As the demand grows for lithium-ion (Li-ion) batteries, their performance requirements and environmental impact increase. Battery performance strongly depends on the composition of the cathode materials, requiring precise elemental ratios. Meanwhile, disposing spent batteries can have a negative environmental impact, which can be greatly reduced through recycling. Inductively coupled plasma–optical emission spectroscopy (ICP-OES) provides solutions in both of these areas. By using high-precision ICP-OES, precise measurements can be made to accurately determine compositions of a variety of different cathode materials. In battery recycling, ICP-OES meets the requirements of being a multielement technique with a wide dynamic range and the ability to handle complex matrices. Therefore, it can measure both high-concentration and impurity elements resulting from the incineration of spent batteries, providing recycling facilities information about the elements present and their levels so that recoveries can be optimized.

The performance of lithium-ion (Li-ion) batteries is strongly influenced by the composition of the cathode materials: different materials have different performance characteristics. Examples of cathodes include LiCoO

, among others, with new ones continuously being developed. However, for a given material, the exact composition impacts battery performance. For example, in LiNixMnyCozO

 (NMC), the molar ratios of Ni:Mn:Co affect battery performance, and they can be adjusted depending on the battery’s intended use (1). Therefore, it is critical to determine the molar ratios of cathode materials elements accurately, precisely, and consistently.

One of the issues facing the Li-ion battery industry is finding enough lithium and other cathode elements to meet the demand (2). Larger quantities can be extracted from the earth, but this process comes with a negative environmental impact (3). To address these challenges, battery recycling is becoming more prominent so that important elements can be reused. Two common methods of battery recycling are pyrometallurgy and hydrometallurgy, both of which produce a complex mixture of carbon, ash, graphite, residual electrolyte, cathode elements, and impurity elements known as “black mass.” To determine the quantities of usable elements that can be extracted, the black mass must be analyzed for cathode material elements and elemental impurities.

Inductively coupled plasma–optical emission spectroscopy (ICP-OES) can help in both of these scenarios. With its multi-element capability, ability to handle complex matrices, and wide dynamic range, ICP-OES is uniquely suited to address the analytical requirements of battery production and recycling. Because of its robust plasma and ability to read multiple wavelengths simultaneously, ICP-OES provides the consistent, high-precision elemental ratios required to characterize cathode materials during battery production. The wide dynamic range allows both cathode and impurity elements to be measured during battery recycling, providing important information so that recoveries can be optimized.

This work shows how ICP-OES can assist in both cathode production and battery recycling.

A 0.1 g quantity of nickel manganese cobalt (NMC) or LiFePO was combined with 3 mL hydrochloric acid (HCl; concentrated) in sample digestion tubes, which were then loosely capped and heated at 120 °C for 30 min in a sample preparation block. After the solutions cooled to room temperature, they were diluted to 50 mL with deionized water and then filtered to remove any graphite. For analysis, the filtered samples were diluted another 10 times with deionized water.

Because of the presence of carbon, ash, and graphite in the black mass sample, it is extremely difficult to digest. Instead, metals were leached from black mass by placing 50 mg of sample in a sample digestion tube, followed by 5 mL of aqua regia. The tubes were loosely capped and heated at 120 °C for 30 min. After the tubes cooled to room temperature, they were diluted to 50 mL with deionized water for analysis.

All analyses were done on a PerkinElmer Avio 550 Max fully simultaneous ICP-OES instrument using the conditions and wavelengths listed in Tables I and II. For the cathode materials, a technique known as high-precision ICP-OES was used (4), which involves using manual integration to measure the internal standard and analyte at exactly the same time by setting their read and integration times to be equal. In this manner, the internal standard compensates in real time for any variation in analyte signal, which can be because of plasma flicker or pulsations from the peristaltic pump. The result is excellent precision, with relative standard deviation (RSD) values of 0.2% or less.

The black mass sample was first analyzed with SmartQuant, a semi-quantitative technique within Syngistix for ICP software, which gives a quick overview of the elemental composition of a sample. Once the major and impurity components present were determined, a comprehensive quantitative analysis was performed on those elements to attain more accurate results.

An important difference between the analyses was the sample 

introduction systems. A standard nebulizer and spray chamber were used for the cathode analysis, but because black mass can contain hydrofluoric acid (HF), an HF-re- sistant sample introduction system was used.

Because the composition of cathode materials is critical to the performance of Li-ion batteries, the elemental ratios of cathode materials must be determined with high precision, both among replicates within a sample and over time as multiple samples are analyzed. To demonstrate high precision between replicates, an NMC sample was measured consecutively three times. As shown in Table III, both the weight percent and precision (RSD) for all elements were <0.1%, demonstrating the precision attainable.

To assess stability, the NMC sample was measured 10 times over 20 min to determine the stability of the measurements. As shown in Figure 1, both the RSDs of the concentrations in solution and the molar ratio are very consistent, with concentration RSDs less than 0.5% and molar ratios less than 0.3%. Taken together, these results demonstrate the ability of ICP-OES to provide the high precision and consistency required for lithium-ion battery production.

