussion is the number of items for the calibration curve formula, andn is the number of points of the available calibration curve. Tis study was the frst to assess fve diferent HNO hair Coefcient of variance (CV) and principal component digestion methods in preparation for inorganic element analysis (PCA) were used to check the repeatability of di- analysis using ICP-MS. A total of 52 elements were quan- gestion methods. Boxplots, two-dimensional projections of titated in this study. Te identifed elements were subdivided the 3D PCA, bar graphs, dot-line graphs, and scatter plots into eight major metal classes such as alkali metals, alkaline were rendered using the ggplot2 R package. Element cov- Earth metals, transition metals, and others (Table S3). Teir erage was compared across the fve digestion protocols and detailed concentrations are displayed in Table S4. Metals displayed using an UpSet plot, constructed using the UpSet detected in each of the classes were evaluated in detail to R package. Te cumulative coefcient of variation was il- assess the performance of the fve diferent digestion lustrated by a horizontal bar chart constructed in Microsoft methods. Te performance of each method was evaluated by PC3 (5.5%) Recovery efficiency (%) PC2 (8.8%) Journal of Analytical Methods in Chemistry 5 UltraS T90 RT MicroD O-MicroD 0 100 200 300 400 500 Alkali metal Metalloid Post-transition metal Lanthanide Actinide Transition metal Alkaline earth metal Non-metal Figure 3: Te horizontal bar chart represents the cumulative coefcient of variations (%) of eight diferent metal classes across fve diferent digestion methods for human hair. Te x-axis is the total CV for all major metal classes. Na Alkali metal Alkaline earth metal Ba Rb Transition metal Mg Mn Post-transition metal Co Zr Metalloid Cr Non-metal Al Fe Actinide Mo Ga Lanthanide TI Zn As Cs Pb 3 Cd Se 2 2 2 2 2 2 K Ce Nd Cu Ag Gd Y Eu Sr 1 La 1 1 1 W Nb 1 1 U Hg Ni Ta Tm Pr Ho Er Ca Re Yb Ge Dy Sm Ti Th UltraS RT O-MicroD T90 MicroD 40 30 20 10 0 Set Size Figure 4: Upset plot of the identifed metal coverage compared across fve diferent digestion methods for human hair. testing internal standard solution stability, digestion re- abnormally high outliers in the T90 and MicroD methods. producibility, metal coverage, and standard solution re- Te RT method demonstrated the best IS stability in this covery efciency. study. 3.1. Internal Standard Solution Stability of the Five Diferent 3.2. Digestion Recovery Efciency of the Standard Solution of Digestion Methods. Te internal standard solution stability Five Diferent Digestion Methods. Te frst criterion which of the fve selected methods was evaluated by comparing was used to evaluate the accuracy of the fve diferent di- levels of the IS elements (Bi, In, and Rh) in the hair samples gestion methods was recovery efciency of the standard following the diferent digestion methods (Figure S1). Te IS solution. Tis is determined according to the recovery levels stability of all digestion methods was between 80% and of diferent elements in a digested standard solution of a 120%, which meets the requirements of the instrument known concentration, as shown in Figure 1. Te RT and measurement. None of the IS elements drifted as an outlier MicroD methods displayed the 99%–100% recovery ef- in the O-MicroD, RT, and UltraS methods, while there were ciency for all eight diferent element classes from the Intersection Size 6 Journal of Analytical Methods in Chemistry Ba Co Cr Fe Mg Mo Na 3 4 3 3 2 2 1 1 0 0 –1 –1 Nd Pb Se U V Zn Cu 3 3 2 3 3 3 2 2 1 2 2 1 1 1 0 1 0 0 –1 0 0 –1 –1 –1 Ni Ag Ta Re Tm Gd Ho 2 2 2 1 1 0 1 0 0 –1 –1 –1 Sm Hg K Sr Yb Ca AI 2 2 1 1 1 2 1 0 0 0 –1 0 0 –1 As Ce Ga La Mn P Pr 2 2 2 1 2 1 1 1 0 1 0 0 0 0 –1 0 Rb TI Zr Dy Nb Ge W 2 1 1 1 1 0 0 0 0 0 –1 0 0 –1 Er Y Ti Cd Cs T Eu 1.0 1.0 2.0 1.0 0.5 2 2 0.5 1.5 0.5 0.0 0.5 1.0 0.0 1 1 –0.5 0.0 0.5 0.0 –0.5 0.0 0 –1.0 –0.5 0 –0.5 –0.5 –1.0 –1.5 Extraction methods Extraction methods O-MicroD T90 RT UltraS MicroD Figure 5: Te dot-line graphs show the element concentrations of each sample across fve diferent digestion methods for human hair. Each dot represents a sample concentration. Te vertical lines are the standard deviation of each digestion method. Te metal concentrations were standardized to Z-scores. CV (%) 6.3 3.6 3.6 (%) 0 100 200 300 400 500 600 700 800 900 1000 103Rh 115In 209Bi Figure 6: Te scatter plot and coefcient of variation (CV) for the internal standard stability from the ICP-MS hair analysis of a thousand maternal hair samples using O-MicroD digestion. Concentration z-score (ug/L) Internal standard stability (%) O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD 103Rh T90 115In RT UltraS 209Bi MicroD Journal of Analytical Methods in Chemistry 7 standard solution; O-MicroD only produced the high simultaneously in the hair matrix than in other studies; Hao recovery yield for alkali metals, post-transition metals, et al. identifed 15 hair elements using HNO /H O at 100 C 3 2 2 metalloids, nonmetals, lanthanide, and actinide; T90 only until dryness using an electric heating plate [28]; Luo et al. performed the high recovery for alkali metals, alkaline quantifed 16 hair elements using HNO and H O digestion 3 2 2 Earth metals, post-transition metals, metalloids, non- at 90 C for 3 h prior to ICP-MS analysis [16]; Tinkov et al. metals, and lanthanide; UltraS was superior for alkali reported 17 trace elements from children’s hair using mi- metals, post-transition metals, metalloids, nonmetals, and crowave digestion (170–180 C) with HNO [29]. We propose lanthanide. We hypothesized that the RT method dis- that this has been achieved by the following two strategies: played the best recovery efciency because it only involves (1) we have established calibration curves for 56 elements in two sample preparation steps (dilution and incubation) a single standard dilution series by combining three sets of and an extensive 24 h digestion period. In addition, the multielement calibration solutions; (2) the Agilent 8900 MicroD method also exhibited high recovery efciency ICP-MS used in our study exhibits high sensitivity and and this is likely because electromagnetic radiation can strong antispectral interference capability through higher penetrate through the hair matrix, promoting molecular mass spectrum scanning frequency (3.0 MHz) and an ad- rotations and efcient heating via friction [24]. Micro- ditional quadrupole mass flter. Furthermore, the Upset plot wave digestion has been found to be one of the most and dot-line graphs illustrate that there are 13 elements that convenient techniques to prepare samples for elemental can be efectively digested and identifed by using any of the determination [25–27]. fve methods (Figures 4 and 5: Na, Ba, Mg, Co, Cr, Fe, Mo, V, Zn, Pb, Se, Nd, and U), 11 elements that can be efectively identifed by using three of the tested methods (Figures 4 and 3.3. Reproducibility of the Five Diferent Digestion Methods. 