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

The 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 (HNO3) digestion is the most common approach for determining inorganic elements from human hair. This 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 different HNO3 (65%) digestion methods were investigated and evaluated for their internal standard solution stability, reproducibility, element coverage, and standard solution recovery efficiency, namely, room temperature for 24 h (RT), 90°C for 4 h (T90), ultrasonic-assisted digestion (UltraS), programmed digestion of microwave digestion (MicroD), and ordinary microwave oven digestion (O-MicroD). Our results demonstrated that O-MicroD, MicroD, and RT were the best performing digestion methods for coefficient of variation (CV) scores, coverage, and recovery efficiency, respectively. In particular, the O-MicroD method detected multiple elements in a small quantity of hair (3 mg), with minimum nitric acid usage (200 μl) and a short digestion time (30 min). The O-MicroD method had excellent reproducibility, as demonstrated by a continuous thousand injections of hair samples with three internal standards (CV: 103Rh = 3.59%, 115In = 3.61%, and 209Bi = 6.31%). Future studies of the elemental content of hair should carefully select their digestion method to meet the primary purpose of their study.


Introduction
Inorganic elements have unique properties that cannot be performed by organic compounds. Some elements are indispensable for humans and essential in biochemical processes. For example, iron (Fe), copper (Cu), and manganese (Mn) are all positioned at the metal-catalytic site of enzymes involved in redox reactions [1]. Calcium, magnesium, and sodium maintain the electrochemical gradient of the cell membranes [1]. Tere are many metal ions that are important for cellular metabolism and mitochondrial function, such as K + , Mg 2+ , and Zn 2+ [2]. Nevertheless, growing evidence suggests that excessive exposure to some elements could cause adverse health outcomes. Te role of cadmium (Cd), mercury (Hg), and copper (Cu) in type 2 diabetes, renal dysfunction, cardiovascular diseases, osteoporosis, and cancers, has been reported in experimental and epidemiological studies [3][4][5]. Excessive aluminium (Al), lead (Pb), and arsenic (As) exposures have also been associated with oxidative stress, intestinal diseases, dyslipidemia, and metabolic diseases [3,6,7]. Exposure to metals can be mediated through food, dermal contact, air pollution, and drinking water [7]. Elements absorbed by the pulmonary tract and the digestive system can be distributed via the bloodstream to a range of diferent organs and also to the hair [8]. Furthermore, some elements might be deposited on the hair through external contamination. Hence, measurements of the composition of the hair can refect both endogenous and exogenous exposures to various inorganic elements.
Hair is a proteinaceous fbre predominantly consisting of keratin proteins [9]. Most inorganic elements have a high afnity for the sulfhydryl group of amino acids in keratin. Terefore, inorganic elements are easily incorporated and retained in human hair [9]. Te accumulation of inorganic elements in hair refects long-term exposure. Since metal concentrations are reduced in urine and blood after days and weeks, respectively, human hair appears to be a more robust specimen for estimating past and ongoing exposure to inorganic elements. Hair also has less background matrix than urine and blood, and the inorganic elements are easier to detect analytically because they are usually present at higher levels in the hair. Moreover, hair samples are collected noninvasively and can be transported and stored without refrigeration or prior processing. Hence, hair has become an attractive clinical specimen for studying the longitudinal exposure of inorganic elements from the external environment.
Numerous instruments have been employed for element detection and quantitation, including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), instrumental neutron activation analysis (INAA), electrothermalatomic absorption spectrometry (ET-AAS), and fame atomic absorption spectrometry (FAAS) [10][11][12][13][14]. For qualitative and quantitative measurement of hair elements, ICP-MS is our selected platform because it has the most superior sensitivity, resolving power, and improved limits of detection (LODs) to measure elements [8]. A variety of hair digestion techniques for element detection via ICP-MS analysis have been published, including using a microwave digestion instrument, leaving the sample at room temperature for 24 h, using a heat block, and using an ultrasonic water bath [15][16][17][18][19]. Tese approaches are generally coupled with efcient acid decomposition procedures such as digestion in concentrated nitric acid (65-68%), and only a few studies use other solutions [8,20,21]. However, these methods often use a large amount of hair biomass (30-150 mg), excessive acid solution (3-10 ml per sample), and extensive digestion time (1 hr-2 days) [15][16][17][18][19]. Tese factors are not conducive to the efcient analysis of a large cohort of samples, and not all participants are willing to donate a large volume of hair. It is clear that the methodology for analysing inorganic elements in hair needs to be improved in order to efectively and efciently analyse hair element concentrations in large cohorts.
Tis study aims to establish a method for analysing the inorganic elements in human hair samples from a large cohort, using low hair mass. We analysed fve diferent sample preparation methods for their reproducibility, element coverage, and extraction efciency. Te results from our fndings have informed the methodology for a large cohort study of hair from pregnant women to investigate the link between exposure to inorganic elements and pregnancy/ fetal outcomes.