, the concentration of each component in the solid is important. Similar to NMC analysis, a LiFePO

 sample was analyzed nine times over 15 min. As shown in Figure 2, the RSDs over the nine measurements were less than 0.5%, demonstrating the stability of the methodology for LiFePO

The black mass sample resulted from the incineration of multiple lithium-ion batteries of various types, so it is a true unknown. However, the sample is expected to contain high concentrations of common cathode elements, but there could be various cathodes used in these batteries. Additionally, a variety of impurities could also be present.

To determine what elements are present, a quick semi-quantitative screening was performed, which showed high levels of expected elements typically present in cathodes (Co, Cu, Fe, Li, Mn, Ni, and P), along with the following impurities: Al, B, S, Ti, W, and Zr. Therefore, quantitative analyses were performed on these 13 elements. The sample was analyzed two times, with the results appearing in Table IV, along with the relative percent differences between the analyses. As expected, the results show percent levels of common cathode elements (Co, Cu, Li, Mn, Ni, and P), along with lower concentrations of several impurity elements (Al, S, Ti, W, and Zr). The relative percent differences between the analyses are less than 3%, demonstrating the consistency of the methodology.

It is surprising that Al was present at percent levels as an impurity, but its presence at this concentration was confirmed through multiple sample preparations and analyses. In addition, sample prep blanks were prepared and analyzed and showed that Al was not being introduced through the sample preparation process.

This work has shown how ICP-OES can be used in the lithium-ion battery industry to help with the development of new battery materials and in recycling spent batteries, because of its versatility. The ability of ICP-OES to provide repeatable high-precision results allows for the accurate characterization of cathode materials, while the wide dynamic range and ability to handle challenging matrices allows it to be used in battery recycling to determine elements important for cathodes and impurity elements in combusted batteries, allowing recovery procedures to be optimized. With this versatility, ICP-OES is 

an important tool for the Li-ion battery industry.

(1) Das, D.; Manna, S.; Purvankara, S. Electrolytes, Additives and Binders for NMC Cathodes in Li-Ion Batteries—A Review. 

(2) Azevedo, M.; Baczynska, M.; Hoffman, K.; Krauze, A. 

Lithium Mining: How New Production Technologies Could Fuel the Global EV Revolution

; Technical Report for McKinsey & Company: London, England, April 2022.

(3) Buchert, M.; Degrief, S.; Dolega, P. 

Ensuring a Sustainable Supply of Raw Materials for Electric Vehicles: A Synthesis Paper on Raw Material Needs for Batteries and Fuel Cells

; Technical Report for Oko-Institut: Darmstadt, Germany, 2018.

(4) PerkinElmer, HP-ICP-OES: Using the Avio 550/560 Max to Achieve the Highest Possible Precisions; Technical Note from PerkinElmer U.S. LLC: Shelton, Connecticut, 2020. 

https://www.perkinelmer.com/libraries/TCH_ Avio-500-ICP-OES-CRTSIS_38131

 is with PerkinElmer LLC, in Shelton, CT. Direct correspondence to: 

Articles

ICP-OES can accurately measure the elemental ratios necessary to optimize battery performance and improve recycling efforts so that battery recoveries are optimized. The authors explore how ICP-OES can help manufacturers meet the increasing performance demands required for lithium-ion batteries.

Using ICP-OES to Improve Lithium-Ion Battery Performance and Reduce Waste

Gas chromatography (GC) coupled to inductively coupled plasma–mass spectrometry (ICP-MS) is a powerful and flexible analytical solution that is well-established for measuring organometallic compounds such as organotin, organomercury, and organolead in environmental samples, foodstuffs, and consumer products. GC–ICP-MS is also used for industrial applications, such as monitoring catalyst poisons and environmental contaminant elements in petrochemical processing and plastics manufacturing. The semiconductor industry uses a range of high-purity specialty gases and volatile liquids as precursors in wafer substrate production, and for processes such as plasma etching and deposition of thin films by chemical vapor deposition (CVD). Semiconductor manufacturers are constantly developing new integrated circuit (IC) chips with smaller sizes, higher speeds, lower power consumptions, and greater transistor densities. This trend means that contaminants must be controlled at even lower levels in process chemicals and precursors. In this article, we show how GC–ICP-MS, particularly using triple quadrupole or tandem ICP-MS (ICP-MS/MS), enables determination of the lowest levels of contaminants in the specialty gases and volatile liquids used to make the most advanced electronic devices.

Integrated circuit (IC) chips play an increasingly important role in modern life, supporting technologies from consumer electronics and self-driving vehicles to the high-speed communications and servers used in accelerated computing and artificial intelligence (AI). The manufacturing of advanced semiconductor chips requires high-purity materials, metals, and a range of process chemicals, such as ultrapure acids and solvents. Wafer manufacturers and chip fabrication plants (FABs) also use a variety of bulk and electronic specialty gases (ESGs) and volatile liquids throughout the manufacturing process (1,2). For example, silane (SiH

) and chlorosilanes are used as feedstocks for gas purification of silicon to the nine to eleven nines purity used to make semiconductor wafers. Nine nines (9N) Si is 99.9999999% pure, meaning there is only 1 part per billion (ppb) of total impurity elements in the solid silicon crystal. Alternative, non-silicon wafers and films are often made from “III-V compound” semiconductors, such as gallium arsenide (GaAs) and indium GaAs (InGaAs), which may use arsine gas (AsH3) as a precursor together with trimethyl gallium (TMG).