5: O-MicroD, MicroD, T90 : Rb, Mn, Zr, Al, Ga, Tl, As, P, Ce, Te O-MicroD method demonstrated the most promising La, and Pr), and four elements which can only can be digestion reproducibility for the trace amount of human hair identifed by using the MicroD method (Figures 4 and 5: Cs, analysed. Te PCA analysis showed that the technical rep- Cd, Eu, and T). Overall, the O-MicroD and MicroD licates of the O-MicroD hair elements (red dots) were methods showed the most comprehensive coverage of clustered the closest to each other along PC1 (74.7%), PC2 identifed elements in this study. (8.8%), and PC3 (5.5%) (Figure 2). Consistent with the Since the O-MicroD method could reproducibly results of the PCA, the O-MicroD digestion method quantitate a large number of elements using only 3 mg of exhibited the most reproducible CV (CV: 23.49%) for the hair biomass with 200μl of nitric acid for 30 min digestion overall metal classes identifed (Figure 3, Table S3 and time, we further tested the reproducibility of O-MicroD by Figure S2). Te MicroD digestion method displayed the least coinjecting three IS with a thousand hair samples. We reproducible CV for hair (CV: 64.37%). Te reproducibility 103 115 209 found that the CV of Rh, In, and Bi were 3.59%, of lanthanide was poor across all fve digestion methods, 3.61%, and 6.31%, respectively, without batch correction with a minimum CV of 36.87% (T90) (Figure 3, Table S3). In (Figure 6). Te excellent reproducibility of O-MicroD, addition, we found that the hair samples that were digested together with minimizing sample usage and preparation using the MicroD method exhibited the highest number of time, makes this approach an ideal choice for a large cohort elements with poor reproducibility (Figure 4). Te possible studies. explanation for the O-MicroD digestion method possessing In summary, our fndings suggest that out of the fve superior reproducibility for most metal elements in a trace digestion methods tested, the O-MicroD method had the amount of human hair could be that the microwave has best reproducibility and digestion time (30 mins) for ele- heating stability and less volume is lost during solution mental analysis of human hair, the MicroD method had the transfer. In contrast, the MicroD method showed the best element coverage, and the RTmethod performed best in poorest reproducibility due to additional preparation steps, element digestion recovery efciency. that is, the microwave-digested hair mixture was further heated and evaporated from 5 ml to 0.2 ml to eliminate the high HNO concentration prior to ICP-MS analysis. Tis 4. Conclusion signifcant fuctuation in volume may greatly increase the variability of element concentrations among individual In conclusion, this is the frst study conducted to investigate samples. the optimal digestion method for analysing the 52 inorganic elements in human hair using ICP-MS analysis. Based on the 3.4.ElementCoverageoftheFiveDiferentDigestionMethods. criteria of reproducibility, detected element coverage, and Another criterion that we used to determine the optimal the digestion recovery yield, the O-MicroD, MicroD, and RT digestion method was the element coverage. Te order of methods were the superior digestion methods for CV, digestion methods ranking from the highest to lowest coverage, and recovery efciency, respectively. However, we number of identifed elements in human hair were as fol- also discovered that diferent digestion methods favor the lows: MicroD (44 elements), O-MicroD (38 elements), T90 digestion of diferent element classes. Future studies should (38 elements), RT (25 elements), and UltraS (22 elements), carefully consider the digestion method they select, choosing as displayed in Table S3. Noticeably, our microwave di- the method that would be best suited to the primary purpose gestion approaches have quantifed more elements of their study. 8 Journal of Analytical Methods in Chemistry Abbreviations Supplementary Materials CLIMB: Complex Lipids in Mothers and Babies Figure S1: the extraction efciency of internal standard el- CV: Coefcient of variation ements (Bi, In, and Rh) in fve diferent digestion methods ET-AAS: Electrothermal-atomic absorption spectrometry for human hair. Figure S2: the bar graphs show the coef- FAAS: Flame atomic absorption spectrometry fcient of variation (%) of each metal across fve diferent ICP-MS: Inductively coupled plasma-mass spectrometry digestion methods for human hair. Table S1: microwave ICP- Inductively coupled plasma-optical emission digestion procedure parameters. Table S2: linear relationship OES: spectrometry and detection limit of each element in the standard solution. INAA: Instrumental neutron activation analysis Table S3: number of identifed elements, concentrations (μg/ LOD: Limits of detection L), and CVs (%) of each element classes in human hair, MicroD: Programmed digestion of microwave digestion compared across fve diferent digestion methods. Table S4: O- Ordinary microwave oven digestion the quantitative concentrations (μg/L) of identifed metals in MicroD: human hairs across the fve diferent digestion methods. PCA: Principal component analysis (Supplementary Materials) PTFE: Polytetrafuoroethylene RE: Relative error References RSE: Relative standard error RT: Room temperature for 24 h [1] J. I. Lachowicz, L. I. Lecca, F. Meloni, and M. Campagna, “Metals and metal-nanoparticles in human pathologies: from T90: 90 C for 4 h exposure to therapy,” Molecules, vol. 26, no. 21, p. 6639, 2021. UltraS: Ultrasonic assisted digestion. [2] X. Wang, P. An, Z. Gu, Y. Luo, and J. Luo, “Mitochondrial metal ion transport in cell metabolism and disease,” Inter- national Journal of Molecular Sciences, vol. 22, no. 14, p. 7525, Data Availability [3] M. Valko, K. Jomova, C. J. Rhodes, K. Kuca, and K. Musilek, Te data used to support the results of this study are in- “Redox- and non-redox-metal-induced formation of free cluded within the article. Any further information is radicals and their role in human disease,” Archives of Toxi- available from corresponding authors upon request. cology, vol. 90, pp. 1–37, 2016. [4] X. F. Hu, M. Lowe, and H. M. Chan, “Mercury exposure, cardiovascular disease, and mortality: a systematic review and dose-response meta-analysis,” Environmental Research, Conflicts of Interest vol. 193, Article ID 110538, 2021. [5] X. Li, Y. Huang, Y. Xing et al., “Association of urinary Te authors declare that they have no conficts of interest. cadmium, circulating fatty acids, and risk of gestational di- abetes mellitus: a nested case-control study in China,” En- Authors’ Contributions vironment International, vol. 137, Article ID 105527, 2020. [6] S. S. Shiek, M. S. Mani, S. P. Kabekkodu, and H. S. Dsouza, Y. L. and T. H. designed the experiment and wrote the “Health repercussions of environmental exposure to lead: manuscript; Y. L., Y. Y., D. Z., and Y-M. L. performed the methylation perspective,” Toxicology, vol. 461, Article ID sample preparation and ICP-MS analysis; Y. L., and T. H. 152927, 2021. performed the data analysis; T. H., J. dS., and Y. X. revised [7] Y. Huang, X. Li, W. Zhang et al., “Aluminum exposure and gestational diabetes mellitus: associations and potential me- the manuscript; T. H., and H. Z. provided funding for the diation by n-6 polyunsaturated fatty acids,” Environmental experimental costs. All authors read and approved the fnal Science & Technology, vol. 54, no. 8, pp. 5031–5040, 2020. version of this manuscript. [8] D. Pozebon, G. L. Schefer, and V. L. Dressler, “Elemental hair analysis: a review of procedures and applications,” Acknowledgments Analytica