Hair Sample Collection.
Hair samples were collected from pregnant women in the Complex Lipids in Mothers and Babies (CLIMB) cohort (Chinese Clinical Trial Register number: ChiCTR-IOR-16007700) [22]. Te 3-6 hair strands were taken from the occipital area, 0.5 cm away from the scalp. Te hair samples were cut into pieces and stored in a self-sealing bag at −20°C. Te collection and segmentation of hair were done with scissors made of polytetrafuoroethylene (PTFE) to avoid elemental contamination. A thousand hair samples collected from CLIMB were used to test the reproducibility of the chosen method. Tese hair samples were collected in accordance with the method published by Delplancke et al. [23] and in full agreement with the principles of the International Conference on Harmonisation Good Clinical Practice E6 (ICH-GCP) and the Declaration of Helsinki. Te study was approved by the Ethics Committee of Chongqing Medical University (No.2014034), and written informed consent was obtained from all participants.

Reagents and Calibration Standard Solutions.
Analytical and internal standards were purchased from Agilent Technologies. ICP-MS-grade nitric acid (65%) and acetone were obtained from ANPEL Laboratory Technologies (Shanghai, China). Ultrapure deionized water (18 mΩ) was obtained from a water purifcation system (Aoside, China).
A working standard solution was prepared by diluting 10 μg/mL of mixed-element standard solutions (Multielement Calibration