Fabricating an advanced IC chip involves upwards of a thousand steps where thin layers of conducting and insulating material are alternately deposited and etched to create the millions of transistors and other features that make up the circuit. Dopant elements are added to modify the conductivity of the silicon substrate or surface film. High-purity gases are used extensively in these “front end” IC fabrication processes. For example, phosphine (PH

) is used to dope the required level of phosphorus into thin-film transistor (TFT) displays. Diborane (B

) is used for boron doping and ion implantation, for growing metal-boron thin films, and as a component of chemical vapor deposition (CVD) of boron-phosphate-silicate-glass (BPSG) films. Fluorocarbon (FC) gases, such as 2-fluorobutane and hexafluoro butadiene, are also used in the electronics industry (for example, in dry etching and for plasma enhanced chemical vapor deposition [PECVD]). The high reactivity and low toxicity of some FC gases means they are also used to clean reaction chambers between fabrication processes. In a typical semiconductor FAB, high-purity gases can make up the second largest material cost after high-purity silicon.

Controlling the level of contaminants in specialty gases is critical to ensuring acceptable chip yield and the performance and reliability of the finished semiconductor device. To understand why contamination is so important, it is useful to consider that advances in semiconductor device technology are driven by reductions in scale. Packing more transistors into a smaller space improves the speed and performance of the chip. The latest chips have circuit features and line widths measured in single nanometers (nm, one millionth of a millimeter), and the presence and position of individual atoms in the silicon crystal lattice can affect the electrical properties of the device. This makes the chips highly susceptible to contamination, so controlling the levels of impurities in the high-purity gases used during fabrication is fundamental to the production process.

Controlling the levels of dopant elements and metallic contaminants in substrate materials, process chemicals, and gases, is a particular industry concern. Dopant elements are deliberately added to the silicon substrate to make it “semiconducting” and control the electrical properties, as illustrated in Figure 1. Silicon has four valence electrons, and pure silicon forms a crystal lattice in which all these electrons are shared with the adjacent silicon atoms. As a result, pure silicon is a poor conductor of electricity. N-type (negative) dopants are Group V elements such as phosphorus, arsenic, antimony, and bismuth, which have five valence electrons, so they add one free electron to the silicon lattice. P-type (positive) dopants are Group III elements, which have three valence electrons, so they contribute a “missing” electron or a positive “hole” in the lattice. The level of doping varies depending on the intended properties, but low-level doping can add as few as one dopant atom per 100 million silicon atoms, so controlling contamination for these elements is critical.

ICP-MS instruments are already widely used in the semiconductor industry to monitor the levels of ultratrace contaminants in high-purity materials and process chemicals. Contaminants in high-purity gases often need to be controlled at lower levels than can be achieved using traditional gas chromatography (GC) detectors, which may not have sufficient selectivity for the required analysis to begin with. Also, for some matrices and compounds, standards are unstable or not available, making calibrating and quantifying contaminants difficult.

As a detector for GC, ICP-MS, particularly with a triple quadrupole or tandem mass spectrometer (MS/MS), can overcome many of the limitations of other GC detectors. GC–ICP-MS can detect and quantify almost every element at low levels in gases and volatile liquids. The technique gives element and isotope-specific analysis, and is highly selective. As a result, matrix components and other compounds have little to no impact on the signal for the target analyte. GC–ICP-MS also gives practically the same elemental signal regardless of the compound, so non-specific or “compound independent” calibration (CIC) can be used, simplifying the quantification of compounds that are unstable or for which standards are not available. GC–ICP-MS is a versatile and sensitive technique that enables semiconductor manufacturers to reliably quantify trace-level contaminants in the wide range of specialty gases and volatile liquids used in semiconductor manufacturing.

Examples of different electronic gas analysis applications are presented in this work. The details of the specific GC columns and methods used for the different sample types and compounds measured are provided in the discussion. Specialty electronic gas analysis using GC–ICP-MS often requires a special approach to sample introduction and analysis, as many of the gases are toxic, flammable, and pyrophoric (spontaneously combust on contact with air or moisture). Silicon compounds such as silanes, siloxanes, and chlorosilanes decompose to form silica, which would deposit in a standard GC injector. Flammable and pyrophoric gases can be analyzed directly from the cylinder, using a switch- ing valve and loop to deliver the required sample volume directly to the column in the GC injector port. A 10-port sampling valve provides the option to automatically add a second gas as an online internal standard, as illustrated in Figure 2. In a similar way, volatile liquids such as trichlorosilane (TCS) can be sampled and injected directly into the GC–ICP-MS system using a high-pressure switching valve (3).