Chimica Acta, vol. 992, pp. 1–23, 2017. [9] C. Popescu and H. Hocker, “Hair - the most sophisticated Tis work was supported by the National Natural Science biological composite material,” Chemical Society Reviews, Foundation of China (No. 81871185, 81971406), Te 111 vol. 36, no. 8, pp. 1282–1291, 2007. [10] M. D. Lakshmi Priya and A. Geetha, “Level of trace elements Project (Yuwaizhuan (2016)32), Kuanren Talents Programs (copper, zinc, magnesium and selenium) and toxic elements of Second Afliated Hospital of Chongqing Medical Uni- (lead and mercury) in the hair and nail of children with versity, Chongqing Science and Technology Commission autism,” Biological Trace Element Research, vol. 142, no. 2, (cstc2021jcyj-msxmX0213), Chongqing Municipal Educa- pp. 148–158, 2011. tion Commission (KJZD-K202100407), Chongqing Health [11] I. Molina-Villalba, M. Lacasana, M. Rodriguez-Barranco et al., Commission and Chongqing Science and Technology “Biomonitoring of arsenic, cadmium, lead, manganese and Commission (2021MSXM121, 2020MSXM101), Smart mercury in urine and hair of children living near mining and Medicine Research Project of Chongqing Medical University industrial areas,” Chemosphere, vol. 124, pp. 83–91, 2015. (No. ZHYX202103), and a Joint Health Research Council [12] J. Kucera and K. Kofronova, “Determination of as by in- New Zealand–National Science Foundation of China Bio- strumental neutron activation analysis in sectioned hair medical Research Fund (19/804). samples for forensic purposes: chronic or acute poisoning?” Journal of Analytical Methods in Chemistry 9 Journal of Radioanalytical and Nuclear Chemistry, vol. 287, [26] S. Kasar, R. Murugan, H. Arae, T. Aono, and S. K. Sahoo, “A microwave digestion technique for the analysis of rare earth no. 3, pp. 769–772, 2011. [13] G. Izydorczyk, M. Mironiuk, S. Basladynska, M. Mikulewicz, elements, thorium and uranium in geochemical certifed reference materials and soils by inductively coupled plasma and K. Chojnacka, “Hair mineral analysis in the population of students living in the Lower Silesia region (Poland) in 2019: mass spectrometry,” Molecules, vol. 25, no. 21, p. 5178, 2020. [27] R. Mohamed, B. H. Zainudin, and A. S. Yaakob, “Method comparison with biomonitoring study in 2009 and literature validation and determination of heavy metals in cocoa beans data,” Environmental Research, vol. 196, Article ID 110441, and cocoa products by microwave assisted digestion tech- nique with inductively coupled plasma mass spectrometry,” [14] S. Grassin-Delyle, M. Martin, O. Hamzaoui et al., “A high- Food Chemistry, vol. 303, Article ID 125392, 2020. resolution ICP-MS method for the determination of 38 in- [28] Z. Hao, Y. Li, Y. Liu, H. Li, W. Wang, and J. Yu, “Hair el- organic elements in human whole blood, urine, hair and ements and healthy aging: a cross-sectional study in Hainan tissues after microwave digestion,” Talanta, vol. 199, Island, China,” Environmental Geochemistry and Health, pp. 228–237, 2019. vol. 38, no. 3, pp. 723–735, 2016. [15] E. Tamburo, D. Varrica, G. Dongarra, and L. M. E. Grimaldi, [29] A. A. Tinkov, A. L. Mazaletskaya, O. P. Ajsuvakova et al., “Trace elements in scalp hair samples from patients with “ICP-MS assessment of hair essential trace elements and relapsing-remitting multiple sclerosis,” PLoS One, vol. 10, minerals in Russian preschool and primary school children no. 4, Article ID e0122142, 2015. with attention-defcit/hyperactivity disorder (ADHD),” Bio- [16] R. Luo, S. Zhang, P. Xiang, B. Shen, X. Zhuo, and D. Ma, logical Trace Element Research, vol. 196, no. 2, pp. 400–409, “Elements concentrations in the scalp hair of methamphet- amine abusers,” Forensic Science International, vol. 249, pp. 112–115, 2015. [17] F. L. Barbieri, A. Cournil, J. E. Souza Sarkis, E. Benefce, and J. Gardon, “Hair trace elements concentration to describe polymetallic mining waste exposure in Bolivian altiplano,” Biological Trace Element Research, vol. 139, no. 1, pp. 10–23, [18] G. Giangrosso, G. Cammilleri, A. Macaluso et al., “Hair mercury levels detection in fshermen from sicily (Italy) by ICP-MS method after microwave-assisted digestion,” Bio- inorganicChemistryandApplications, vol. 2016, pp. 1–5, 2016. [19] F. Domanico, G. Forte, C. Majorani et al., “Determination of mercury in hair: comparison between gold amalgamation- atomic absorption spectrometry and mass spectrometry,” Journal of Trace Elements in Medicine & Biology, vol. 43, pp. 3–8, 2017. [20] H. Yasuda, K. Yoshida, M. Segawa et al., “Metallomics study using hair mineral analysis and multiple logistic regression analysis: relationship between cancer and minerals,” Envi- ronmental Health and Preventive Medicine, vol. 14, no. 5, pp. 261–266, 2009. [21] M. F. Mesko, R. M. Pereira, P. T. Scaglioni, and D. L. R. Novo, “Single analysis of human hair for determining halogens and sulfur after sample preparation based on combustion reac- tion,” Analytical and Bioanalytical Chemistry, vol. 411, no. 19, pp. 4873–4881, 2019. [22] S. Huang, T. T. Mo, T. Norris et al., “Te CLIMB (Complex Lipids in Mothers and Babies) study: protocol for a multi- centre, three-group, parallel randomised controlled trial to investigate the efect of supplementation of complex lipids in pregnancy, on maternal ganglioside status and subsequent cognitive outcomes in the ofspring,” BMJOpen, vol. 7, no. 10, Article ID e016637, 2017. [23] T. D. J. Delplancke, J. V. de Seymour, C. Tong et al., “Analysis of sequential hair segments refects changes in the metab- olome across the trimesters of pregnancy,” Scientifc Reports, vol. 8, no. 1, p. 36, 2018. [24] Y. Wu, R. Mu, G. Li, M. Li, and W. Lv, “Research progress in fuid and semifuid microwave heating technology in food processing,”ComprehensiveReviewsinFoodScienceandFood Safety, vol. 21, no. 4, pp. 3436–3454, 2022. [25] H. Altundag and M. Tuzen, “Comparison of dry, wet and microwave digestion methods for the multi element deter- mination in some dried fruit samples by ICP-OES,” Food and Chemical Toxicology, vol. 49, no. 11, pp. 2800–2807, 2011. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Analytical Methods in Chemistry Hindawi Publishing Corporation

An Evaluation of Different Digestion Methods for the Quantitation of Inorganic Elements in Human Hair Using ICP-MS