Sample Preparation.
Te hair strands were washed two times with acetone and then once with deionized water, prior to drying in an oven at 37°C. Hair segments were mixed together in 2 mL eppendorf tubes with PTFE beads to break the hairs into powder using tissue lysis U (QIAGEN), to ensure homogeneity of the hairs for all digest methods under investigation. Four replicates of 3 mg ± 0.5 mg of mixed hair were weighed into 15 mL PTFE digestion tubes. Next, 200 μL of concentrated nitric acid was added, and the tubes were capped to carry out the digestion reaction. Importantly, the MicroD digestion method required adding 3 mg ± 0.5 mg hair sample and 5 ml concentrated nitric acid to the matching digestion tank and then concentrated the acid into 200 μl before transfer. After the samples had dissolved, they were transferred to 15 mL centrifuge tubes and made up to a volume of 3 mL with deionized water. A 100 μl aliquot of each of the three mixed standard solutions was transferred into the PTFE tube, and then 200 μl of the concentrated nitric acid solution was added. After digestion, using the fve diferent methods detailed above, the digested solution was transferred to 15 ml centrifuge tubes and made up to a volume of 10 mL with deionized water.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)
Analysis. Te samples were handled using a SPS 4 autosampler (Agilent Technologies). Te ICP-MS/MS instrument was an Agilent 8900 controlled with MassHunter 4.6 Workstation Software 8900 ICP-QQQ Top (C.01.06). Te 8900 ICP-MS/MS uses a tandem mass spectrometer layout, with two quadrupole mass spectrometers enabling it to operate in the MS/MS mode. ICP-QQQ ofers an additional quadrupole mass flter in front of the collision reaction cell to resolve spectral interferences. Te 8900 has higher sensitivity and a specialized fow path with argon gas to provide lower backgrounds (sensitivity: 59 Co ≥ 22 × 106 cps/ppm (<10% RSD, relative standard deviation) and background (on mass) mass 78 ≤ 400 cps). Te operating conditions (lenses, torch position, detector voltage, and gas fows) were optimized daily (the count of mass 7, 89, and 205 needs to reach more than 3000, 10000, and 6000, respectively; oxide <2%; doubly charged <3%; peak width -10%: 0.65-0.80), using a 1 μg/L tuning solution (Ce, Co, Li, Mg, Tl, and Y; Agilent Technologies). Te main ICP-MS detection parameters were as follows: radio-frequency (RF) power: 1550.0 w, RF matching: 1.80 V, sampling depth: 10 (Table S2). Only elements not in the calibration standard solution were selected as ISs ( 103 Rh, 115 In, 209 Bi). Te measuring elements were normalized by the most appropriate IS according to the following mass range: 103 Rh for mass 115 In for mass 111-169, and 209 Bi for mass 172-238. Te blank deduction was performed by subtracting the blank from the sample results. Calibration curves were then set up by external calibration, linear ft, and blank ofset. Te y value was obtained by dividing the count of each point of the correction curve by the count of the unit concentration of the internal standard of the same level as follows: when x i � 0, y � y σ ; x i is the IS element concentration, y i is the IS element count, and y σ is the sample data count. Te correlation coefcient of the linear regression correction curve was calculated using the following formula: where x is the average of x i , y is the average of y i , x i is the determination value of x, and y i is the determination value of y. Te relative error (%RE) was calculated according to the following formula: where x i is the actual value of the calibration standard and x i ′ is the concentration of the collected corrected standard. Te relative standard error (%RSE) represented the ftting index of the correction curve, a value calculated using the following formula: Journal of Analytical Methods in Chemistry 3 where x i is the actual value of the calibration curve level i, x i ′ is the collection concentrations of the calibration curve level i, p is the number of items for the calibration curve formula, and n is the number of points of the available calibration curve.
Coefcient of variance (CV) and principal component analysis (PCA) were used to check the repeatability of digestion methods. Boxplots, two-dimensional projections of the 3D PCA, bar graphs, dot-line graphs, and scatter plots were rendered using the ggplot2 R package. Element coverage was compared across the fve digestion protocols and displayed using an UpSet plot, constructed using the UpSet R package. Te cumulative coefcient of variation was illustrated by a horizontal bar chart constructed in Microsoft Excel version 2019. Te recovery efciency of elements in the standard solution was compared and displayed using a Microsoft Excel bar chart.

Results and Discussion
Tis study was the frst to assess fve diferent HNO 3 hair digestion methods in preparation for inorganic element analysis using ICP-MS. A total of 52 elements were quantitated in this study. Te identifed elements were subdivided into eight major metal classes such as alkali metals, alkaline Earth metals, transition metals, and others (Table S3). Teir detailed concentrations are displayed in Table S4. Metals detected in each of the classes were evaluated in detail to assess the performance of the fve diferent digestion methods. Te performance of each method was evaluated by testing internal standard solution stability, digestion reproducibility, metal coverage, and standard solution recovery efciency.

Internal Standard Solution Stability of the Five Diferent
Digestion Methods. Te internal standard solution stability of the fve selected methods was evaluated by comparing levels of the IS elements (Bi, In, and Rh) in the hair samples following the diferent digestion methods ( Figure S1). Te IS stability of all digestion methods was between 80% and 120%, which meets the requirements of the instrument measurement. None of the IS elements drifted as an outlier in the O-MicroD, RT, and UltraS methods, while there were abnormally high outliers in the T90 and MicroD methods. Te RT method demonstrated the best IS stability in this study.