A further consideration when GC– ICP-MS is used to analyze samples such as silanes, chlorosilanes, AsH

) is that these compounds are inorganic. This means the matrix is not easily decomposed and volatilized in the ICP instrument, unlike with hydrocarbon matrices. To successfully analyze these inorganic matrices, a low-volume Deans Switch microfluidic flow control device was used to divert the matrix to vent, preventing excessive matrix load from reaching the ICP. The Deans Switch was positioned in the GC oven in the flow path between the column exit and the ICP-MS transfer line. Once the retention time of the matrix peaks were established using a low volume injection, the Deans Switch was used to divert the matrix to vent during the GC separation.

Both single and triple quadrupole (QQQ) Agilent ICP-MS systems were used for elemental detection. ICP-QQQ is a tandem MS configuration (MS/MS); as a result, it supports the use of reactive cell gases to give more effective control of spectral overlaps. In many cases, analysis can be performed on either type of mass spectrometer, with ICP-MS/MS typically giving detection limits (DLs) that are approximately an order of magnitude lower or more, depending on the element. Because the ICP-MS plasma is “dry” (no aqueous solution), many spectral backgrounds are much lower with GC–ICP-MS than with the conventional liquid sample introduction. As a result, non-typical ICP-MS elements, such as silicon and sulfur, can be measured at their major isotopes using single quadrupole ICP-MS. However, lower DLs are possible if an ICP-MS/MS method is used with a reactive cell gas to further reduce spectral overlaps. For more typical ICP-MS elements, such as germanium and arsenic, part per trillion (ppt) level DLs are routinely achievable. As a multi-element detector, GC–ICP-MS can be used to analyze hydride gas contaminants such as SiH

 at trace level (single ppb down to 10 ppt DLs) in other ultrapure hydride gases (4).

The sulfur compounds, hydrogen sulfide (H

S) and carbonyl sulfide (COS), were determined as ppb-level contaminants in PH

 using a 60-m (2 x 30 m) x 0.53 mm i.d. x 20 μm film mega-bore Plot U GC column at 50 

C with helium carrier gas at 17.5 psig. Sulfur was measured as the 

P matrix peak. The chromatogram is illustrated in Figure 3, demonstrating the complete separation of the sulfur compounds from the PH

 gas. The shaded region of the time plot shows when the PH

 matrix was diverted to vent using the Deans Switch.

 matrix is difficult for organic MS as the two compounds both have their large molecular ion peak at 

 34 and their fragmentation patterns are similar. As illustrated in Figure 4, the only major difference in the GC–TOF-MS spectra is the 

 31, which is not useful for quantifying sulfur. Although H2S is separated from the PH

 matrix peak chromatographically, the tail of the matrix peak causes a raised background for H

S. By contrast, low-level determination of H

 matrix is straightforward using ICP-MS, which is a mass-specific elemental detector that determines 

 is a critical high-purity process gas, in which sulfur compounds and other hydride contaminants must be monitored at trace levels. However, PH

 also needs to be measured as a trace-level contaminant in other gases and volatile liquids used in the semiconductor industry, to avoid unwanted contamination by phosphorus, an 

-type dopant element. Dichlorosilane (DCS) is used as a precursor for high-purity silicon crystal growth, and in combination with ammonia (NH

) to grow silicon nitride (SiN) thin films by CVD. The purity of the silicon crystal or SiN film depends on the purity of the precursor gases, so contaminants including PH

 must be monitored at ppb levels in the DCS. PH

 was determined in DCS by GC–ICP-MS/MS using a 60 m x 0.32 mm x 5.0 μm film DB-1 column using helium carrier gas at 20 psig. The oven program had a starting temperature of 35 

C/min ramp and a final temperature of 220 

Figure 5 shows overlaid chromatograms for a PH

 being determined as a trace contaminant in DCS. The PH

 contaminant was quantified at 4.8 ppb. The use of ICP-MS/MS with O

 47]) enabled low-level phosphorus analysis with minimal contribution from the silicon matrix.

GC–ICP-MS was used to measure higher boranes as trace impurities in high-purity diborane (B

) using a 60 m x 0.32 mm x 1.5 m Rtx-Volatiles column. The oven program used a 35 

C/min ramp and a final temperature of 240 

C. Tolerances for diborane purity are extremely tight (<1% variation), so accurate measurement of higher boranes is essential for production control. Standards are not available or stable for higher boranes, but GC–ICP-MS provides a solution, as illustrated in Figure 6. The chromatogram shows the separation and quantification of tetraborane (B

 matrix. The higher boranes were quantified at single ppm levels in 2% B

 using CIC against a diborane standard. Quantification by CIC is based on the consistent response for elemental boron and a simple mathematical correction for the different amounts of boron in the different compounds. ICP-MS also offers isotope specific measurement, which is beneficial in this application, as some uses of borane require B

 that is isotopically enriched up to 99.99% B-11. The 

B isotopic abundances can easily be measured using GC–ICP-MS.