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Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 5742468, 9 pages https://doi.org/10.1155/2022/5742468 Research Article An Evaluation of Different Digestion Methods for the Quantitation of Inorganic Elements in Human Hair Using ICP-MS 1,2 3,4 2 5 6 Yue Liu , Yang Yang, Yin-Yin Xia, Jamie V. de Seymour , De-Zhang Zhao, 7 3,4 1,4 Yang-Mei Li, Hua Zhang , and Ting-Li Han Department of Obstetrics andGynaecology, TeSecond Afliated Hospital of Chongqing Medical University, Chongqing, China Department of Occupational and Environmental Hygiene, School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, China Department of Obstetrics and Gynaecology, Te First Afliated Hospital of Chongqing Medical University, Chongqing, China Mass Spectrometry Center of Maternal-Fetal Medicine, Chongqing Medical University, Chongqing, China College of Health, Massey University, Auckland, New Zealand College of Pharmacy, Chongqing Medical University, Chongqing, China School of Public Health and Management, Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, China Correspondence should be addressed to Hua Zhang; zh2844@gmail.com and Ting-Li Han; tinglihan@cqmu.edu.cn Received 23 August 2022; Revised 28 October 2022; Accepted 21 November 2022; Published 1 December 2022 Academic Editor: Radosław Kowalski Copyright © 2022 Yue Liu et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te inorganic elements have unique properties in biochemical processes in humans. An increasing number of pathologies have been associated with essential element ions, such as lead, mercury, and cadmium. Hair has become an attractive clinical specimen for studying the longitudinal exposure to elements from the external environment. Inductively coupled plasma-mass spectrometry (ICP- MS) coupled with nitric acid (HNO ) digestion is the most common approach for determining inorganic elements from human hair. Tis study aims to optimize the digestion method for the absolute quantitation of 52 elements using ICP-MS, for a large cohort study in human hair. Five diferent HNO (65%) digestion methods were investigated and evaluated for their internal standard solution stability, reproducibility, element coverage, and standard solution recovery efciency, namely, room temperature for 24 h (RT), 90 C for 4h (T90), ultrasonic-assisted digestion (UltraS), programmed digestion of microwave digestion (MicroD), and ordinary mi- crowave oven digestion (O-MicroD). Our results demonstrated that O-MicroD, MicroD, and RTwere the best performing digestion methods for coefcient of variation (CV) scores, coverage, and recovery efciency, respectively. In particular, the O-MicroD method detected multiple elements in a small quantity of hair (3 mg), with minimum nitric acid usage (200μl) and a short digestion time (30 min). Te O-MicroD method had excellent reproducibility, as demonstrated by a continuous thousand injections of hair samples 103 115 209 with three internal standards (CV: Rh � 3.59%, In � 3.61%, and Bi � 6.31%). Future studies of the elemental content of hair should carefully select their digestion method to meet the primary purpose of their study. sodium maintain the electrochemical gradient of the cell 1. Introduction membranes [1]. Tere are many metal ions that are im- Inorganic elements have unique properties that cannot be portant for cellular metabolism and mitochondrial function, + 2+ 2+ performed by organic compounds. Some elements are in- such as K , Mg , and Zn [2]. Nevertheless, growing dispensable for humans and essential in biochemical pro- evidence suggests that excessive exposure to some elements cesses. For example, iron (Fe), copper (Cu), and manganese could cause adverse health outcomes. Te role of cadmium (Mn) are all positioned at the metal-catalytic site of enzymes (Cd), mercury (Hg), and copper (Cu) in type 2 diabetes, involved in redox reactions [1]. Calcium, magnesium, and renal dysfunction, cardiovascular diseases, osteoporosis, and 2 Journal of Analytical Methods in Chemistry Tis study aims to establish a method for analysing the cancers, has been reported in experimental and epidemio- logical studies [3–5]. Excessive aluminium (Al), lead (Pb), inorganic elements in human hair samples from a large cohort, using low hair mass. We analysed fve diferent and arsenic (As) exposures have also been associated with oxidative stress, intestinal diseases, dyslipidemia, and met- sample preparation methods for their reproducibility, ele- abolic diseases [3, 6, 7]. Exposure to metals can be mediated ment coverage, and extraction efciency. Te results from through food, dermal contact, air pollution, and drinking our fndings have informed the methodology for a large water [7]. Elements absorbed by the pulmonary tract and the cohort study of hair from pregnant women to investigate the digestive system can be distributed via the bloodstream to a link between exposure to inorganic elements and pregnancy/ range of diferent organs and also to the hair [8]. Fur- fetal outcomes. thermore, some elements might be deposited on the hair through external contamination. Hence, measurements of 2. Experimental the composition of the hair can refect both endogenous and exogenous exposures to various inorganic elements. 2.1. Hair Sample Collection. Hair samples were collected Hair is a proteinaceous fbre predominantly consisting of from pregnant women in the Complex Lipids in Mothers keratin proteins [9]. Most inorganic elements have a high and Babies (CLIMB) cohort (Chinese Clinical Trial Register afnity for the sulfhydryl group of amino acids in keratin. number: ChiCTR-IOR-16007700) [22]. Te 3–6 hair strands Terefore, inorganic elements are easily incorporated and were taken from the occipital area, 0.5 cm away from the retained in human hair [9]. Te accumulation of inorganic scalp. Te hair samples were cut into pieces and stored in a elements in hair refects long-term exposure. Since metal self-sealing bag at −20 C. Te collection and segmentation of concentrations are reduced in urine and blood after days and hair were done with scissors made of polytetrafuoroethylene weeks, respectively, human hair appears to be a more robust (PTFE) to avoid elemental contamination. A thousand hair specimen for estimating past and ongoing exposure to inor- samples collected from CLIMB were used to test the re- ganic elements. Hair also has less background matrix than producibility of the chosen method. Tese hair samples were urine and blood, and the inorganic elements are easier to detect collected in accordance with the method published by analytically because they are usually present at higher levels in Delplancke et al. [23] and in full agreement with the the hair. Moreover, hair samples are collected noninvasively principles of the International Conference on Harmo- and can be transported and stored without refrigeration or nisation Good Clinical Practice E6 (ICH-GCP) and the prior processing. Hence, hair has become an attractive clinical Declaration of Helsinki. Te study was approved by the specimen for studying the longitudinal exposure of inorganic Ethics Committee of Chongqing Medical University elements from the external environment. (No.2014034), and written informed consent was obtained Numerous instruments have been employed for element from all participants. detection and quantitation, including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), instru- 2.2. Reagents and Calibration Standard Solutions. mental neutron activation analysis (INAA), electrothermal- Analytical and internal standards were purchased from atomic absorption spectrometry (ET-AAS), and fame Agilent Technologies. ICP-MS-grade nitric acid (65%) and atomic absorption spectrometry (FAAS) [10–14]. For acetone were obtained from ANPEL Laboratory Technol- qualitative and quantitative measurement of hair elements, ogies (Shanghai, China). Ultrapure deionized water (18 mΩ) ICP-MS is our selected platform because it has the most was obtained from a water purifcation system (Aoside, superior sensitivity, resolving power, and improved limits of China). detection (LODs) to measure elements [8]. A variety of hair A working standard solution was prepared by diluting digestion techniques for element detection via ICP-MS 10μg/mL of mixed-element standard solutions (Multiele- analysis have been published, including using a microwave ment Calibration Standard 2A: Ag, Al, As, Ba, Be, Ca, Cd, digestion instrument, leaving the sample at room temper- Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, ature for 24 h, using a heat block, and using an ultrasonic Tl, U, V, and Zn; Multielement Calibration Standard 4: B, water bath [15–19]. Tese approaches are generally coupled Ge, Mo, Nb, P, Re, S, Si, Ta, Ti, W, and Zr; Multielement with efcient acid decomposition procedures such as di- Calibration Standard 1: Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, gestion in concentrated nitric acid (65–68%), and only a few Pr, Sc, Sm, Tb, T, Tm, Y, and Yb) or a single-element studies use other solutions [8, 20, 21]. However, these standard solution (Multielement Calibration Standard 2A- methods often use a large amount of hair biomass HG: Hg). Two sets of seven standard solutions for cali- (30–150 mg), excessive acid solution (3–10 ml per sample), bration curves were prepared. Te frst set contained all the and extensive digestion time (1 hr–2 days) [15–19]. Tese elements (except Hg) diluted in 5% HNO in 15 ml cen- factors are not conducive to the efcient analysis of a large trifuge tubes (concentration gradient: 0.0μg/L, 0.1μg/L, cohort of samples, and not all participants are willing to 0.5μg/L, 2.5μg/L, 25μg/L, 250μg/L, and 2500μg/L). Te donate a large volume of hair. It is clear that the method- second set contained the low concentration Hg (<5μg/L) ology for analysing inorganic elements in hair needs to be diluted in 5% HNO in 15 ml centrifuge tubes (concentra- improved in order to efectively and efciently analyse hair tion gradient: 0.0μg/L, 0.02μg/L, 0.1μg/L, 0.5μg/L, and element concentrations in large cohorts. 2.5μg/L). An internal standard (IS) solution was also Journal of Analytical Methods in Chemistry 3 prepared by diluting 100μg/mL ICP-MS Internal Standard reach more than 3000, 10000, and 6000, respectively; oxide Mix in 5% HNO (Bi, Ge, In, Li, Lu, Rh, Sc, and Tb) (ICP-MS <2%; doubly charged <3%; peak width -10%: 0.65–0.80), Internal Standard Mix: 5% HNO �1 : 200). using a 1μg/L tuning solution (Ce, Co, Li, Mg, Tl, and Y; Agilent Technologies). Te main ICP-MS detection pa- rameters were as follows: radio-frequency (RF) power: 2.3. Hair Digestion and Element Extraction Methods. Five 1550.0 w, RF matching: 1.80 V, sampling depth: 10.0 mm, diferent extraction methods were chosen based on previ- nebulizer gas fow: 1.05 L/min, peristaltic pump speed: ously reported studies analysing human hair acid decom- 0.1 rps, atomization chamber temperature: 2 C, extract 2: position. Te extraction methods were as follows: (1) HNO −230.0 V, omega bias: −105.0 V, omega lens: 8.8 V, collision digestion at room temperature for 24 h (RT), (2) HNO cell entrance: −50.0 V, collision cell exit: −60.0 V, Q1 en- digestion at 90 C for 4 h in a constant temperature drying trance: −50.0 V, Q1 exit: 1.0 V, Q1 bias: −1.0 V, fow rate of oven (T90) (Termo Scientifc), (3) ultrasonic-assisted acid argon cell for collision cell: 5.5 mL/min, OctP RF: 150.0 V, digestion with HNO (UltraS) (SCIENTZ, SB-5200DT), (4) and OctP bias: −18.0 V. An internal standard solution was programmed digestion using microwave digestion with online to join by peristaltic pump. HNO (MicroD, Table S1) (PreeKem, WX-6000), and (5) ordinary microwave oven digestion with HNO (O-MicroD, 100% power, 30 min) (Galanz, G70D20CN1P-D2(S0)). 2.6. Elemental Quantitation, Normalization, and Statistical Analysis. Inorganic element identifcation and absolute quantitation were performed using MassHunter 4.6 2.4. Sample Preparation. Te hair strands were washed two Workstation Software 8900 ICP-QQQ Data Analysis times with acetone and then once with deionized water, (C.01.06). Te calibration standard’s regression equation prior to drying in an oven at 37 C. Hair segments were mixed and correlation coefcient (r) for individual elements were together in 2 mL eppendorf tubes with PTFE beads to break determined (Table S2). Only elements not in the calibration the hairs into powder using tissue lysis U (QIAGEN), to 103 115 209 standard solution were selected as ISs ( Rh, In, Bi). ensure homogeneity of the hairs for all digest methods under Te measuring elements were normalized by the most ap- investigation. Four replicates of 3 mg ± 0.5 mg of mixed hair propriate IS according to the following mass range: Rh for were weighed into 15 mL PTFE digestion tubes. Next, 200μL 115 209 mass 23–107, In for mass 111–169, and Bi for mass of concentrated nitric acid was added, and the tubes were 172–238. Te blank deduction was performed by subtracting capped to carry out the digestion reaction. Importantly, the the blank from the sample results. Calibration curves were MicroD digestion method required adding 3 mg ± 0.5 mg then set up by external calibration, linear ft, and blank ofset. hair sample and 5 ml concentrated nitric acid to the Te y value was obtained by dividing the count of each point matching digestion tank and then concentrated the acid into of the correction curve by the count of the unit concen- 200μl before transfer. After the samples had dissolved, they tration of the internal standard of the same level as follows: were transferred to 15 mL centrifuge tubes and made up to a volume of 3 mL with deionized water. A 100μl aliquot of y � , each of the three mixed standard solutions was transferred (1) y /x􏼁 i i into the PTFE tube, and then 200μl of the concentrated nitric acid solution was added. After digestion, using the fve whenx � 0,y �y ;x is the IS element concentration,y is the i σ i i diferent methods detailed above, the digested solution was IS element count, and y is the sample data count. transferred to 15 ml centrifuge tubes and made up to a Te correlation coefcient of the linear regression cor- volume of 10 mL with deionized water. rection curve was calculated using the following formula: 􏽐 􏼈 x − x􏼁 y − y􏼁 􏼉 2.5.InductivelyCoupledPlasma-MassSpectrometry(ICP-MS) i i i r � , (2) 1/2 2 2 Analysis. Te samples were handled using a SPS 4 auto- 􏽐 x − x y − y 􏽮􏽨 􏼁 􏽩􏽨􏽐 􏼁 􏽩􏽯 i i i i sampler (Agilent Technologies). Te ICP-MS/MS instru- ment was an Agilent 8900 controlled with MassHunter 4.6 where x is the average of x , y is the average of y , x is the i i i Workstation Software 8900 ICP-QQQ Top (C.01.06). Te determination value ofx, and y is the determination value ofy. 8900 ICP-MS/MS uses a tandem mass spectrometer layout, Te relative error (%RE) was calculated according to the with two quadrupole mass spectrometers enabling it to following formula: operate in the MS/MS mode. ICP-QQQ ofers an additional quadrupole mass flter in front of the collision reaction cell x − x i i (3) %RE � × 100, to resolve spectral interferences. Te 8900 has higher sen- sitivity and a specialized fow path with argon gas to provide lower backgrounds (sensitivity: Co ≥ 22 ×106 cps/ppm wherex is the actual value of the calibration standard and x (<10% RSD, relative standard deviation) and background is the concentration of the collected corrected standard. (on mass) mass 78 ≤ 400 cps). Te operating conditions Te relative standard error (%RSE) represented the (lenses, torch position, detector voltage, and gas fows) were ftting index of the correction curve, a value calculated using optimized daily (the count of mass 7, 89, and 205 needs to the following formula: 4 Journal of Analytical Methods in Chemistry Alkali Alkaline earth Transition Post-transition Metalloid Non-metal Actinide Lanthanide metal metal metal metal O-MicroD T90 MicroD UltraS RT Figure 1: Te bar chart represents the recovery efciency (%) of eight diferent element classes across fve diferent digestion methods for the standard solution. -0.3 -0.2 -0.1 0.0 0.1 0.2 0.2 0.3 0.1 0.0 -0.1 -0.2 0.20 0.22 -0.3 0.24 0.26 PC1 (74.7%) Figure 2: Principal component analysis (PCA) of the human hair elements, digested using fve diferent methods. Each color represents a digestion method. An ellipse with dotted lines indicates the 95% confdence interval calculated using Hotelling’s T2 statistics. 