Digestion Recovery Efciency of the Standard Solution of
Five Diferent Digestion Methods. Te frst criterion which was used to evaluate the accuracy of the fve diferent digestion methods was recovery efciency of the standard solution. Tis is determined according to the recovery levels of diferent elements in a digested standard solution of a known concentration, as shown in Figure 1. Te RT and MicroD methods displayed the 99%-100% recovery efciency for all eight diferent element classes from the   MicroD only produced the high  recovery yield for alkali metals, post-transition metals,  metalloids, nonmetals, lanthanide, and actinide; T90 only  performed the high recovery for alkali metals, alkaline Earth metals, post-transition metals, metalloids, nonmetals, and lanthanide; UltraS was superior for alkali metals, post-transition metals, metalloids, nonmetals, and lanthanide. We hypothesized that the RT method displayed the best recovery efciency because it only involves two sample preparation steps (dilution and incubation) and an extensive 24 h digestion period. In addition, the MicroD method also exhibited high recovery efciency and this is likely because electromagnetic radiation can penetrate through the hair matrix, promoting molecular rotations and efcient heating via friction [24]. Microwave digestion has been found to be one of the most convenient techniques to prepare samples for elemental determination [25][26][27].  Table S3 and Figure S2). Te MicroD digestion method displayed the least reproducible CV for hair (CV: 64.37%). Te reproducibility of lanthanide was poor across all fve digestion methods, with a minimum CV of 36.87% (T90) (Figure 3, Table S3). In addition, we found that the hair samples that were digested using the MicroD method exhibited the highest number of elements with poor reproducibility (Figure 4). Te possible explanation for the O-MicroD digestion method possessing superior reproducibility for most metal elements in a trace amount of human hair could be that the microwave has heating stability and less volume is lost during solution transfer. In contrast, the MicroD method showed the poorest reproducibility due to additional preparation steps, that is, the microwave-digested hair mixture was further heated and evaporated from 5 ml to 0.2 ml to eliminate the high HNO 3 concentration prior to ICP-MS analysis. Tis signifcant fuctuation in volume may greatly increase the variability of element concentrations among individual samples.

Element Coverage of the Five Diferent Digestion Methods.
Another criterion that we used to determine the optimal digestion method was the element coverage.  [29]. We propose that this has been achieved by the following two strategies: Since the O-MicroD method could reproducibly quantitate a large number of elements using only 3 mg of hair biomass with 200 μl of nitric acid for 30 min digestion time, we further tested the reproducibility of O-MicroD by coinjecting three IS with a thousand hair samples. We found that the CV of 103 Rh, 115 In, and 209 Bi were 3.59%, 3.61%, and 6.31%, respectively, without batch correction ( Figure 6). Te excellent reproducibility of O-MicroD, together with minimizing sample usage and preparation time, makes this approach an ideal choice for a large cohort studies.
In summary, our fndings suggest that out of the fve digestion methods tested, the O-MicroD method had the best reproducibility and digestion time (30 mins) for elemental analysis of human hair, the MicroD method had the best element coverage, and the RT method performed best in element digestion recovery efciency.

Conclusion
In conclusion, this is the frst study conducted to investigate the optimal digestion method for analysing the 52 inorganic elements in human hair using ICP-MS analysis. Based on the criteria of reproducibility, detected element coverage, and the digestion recovery yield, the O-MicroD, MicroD, and RT methods were the superior digestion methods for CV, coverage, and recovery efciency, respectively. However, we also discovered that diferent digestion methods favor the digestion of diferent element classes. Future studies should carefully consider the digestion method they select, choosing the method that would be best suited to the primary purpose of their study. Figure S1: the extraction efciency of internal standard elements (Bi, In, and Rh) in fve diferent digestion methods for human hair. Figure S2: the bar graphs show the coeffcient of variation (%) of each metal across fve diferent digestion methods for human hair. Table S1: microwave digestion procedure parameters. Table S2: linear relationship and detection limit of each element in the standard solution. Table S3: number of identifed elements, concentrations (μg/ L), and CVs (%) of each element classes in human hair, compared across fve diferent digestion methods. Table S4: the quantitative concentrations (μg/L) of identifed metals in human hairs across the fve diferent digestion methods. (Supplementary Materials)