ICP-MS and ICP-QQQ are well-established techniques that are widely used in the semiconductor industry for measuring ultratrace levels of elemental contaminants in wafer substrate materials, metals, and process chemicals. Combining ICP-MS with GC extends the use of the technique to monitoring contaminants in bulk, specialty gases, and volatile liquids. These sample types require special GC sampling approaches, as many of the samples are toxic, flammable, or pyrophoric. Such samples can be handled safely using an appropriate switching valve or loop injection gas sampling system.

As a sensitive, multi-elemental detector for GC, ICP-MS gives low detection limits, mass-specific measurements, and excellent selectivity. GC coupled to a triple quadrupole ICP-MS system enables trace-level analysis of compounds containing elements traditionally considered difficult to determine by ICP-MS, such as silicon and phosphorus. The high temperature of the ICP ion source means that ICP-MS gives nearly equal elemental response regardless of the compound’s composition. This enables the use of compound independent calibration, which is useful when 

compound-specific calibration standards are unstable or unavailable.

Trace Analysis of Specialty and Electronic Gases

(2) Linde. Electronic Special Gases Index. 

High-Pressure Liquid Injection Device for Agilent Gas Chromatographs

(4) Geiger, W. M.; McElmurry, B.; Anguiano, J.; Kelinske, M. 

Determination of Trace Impurities in Electronic Grade Arsine by GC-ICP-QQQ

 is with Consolidated Sciences (CONSCI) Ltd., in Pasadena, Texas. 

 are with Agilent Technologies, Inc. Direct correspondence to 

Manufacturing advanced electronic devices requires the production of high-quality semiconductors and integrated circuit chips. In this article, the authors explain how GC, when coupled with ICP-MS, enables the detection of elements that are essential in semiconductor production.

GC-ICP-MS for Process and Quality Control in Semiconductor Manufacturing

In this study, 48 different wild and cultivated mushrooms were purchased in local markets in Madrid and provided by trusted specialists from the Mycological Society of Madrid. Cadmium, mercury, and lead were determined by inductively coupled plasma–mass spectrometry (ICP-MS) in samples previously digested in a microwave oven. Methylmercury (MeHg) was determined by liquid chromatography separation (HPLC) combined with ICP-MS after microwave-assisted extraction. Regarding the cadmium and lead contents, all the samples complied with the maximum permitted limits. For mercury, a single sample exceeded the maximum value established in Regulation (EU) 2018/73 for wild mushrooms. In the case of methylmercury, the highest amount represents 13% of the total mercury in one sample. In the rest of the cases, the concentration found was negligible.

Historically, fungi have been classified as inferior plants in the division 

 because of their similarities to other members of that group (no roots, no leaves). However, later studies demonstrated unique features that led to the formation of the new kingdom, the Kingdom Myceteae, which includes microscopic and macroscopic species of fungi. Mushrooms are described as large multicellular fungi with stems and caps. According to Chang and Milles, mushrooms can be simply defined as “macro fungi with a distinctive fruiting body and large enough to be seen with the naked eye and to be picked by hand” (1).

Edible mushrooms have always been a popular food in diets around the world. Their texture and flavor make them highly appreciated in the kitchen. Consuming mushrooms brings multiple benefits because they are rich in protein, dietary fiber, vitamins and minerals, and low in fat. In addition, they contain antioxidant compounds such as polysaccharides, terpenoids, and polyphenols, all of which have a positive impact on health (2,3)

However, the scientific literature shows that mushrooms can accumulate heavy metals present in the soil. The fungal mycelium plays an essential role in this, which spreads over the available area and incorporates metal ions into its cytosol under suitable conditions. Mushrooms are able to accumulate higher concentrations than plants because, unlike their roots, their mycelium develops mainly horizontally, normally occupying the most superficial part (5–10 cm) (4). The mycelium contains the highest concentration of heavy metals (5). Although this characteristic makes them extremely useful as bioindicators of contaminated areas (6), edible mushrooms may pose a risk to consumers if the heavy metal content exceeds reference levels.

The main factors that influence the bioaccumulation of heavy metals in these organisms are environmental factors, which are the concentrations of metals in the soil, the pH, the amount of organic matter and contamination by atmospheric deposition, and intrinsic fungal factors of the species, such as its structure, biochemical composition, decomposition activity, and development of mycelium and fruit bodies (7,8).

Among the heavy metals, it is worth mentioning mercury, cadmium, and lead, which are considered toxic elements for human health by the European Food Safety Authority (EFSA).

Mercury is a toxic heavy metal that can bioaccumulate not only in marine organisms (mainly swordfish and sharks), but also in fungi. There has always been significant concern about its exposures, intakes, and toxicity. In the environment, mercury can be found in three different chemical forms: elemental/metallic mercury (Hg

), and organic mercury (organometallic compounds where one or two alkyl/aryl substituents are attached to the mercury atom). These forms present different bioavailabilities and toxicity, with methylmercury being the most dangerous in the food chain (9).

To perform an accurate risk assessment, it is necessary to collect as much information as possible on the presence of these forms in food because exposure to methylmercury can cause harm by interacting with the sulfhydryl (-SH) groups distributed throughout the body, causing the well- known Minamata disease (6).