􏽳��������������� � Excel version 2019. Te recovery efciency of elements in the n 2 􏽐 􏼂 x − x􏼁 /x 􏼃 i− 1 i i i (4) standard solution was compared and displayed using a %RSE � 100 × , (n − p) Microsoft Excel bar chart. where x is the actual value of the calibration curve leveli, x is i i the collection concentrations of the calibration curve level i, p 3. Results and Discussion is the number of items for the calibration curve formula, andn is the number of points of the available calibration curve. Tis study was the frst to assess fve diferent HNO hair Coefcient of variance (CV) and principal component digestion methods in preparation for inorganic element analysis (PCA) were used to check the repeatability of di- analysis using ICP-MS. A total of 52 elements were quan- gestion methods. Boxplots, two-dimensional projections of titated in this study. Te identifed elements were subdivided the 3D PCA, bar graphs, dot-line graphs, and scatter plots into eight major metal classes such as alkali metals, alkaline were rendered using the ggplot2 R package. Element cov- Earth metals, transition metals, and others (Table S3). Teir erage was compared across the fve digestion protocols and detailed concentrations are displayed in Table S4. Metals displayed using an UpSet plot, constructed using the UpSet detected in each of the classes were evaluated in detail to R package. Te cumulative coefcient of variation was il- assess the performance of the fve diferent digestion lustrated by a horizontal bar chart constructed in Microsoft methods. Te performance of each method was evaluated by PC3 (5.5%) Recovery efficiency (%) PC2 (8.8%) Journal of Analytical Methods in Chemistry 5 UltraS T90 RT MicroD O-MicroD 0 100 200 300 400 500 Alkali metal Metalloid Post-transition metal Lanthanide Actinide Transition metal Alkaline earth metal Non-metal Figure 3: Te horizontal bar chart represents the cumulative coefcient of variations (%) of eight diferent metal classes across fve diferent digestion methods for human hair. Te x-axis is the total CV for all major metal classes. Na Alkali metal Alkaline earth metal Ba Rb Transition metal Mg Mn Post-transition metal Co Zr Metalloid Cr Non-metal Al Fe Actinide Mo Ga Lanthanide TI Zn As Cs Pb 3 Cd Se 2 2 2 2 2 2 K Ce Nd Cu Ag Gd Y Eu Sr 1 La 1 1 1 W Nb 1 1 U Hg Ni Ta Tm Pr Ho Er Ca Re Yb Ge Dy Sm Ti Th UltraS RT O-MicroD T90 MicroD 40 30 20 10 0 Set Size Figure 4: Upset plot of the identifed metal coverage compared across fve diferent digestion methods for human hair. testing internal standard solution stability, digestion re- abnormally high outliers in the T90 and MicroD methods. producibility, metal coverage, and standard solution re- Te RT method demonstrated the best IS stability in this covery efciency. study. 3.1. Internal Standard Solution Stability of the Five Diferent 3.2. Digestion Recovery Efciency of the Standard Solution of Digestion Methods. Te internal standard solution stability Five Diferent Digestion Methods. Te frst criterion which of the fve selected methods was evaluated by comparing was used to evaluate the accuracy of the fve diferent di- levels of the IS elements (Bi, In, and Rh) in the hair samples gestion methods was recovery efciency of the standard following the diferent digestion methods (Figure S1). Te IS solution. Tis is determined according to the recovery levels stability of all digestion methods was between 80% and of diferent elements in a digested standard solution of a 120%, which meets the requirements of the instrument known concentration, as shown in Figure 1. Te RT and measurement. None of the IS elements drifted as an outlier MicroD methods displayed the 99%–100% recovery ef- in the O-MicroD, RT, and UltraS methods, while there were ciency for all eight diferent element classes from the Intersection Size 6 Journal of Analytical Methods in Chemistry Ba Co Cr Fe Mg Mo Na 3 4 3 3 2 2 1 1 0 0 –1 –1 Nd Pb Se U V Zn Cu 3 3 2 3 3 3 2 2 1 2 2 1 1 1 0 1 0 0 –1 0 0 –1 –1 –1 Ni Ag Ta Re Tm Gd Ho 2 2 2 1 1 0 1 0 0 –1 –1 –1 Sm Hg K Sr Yb Ca AI 2 2 1 1 1 2 1 0 0 0 –1 0 0 –1 As Ce Ga La Mn P Pr 2 2 2 1 2 1 1 1 0 1 0 0 0 0 –1 0 Rb TI Zr Dy Nb Ge W 2 1 1 1 1 0 0 0 0 0 –1 0 0 –1 Er Y Ti Cd Cs T Eu 1.0 1.0 2.0 1.0 0.5 2 2 0.5 1.5 0.5 0.0 0.5 1.0 0.0 1 1 –0.5 0.0 0.5 0.0 –0.5 0.0 0 –1.0 –0.5 0 –0.5 –0.5 –1.0 –1.5 Extraction methods Extraction methods O-MicroD T90 RT UltraS MicroD Figure 5: Te dot-line graphs show the element concentrations of each sample across fve diferent digestion methods for human hair. Each dot represents a sample concentration. Te vertical lines are the standard deviation of each digestion method. Te metal concentrations were standardized to Z-scores. CV (%) 6.3 3.6 3.6 (%) 0 100 200 300 400 500 600 700 800 900 1000 103Rh 115In 209Bi Figure 6: Te scatter plot and coefcient of variation (CV) for the internal standard stability from the ICP-MS hair analysis of a thousand maternal hair samples using O-MicroD digestion. Concentration z-score (ug/L) Internal standard stability (%) O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD T90 RT UltraS MicroD O-MicroD 103Rh T90 115In RT UltraS 209Bi MicroD Journal of Analytical Methods in Chemistry 7 standard solution; O-MicroD only produced the high simultaneously in the hair matrix than in other studies; Hao recovery yield for alkali metals, post-transition metals, et al. identifed 15 hair elements using HNO /H O at 100 C 3 2 2 metalloids, nonmetals, lanthanide, and actinide; T90 only until dryness using an electric heating plate [28]; Luo et al. performed the high recovery for alkali metals, alkaline quantifed 16 hair elements using HNO and H O digestion 3 2 2 Earth metals, post-transition metals, metalloids, non- at 90 C for 3 h prior to ICP-MS analysis [16]; Tinkov et al. metals, and lanthanide; UltraS was superior for alkali reported 17 trace elements from children’s hair using mi- metals, post-transition metals, metalloids, nonmetals, and crowave digestion (170–180 C) with HNO [29]. We propose lanthanide. We hypothesized that the RT method dis- that this has been achieved by the following two strategies: played the best recovery efciency because it only involves (1) we have established calibration curves for 56 elements in two sample preparation steps (dilution and incubation) a single standard dilution series by combining three sets of and an extensive 24 h digestion period. In addition, the multielement calibration solutions; (2) the Agilent 8900 MicroD method also exhibited high recovery efciency ICP-MS used in our study exhibits high sensitivity and and this is likely because electromagnetic radiation can strong antispectral interference capability through higher penetrate through the hair matrix, promoting molecular mass spectrum scanning frequency (3.0 MHz) and an ad- rotations and efcient heating via friction [24]. Micro- ditional quadrupole mass flter. Furthermore, the Upset plot wave digestion has been found to be one of the most and dot-line graphs illustrate that there are 13 elements that convenient techniques to prepare samples for elemental can be efectively digested and identifed by using any of the determination [25–27]. fve methods (Figures 4 and 5: Na, Ba, Mg, Co, Cr, Fe, Mo, V, Zn, Pb, Se, Nd, and U), 11 elements that can be efectively identifed by using three of the tested methods (Figures 4 and 3.3. Reproducibility of the Five Diferent Digestion Methods. 