The central nervous system is the most important target of methylmercury, which can cause different symptoms, including sensory disturbances in the hands and feet, ataxia, and visual impairment. In addition, organic mercury can reduce the activity of natural killer (NK) cells and generate free radicals. Acute inorganic mercury poisoning affects the gastrointestinal tract and kidneys, causing severe diarrhea, vomiting, and renal tubular necrosis with anuria (6).

In the 2012 EFSA opinion, the tolerable weekly intake (TLWI) for methylmercury was updated to 1.3 μg/kg bw and 4.0 μg/kg bw for inorganic mercury, which is the maximum amount that a person can ingest weekly throughout their lives without adverse effects (9).

European legislation establishes maximum Hg contents in mushrooms. Commission Regulation (EU) 2018/73 distinguishes between cultivated and wild mushrooms, establishing maximum limits of 0.050 mg/kg and 0.50 mg/kg of Hg, respectively. It also establishes a maximum limit of 0.90 mg/kg in 

Cadmium is also a heavy metal that poses serious risks to human health. Until recently, it has not been possible to demonstrate that cadmium has any physiological function, so its main interest lies in its high toxicity in humans, especially in its inorganic forms (11).

Various studies have shown that exposure to cadmium through diet can cause harmful effects on health, mainly in the kidney, spleen, and liver where it accumulates (12). In addition, it is currently classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1), as an association between its intake and the risk of postmenopausal endometrial cancer has been demonstrated. As a result of its ability to induce oxidative stress, it can also affect male fertility by decreasing sperm quality (13).

Prolonged ingestion of cadmium also poses a serious health risk, by causing Itai-Itai disease, the best-known case of which was the mass poisoning that took place in Japan in the last century. This disease is characterized by creating bone damage due to osteoporosis and osteomalacia, in addition to severe kidney damage (14,15). Derived from a comprehensive toxicological assessment, EFSA has established a tolerable weekly intake (TSI) for cadmium of 2.5 μg/kg body weight (16).

In 2023, the maximum cadmium content in mushrooms has been updated by Regulation (EC) No 2023/915, establishing the maximum limit at 0.050 mg/kg for cultivated mushrooms, at 0.15 mg/kg for the mushrooms 

 (oyster mushroom), and 0.50 mg/kg in wild mushrooms (17).

Finally, lead is another heavy metal to consider. Lead contamination arises primarily from the environment, through atmospheric lead through deposition on agricultural crops, from water, or from food processing, handling, and packaging. Although lead exists in both organic and inorganic forms, only inorganic lead has been detected in food (18). When lead is ingested, it is distributed to certain organs such as the brain, kidneys, liver, and bones, causing tissue damage. After several weeks, most of it moves to the bones and teeth, where it can be stored for decades (19,20).

Accumulated lead can return to the blood, reaching vital organs, resulting in episodes of broken bones in the elderly. During pregnancy, accumulation of lead can lead to possible effects on the fetus and lactation (20).

Lead poisoning produces adverse effects at the central nervous system level, which can affect brain development and cause behavioral changes such as reduced attention span and increased antisocial behavior. In addition, it can lead to anemia, hypertension, renal failure, immunotoxicity, and toxicity to reproductive organs (19).

Due to the lack of sufficient scientific information, in 2010 the existing toxicological safety threshold for lead was withdrawn, and a new one could not be determined. Therefore, there is currently no recommended tolerable intake for lead (21).

As of this year, the maximum lead content in mushrooms has been updated by Regulation (EC) No. 2023/915, establishing the maximum limit at 0.30 mg/kg for 

 (shiitake mushroom) mushrooms, and 0.80 mg/kg in wild mushrooms (17).

The main objective for this study is to provide information on the content of cadmium, lead, mercury, and methylmercury in both cultivated and wild mushrooms and to compare the results obtained with the limits established in current legislation. For this, a total of 48 mushrooms were obtained and analyzed.

For the analysis of cadmium, lead, and total mercury, the inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies model 7900) was used. In addition, an integrated sample introduction system (ISIS 3) and an automatic sampler (SPS 4) were used in the determination together with the ICP-MS. The operating parameters of the ICP-MS analysis are shown in Table I.

Methyl-mercury determinations were performed using a liquid chromato- graph (HPLC) (Agilent Technologies 1200 Infinity), consisting of a model 1260 Quaternary Pump VL, a model 1260 ALS injector, and a model 1260 TCC column compartment. This HPLC system is coupled to the ICP-MS described above. The characteristics are detailed in Table II.

For digestions and extractions, a DigiPREP Jr digestion block from SCP Science with 24 positions was used. The volumetric material used for all the solutions were DigiTubes, polypropylene tubes single-use, except in the case of MeHg analysis, which opted for centrifuge tubes, also single-use, VWR Metal Free, 15 mL, and volumetric flasks of 1 L and 200 mL capacity. To take the cor

responding aliquots, Eppendorf brand micropipettes were used.

For centrifugation of the samples, a Lisa refrigerated centrifuge of AFI groups was used.

For the preparation of the reagents, dilutions, washing, and rinsing solution of the work material, deionized water (H

O) of Class Type I (according to the UNE-EN-ISO 3696 Standard) was used, purified with the Milli-Merck’s Q Comprehensive. Nitric acid (HNO

, 65%) from Scharlau was distilled in the laboratory using Savillex equipment, model DST-1000. Suprapur hydrochloric acid for trace analysis (HCl, 37%) supplied by Scharlau and hydrogen peroxide (H

Regarding the ICP-MS, the tuning solution containing 1 μg/l Ce, Co, Li, Mg, Tl, and Y was used. Finally, argon and helium (99.999%) for plasma generation and interference removal.

Standards of 1000 mg/L of Cd (Scharlau), Pb (VHG-Labs), Hg (Scharlau), and MeHgCl (VHG-Labs) were also used. For the determination of cadmium, lead, and total mercury, the intermediate solutions and the calibration curve were prepared with a 2% HNO

As an internal standard, a 0.4 mg/L iridium and rhodium solution (from a 1000 mg/L Scharlau standard) was prepared in a 2% HNO

, 1% HCl, and 5% acetic acid solution for the determination of cadmium, lead, and total mercury. To stabilize the signals, 0.4 mg/L Au from SCP Science was added. No internal standard was used for the methylmercury speciation method.

The composition of the mobile phase was 1 g/L L-cysteine/20 mM CH

/20 mL MeOH in deionized water, with a pH of 6.9, using L-cysteine from Acros Organics (minimum purity 98%), ammonium acetate (CH

) from PanReac AppliChem (96% purity), and methanol (MeOH) from Scharlau (GC residue analysis grade, 

In the case of the mercury speciation analysis, the solution used for the extraction was 5 g/L L-cysteine/0.5% HCl, and the solution used for the preparation of all the intermediate solutions and the calibration curve was the mobile phase.

The samples were homogenized by crushing with a mincer, until a representative sample of the product to be tested was obtained. Two different methods were carried out in this study. Both represented in Figure 1.

Regarding the total analysis of mercury, cadmium, and lead, first 0.5 ± 0.05 g of sample were weighed in a DigiTube, then 3 mL HNO

 were added, and the samples were digested in the DigiPrep afterwards. Once the digestion was complete and the samples had reached room temperature, they were made up to volume in 50 mL with deionized water. The sample collection were analyzed by ICP-MS, samples with high analyte contents that were diluted previously.

For methylmercury speciation, 0.2 ± 0.02 g of sample was weighed into a DigiTube, 10 mL of extraction solution (1 g L-cysteine; 5% HCl) was added, and extraction was performed on the DigiPrep for 30 min at 60 

C (Figure 2). Once the process was finished, the samples were allowed to cool to room temperature, transferred to 15 mL tubes, and centrifuged for 10 min at ≥ 4000 rpm and 4 

C. Subsequently, the supernatant was filtered using 0.45 μm pore size filters and placed in 1-mL polypropylene vials to be injected into the HPLC system.

Figure 3 shows a chromatogram where you can see the separation of the peaks obtained, where the first corresponds to Hg, with a retention time of 2.384 min, and the second to MeHg, with a retention time of 3.868 min.

For the calibration curves used in the quantification of the analytes, the 

-axis represents the value of the ratio, calculated as the number of counts of the analyte divided by the number of counts of the internal standard, whereas the 

-axis represents the concentration in μg/L. In the case of MeHg, the 

-axis represents the number of counts per area of the chromatographic peak.

Figures 4, 5, 6, and 7 show the calibration curves used in the quantification of the samples.

The test method used for the determination of heavy metals meets the criteria of Regulation (EC) 333/2007 of the Commission of March 28, 2007, which establishes the sampling and analysis methods for the control of levels trace elements and process contaminants from food products and their modifications. The validation was carried out according to the requirements of the UNE/EN-ISO 17025 Standard for Testing Laboratories, and it is accredited by the National Accreditation Entity (ENAC). For MeHg determination, the test method is in the accreditation process.

A total of 48 wild and cultivated mushrooms were acquired and analyzed. All of them were purchased in local markets in Madrid and provided by trusted specialists in the manual collection of mushrooms from the Mycological Society of Madrid.

The results obtained in the study of cadmium, lead, and total mercury of the 48 samples analyzed are shown in Table III.

Regarding the cadmium and lead contents, all the samples comply with the maximum permitted limits, and do not seem to pose a risk to the population.

Regarding the total mercury content, it should be noted that a single sample (

 with a content of 0.97 mg/kg) exceeded the maximum value established in Regulation (EU) 2018/73, set at 0.50 mg/kg for wild mushrooms. No sample of 

 reached the maximum allowed limit of 0.90 mg/kg for said species. In the case of samples of cultivated mushrooms, it is observed that their Hg content is less than 0.050 mg/kg established by the aforementioned regulation.

According to the data obtained, it can be observed that cultivated mushrooms have significantly lower concentrations of metals than wild mushrooms.

The results of the methylmercury determination are shown in Table IV. In this case, only the samples with a significant total mercury content were analyzed. Speciation was performed in the 10 samples that exceeded 0.075 mg/kg of total Hg, all of them wild. The highest amount of MeHg found was 0.12 mg/kg in a sample of 

, which represents 13% of the total mercury in the sample.