5: O-MicroD, MicroD, T90 : Rb, Mn, Zr, Al, Ga, Tl, As, P, Ce, Te O-MicroD method demonstrated the most promising La, and Pr), and four elements which can only can be digestion reproducibility for the trace amount of human hair identifed by using the MicroD method (Figures 4 and 5: Cs, analysed. Te PCA analysis showed that the technical rep- Cd, Eu, and T). Overall, the O-MicroD and MicroD licates of the O-MicroD hair elements (red dots) were methods showed the most comprehensive coverage of clustered the closest to each other along PC1 (74.7%), PC2 identifed elements in this study. (8.8%), and PC3 (5.5%) (Figure 2). Consistent with the Since the O-MicroD method could reproducibly results of the PCA, the O-MicroD digestion method quantitate a large number of elements using only 3 mg of exhibited the most reproducible CV (CV: 23.49%) for the hair biomass with 200μl of nitric acid for 30 min digestion overall metal classes identifed (Figure 3, Table S3 and time, we further tested the reproducibility of O-MicroD by Figure S2). Te MicroD digestion method displayed the least coinjecting three IS with a thousand hair samples. We reproducible CV for hair (CV: 64.37%). Te reproducibility 103 115 209 found that the CV of Rh, In, and Bi were 3.59%, of lanthanide was poor across all fve digestion methods, 3.61%, and 6.31%, respectively, without batch correction with a minimum CV of 36.87% (T90) (Figure 3, Table S3). In (Figure 6). Te excellent reproducibility of O-MicroD, addition, we found that the hair samples that were digested together with minimizing sample usage and preparation using the MicroD method exhibited the highest number of time, makes this approach an ideal choice for a large cohort elements with poor reproducibility (Figure 4). Te possible studies. explanation for the O-MicroD digestion method possessing In summary, our fndings suggest that out of the fve superior reproducibility for most metal elements in a trace digestion methods tested, the O-MicroD method had the amount of human hair could be that the microwave has best reproducibility and digestion time (30 mins) for ele- heating stability and less volume is lost during solution mental analysis of human hair, the MicroD method had the transfer. In contrast, the MicroD method showed the best element coverage, and the RTmethod performed best in poorest reproducibility due to additional preparation steps, element digestion recovery efciency. that is, the microwave-digested hair mixture was further heated and evaporated from 5 ml to 0.2 ml to eliminate the high HNO concentration prior to ICP-MS analysis. Tis 4. Conclusion signifcant fuctuation in volume may greatly increase the variability of element concentrations among individual In conclusion, this is the frst study conducted to investigate samples. the optimal digestion method for analysing the 52 inorganic elements in human hair using ICP-MS analysis. Based on the 3.4.ElementCoverageoftheFiveDiferentDigestionMethods. criteria of reproducibility, detected element coverage, and Another criterion that we used to determine the optimal the digestion recovery yield, the O-MicroD, MicroD, and RT digestion method was the element coverage. Te order of methods were the superior digestion methods for CV, digestion methods ranking from the highest to lowest coverage, and recovery efciency, respectively. However, we number of identifed elements in human hair were as fol- also discovered that diferent digestion methods favor the lows: MicroD (44 elements), O-MicroD (38 elements), T90 digestion of diferent element classes. Future studies should (38 elements), RT (25 elements), and UltraS (22 elements), carefully consider the digestion method they select, choosing as displayed in Table S3. Noticeably, our microwave di- the method that would be best suited to the primary purpose gestion approaches have quantifed more elements of their study. 8 Journal of Analytical Methods in Chemistry Abbreviations Supplementary Materials CLIMB: Complex Lipids in Mothers and Babies Figure S1: the extraction efciency of internal standard el- CV: Coefcient of variation ements (Bi, In, and Rh) in fve diferent digestion methods ET-AAS: Electrothermal-atomic absorption spectrometry for human hair. Figure S2: the bar graphs show the coef- FAAS: Flame atomic absorption spectrometry fcient of variation (%) of each metal across fve diferent ICP-MS: Inductively coupled plasma-mass spectrometry digestion methods for human hair. Table S1: microwave ICP- Inductively coupled plasma-optical emission digestion procedure parameters. Table S2: linear relationship OES: spectrometry and detection limit of each element in the standard solution. INAA: Instrumental neutron activation analysis Table S3: number of identifed elements, concentrations (μg/ LOD: Limits of detection L), and CVs (%) of each element classes in human hair, MicroD: Programmed digestion of microwave digestion compared across fve diferent digestion methods. Table S4: O- Ordinary microwave oven digestion the quantitative concentrations (μg/L) of identifed metals in MicroD: human hairs across the fve diferent digestion methods. PCA: Principal component analysis (Supplementary Materials) PTFE: Polytetrafuoroethylene RE: Relative error References RSE: Relative standard error RT: Room temperature for 24 h [1] J. I. Lachowicz, L. I. Lecca, F. Meloni, and M. Campagna, “Metals and metal-nanoparticles in human pathologies: from T90: 90 C for 4 h exposure to therapy,” Molecules, vol. 26, no. 21, p. 6639, 2021. UltraS: Ultrasonic assisted digestion. [2] X. Wang, P. An, Z. Gu, Y. Luo, and J. Luo, “Mitochondrial metal ion transport in cell metabolism and disease,” Inter- national Journal of Molecular Sciences, vol. 22, no. 14, p. 7525, Data Availability [3] M. Valko, K. Jomova, C. J. Rhodes, K. Kuca, and K. Musilek, Te data used to support the results of this study are in- “Redox- and non-redox-metal-induced formation of free cluded within the article. Any further information is radicals and their role in human disease,” Archives of Toxi- available from corresponding authors upon request. cology, vol. 90, pp. 1–37, 2016. [4] X. F. Hu, M. Lowe, and H. M. Chan, “Mercury exposure, cardiovascular disease, and mortality: a systematic review and dose-response meta-analysis,” Environmental Research, Conflicts of Interest vol. 193, Article ID 110538, 2021. [5] X. Li, Y. Huang, Y. Xing et al., “Association of urinary Te authors declare that they have no conficts of interest. cadmium, circulating fatty acids, and risk of gestational di- abetes mellitus: a nested case-control study in China,” En- Authors’ Contributions vironment International, vol. 137, Article ID 105527, 2020. [6] S. S. Shiek, M. S. Mani, S. P. Kabekkodu, and H. S. Dsouza, Y. L. and T. H. designed the experiment and wrote the “Health repercussions of environmental exposure to lead: manuscript; Y. L., Y. Y., D. Z., and Y-M. L. performed the methylation perspective,” Toxicology, vol. 461, Article ID sample preparation and ICP-MS analysis; Y. L., and T. H. 152927, 2021. performed the data analysis; T. H., J. dS., and Y. X. revised [7] Y. Huang, X. Li, W. Zhang et al., “Aluminum exposure and gestational diabetes mellitus: associations and potential me- the manuscript; T. H., and H. Z. provided funding for the diation by n-6 polyunsaturated fatty acids,” Environmental experimental costs. All authors read and approved the fnal Science & Technology, vol. 54, no. 8, pp. 5031–5040, 2020. version of this manuscript. [8] D. Pozebon, G. L. Schefer, and V. L. Dressler, “Elemental hair analysis: a review of procedures and applications,” Acknowledgments Analytica Chimica Acta, vol. 992, pp. 1–23, 2017. [9] C. Popescu and H. Hocker, “Hair - the most sophisticated Tis work was supported by the National Natural Science biological composite material,” Chemical Society Reviews, Foundation of China (No. 81871185, 81971406), Te 111 vol. 36, no. 8, pp. 1282–1291, 2007. [10] M. D. Lakshmi Priya and A. Geetha, “Level of trace elements Project (Yuwaizhuan (2016)32), Kuanren Talents Programs (copper, zinc, magnesium and selenium) and toxic elements of Second Afliated Hospital of Chongqing Medical Uni- (lead and mercury) in the hair and nail of children with versity, Chongqing Science and Technology Commission autism,” Biological Trace Element Research, vol. 142, no. 2, (cstc2021jcyj-msxmX0213), Chongqing Municipal Educa- pp. 148–158, 2011. tion Commission (KJZD-K202100407), Chongqing Health [11] I. Molina-Villalba, M. Lacasana, M. Rodriguez-Barranco et al., Commission and Chongqing Science and Technology “Biomonitoring of arsenic, cadmium, lead, manganese and Commission (2021MSXM121, 2020MSXM101), Smart mercury in urine and hair of children living near mining and Medicine Research Project of Chongqing Medical University industrial areas,” Chemosphere, vol. 124, pp. 83–91, 2015. (No. ZHYX202103), and a Joint Health Research Council [12] J. Kucera and K. Kofronova, “Determination of as by in- New Zealand–National Science Foundation of China Bio- strumental neutron activation analysis in sectioned hair medical Research Fund (19/804). samples for forensic purposes: chronic or acute poisoning?” Journal of Analytical Methods in Chemistry 9 Journal of Radioanalytical and Nuclear Chemistry, vol. 287, [26] S. Kasar, R. Murugan, H. Arae, T. Aono, and S. K. Sahoo, “A microwave digestion technique for the analysis of rare earth no. 3, pp. 769–772, 2011. [13] G. Izydorczyk, M. Mironiuk, S. Basladynska, M. Mikulewicz, elements, thorium and uranium in geochemical certifed reference materials and soils by inductively coupled plasma and K. Chojnacka, “Hair mineral analysis in the population of students living in the Lower Silesia region (Poland) in 2019: mass spectrometry,” Molecules, vol. 25, no. 21, p. 5178, 2020. [27] R. Mohamed, B. H. Zainudin, and A. S. Yaakob, “Method comparison with biomonitoring study in 2009 and literature validation and determination of heavy metals in cocoa beans data,” Environmental Research, vol. 196, Article ID 110441, and cocoa products by microwave assisted digestion tech- nique with inductively coupled plasma mass spectrometry,” [14] S. Grassin-Delyle, M. Martin, O. Hamzaoui et al., “A high- Food Chemistry, vol. 303, Article ID 125392, 2020. resolution ICP-MS method for the determination of 38 in- [28] Z. Hao, Y. Li, Y. Liu, H. Li, W. Wang, and J. Yu, “Hair el- organic elements in human whole blood, urine, hair and ements and healthy aging: a cross-sectional study in Hainan tissues after microwave digestion,” Talanta, vol. 199, Island, China,” Environmental Geochemistry and Health, pp. 228–237, 2019. vol. 38, no. 3, pp. 723–735, 2016. [15] E. Tamburo, D. Varrica, G. Dongarra, and L. M. E. Grimaldi, [29] A. A. Tinkov, A. L. Mazaletskaya, O. P. Ajsuvakova et al., “Trace elements in scalp hair samples from patients with “ICP-MS assessment of hair essential trace elements and relapsing-remitting multiple sclerosis,” PLoS One, vol. 10, minerals in Russian preschool and primary school children no. 4, Article ID e0122142, 2015. with attention-defcit/hyperactivity disorder (ADHD),” Bio- [16] R. Luo, S. Zhang, P. Xiang, B. Shen, X. Zhuo, and D. Ma, logical Trace Element Research, vol. 196, no. 2, pp. 400–409, “Elements concentrations in the scalp hair of methamphet- amine abusers,” Forensic Science International, vol. 249, pp. 112–115, 2015. [17] F. L. Barbieri, A. Cournil, J. E. Souza Sarkis, E. Benefce, and J. Gardon, “Hair trace elements concentration to describe polymetallic mining waste exposure in Bolivian altiplano,” Biological Trace Element Research, vol. 139, no. 1, pp. 10–23, [18] G. Giangrosso, G. Cammilleri, A. Macaluso et al., “Hair mercury levels detection in fshermen from sicily (Italy) by ICP-MS method after microwave-assisted digestion,” Bio- inorganicChemistryandApplications, vol. 2016, pp. 1–5, 2016. [19] F. Domanico, G. Forte, C. Majorani et al., “Determination of mercury in hair: comparison between gold amalgamation- atomic absorption spectrometry and mass spectrometry,” Journal of Trace Elements in Medicine & Biology, vol. 43, pp. 3–8, 2017. [20] H. Yasuda, K. Yoshida, M. Segawa et al., “Metallomics study using hair mineral analysis and multiple logistic regression analysis: relationship between cancer and minerals,” Envi- ronmental Health and Preventive Medicine, vol. 14, no. 5, pp. 261–266, 2009. [21] M. F. Mesko, R. M. Pereira, P. T. Scaglioni, and D. L. R. Novo, “Single analysis of human hair for determining halogens and sulfur after sample preparation based on combustion reac- tion,” Analytical and Bioanalytical Chemistry, vol. 411, no. 19, pp. 4873–4881, 2019. [22] S. Huang, T. T. Mo, T. Norris et al., “Te CLIMB (Complex Lipids in Mothers and Babies) study: protocol for a multi- centre, three-group, parallel randomised controlled trial to investigate the efect of supplementation of complex lipids in pregnancy, on maternal ganglioside status and subsequent cognitive outcomes in the ofspring,” BMJOpen, vol. 7, no. 10, Article ID e016637, 2017. [23] T. D. J. Delplancke, J. V. de Seymour, C. Tong et al., “Analysis of sequential hair segments refects changes in the metab- olome across the trimesters of pregnancy,” Scientifc Reports, vol. 8, no. 1, p. 36, 2018. [24] Y. Wu, R. Mu, G. Li, M. Li, and W. Lv, “Research progress in fuid and semifuid microwave heating technology in food processing,”ComprehensiveReviewsinFoodScienceandFood Safety, vol. 21, no. 4, pp. 3436–3454, 2022. [25] H. Altundag and M. Tuzen, “Comparison of dry, wet and microwave digestion methods for the multi element deter- mination in some dried fruit samples by ICP-OES,” Food and Chemical Toxicology, vol. 49, no. 11, pp. 2800–2807, 2011.

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