It can be determined that the test method used for the analysis of Cd, Hg, and Pb by acid digestion and determination by inductively coupled plasma–mass spectrometry is considered a valid option to carry out a study, such as the one presented here, obtaining results in accordance with the requirements of ISO 17025.

In the same way, the coupling of a liquid chromatograph to an ICP-MS is a recommended method to perform the chromatographic separation of the different mercury species, and to be able to achieve the methylmercury analysis. This method is in the accreditation process.

After determining the content of cadmium, lead, and total mercury and the speciation of methylmercury in the samples analyzed, it can be concluded that the amounts found in the case of cultivated mushrooms are lower than the limits established in European legislation, whereas in the case of wild mushrooms, a result was obtained above the established limits, which may mean a risk to the population.

The values obtained by performing the mercury speciation reveal that the mercury content in the mushrooms is almost entirely inorganic mercury and the amounts of methyl mercury found are practically negligible, becoming, in the worst case, less than 13% of total Hg.

We would like to thank the Mycological Society of Madrid for its invaluable role in providing us with samples of wild mushrooms in order to develop the study of their metal content.

Mushroom: Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact

(2) Gupta, S.; Summuna, B.; Gupta, M.; Annepu, S. K. 

; Springer International Publishing AG, 2018; pp 1815–1847.

(3) Heleno, S. A.; Barros, L.; Martins, A.; et al. Chemical Composition, Antioxidant Activity, and Bioaccessibility Studies in Phenolic Extracts of Two Hericium Wild Edible Species. 

(5) Berthelsen, B. O.; Steinnes, E. Accumulation Patterns of Heavy Metals in Soil Profiles as Affected by Forest Clear-Cutting. 

(6) Bernhoft, R. A. Mercury Toxicity and Treatment: A Review of the Literature. 

(7) García, M. A.; Alonso, J.; Fernández, M. I.; Melga, M. J. Lead Content in Edible Wild Mushrooms in Northwest Spain as Indicator of Environmental Contamination. 

(8) Lalotra, P.; Gupta, D.; Yangdol, R.; Sharma, Y. P.; Gupta, S. K. Bioaccumulation of Heavy Metals in the Sporocarps of Some Wild Mushrooms. 

https://www.efsa.europa.eu/en/efsajournal/pub/2985

(10) European Commission. Commission Regulation (EU) 2018/73 of 16 January 2018 Amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as Regards Maximum Residue Levels for Mercury Compounds in or on Certain Products. 

https://eur-lex.europa.eu/collection/eu-law/consleg.html

(11) Godt, J.; Scheidig, F.; Grosse-Siestrup, C.; et al. The Toxicity of Cadmium and Resulting Hazards for Human Health. 

(12) Kalač, P.; Svoboda, L.; Havlíčková, B. Contents of Cadmium and Mercury in Edible Mushrooms. 

(13) Ciobanu, C.; Şlencu, B. G.; Cuciureanu, R. Estimation of Dietary Intake of Cadmium and Lead through Food Consumption. 

(14) Satarug, S. Dietary Cadmium Intake and Its Effects on Kidneys. 

(15) Inaba, T.; Kobayashi, E.; Suwazono, Y.; et al. Estimation of Cumulative Cadmium Intake Causing Itai–Itai Disease. 

(16) Cadmium in Food—Scientific Opinion of the Panel on Contaminants in the Food Chain. 

(17) European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No. 1881/2006. 

Evaluation of Certain Food Additives and Contaminants: Seventy-Third Report of the Joint FAO/WHO Expert Committee on Food Additives

; WHO Technical Report Series; 960; Geneva, Switzerland, 2011.

(19) World Health Organization. Lead Poisoning and Health. 2022. 

https://www.who.int/news-room/fact-sheets/detail/lead-poisoning-and-health

(20) Agency for Toxic Substances and Disease. Public Health Statement Lead CAS# 7439-92-1. 

https://wwwn.cdc.gov/TSP/PHS/PHS. aspx?phsid=92&toxid=22, 2007

(21) Scientific Opinion on Lead in Food. 

 is with the Public Health Laboratory, in Madrid, Spain. Direct correspondence to: 

The bioaccumulation of heavy metals can lead to disastrous effects on the environment and human health. Here, ICP-MS was applied to study 48 different mushrooms collected at local markets in Madrid, Spain, showcasing its effectiveness at detecting heavy metals in edible fungi.

Study of Heavy Metals and Methyl-Mercury in Fungi in Markets of Madrid

Because cells are heterogeneous materials appearing in many sizes and containing many different elements depending on the model, inductively coupled plasma–mass spectrometry (ICP-MS) is often used to conduct element analysis of single cells. Maria Montes-Bayón of the Faculty of Chemistry at the University of Oviedo (Asturias, Spain) has been working with single cell ICP-MS (sc-ICP-MS) to study the uptake and apoptotic status of nanoplatinum (IV) treated cells, specifically selenized yeast. Montes-Bayón recently spoke to 

What makes ICP-MS an effective technique in your experiments for single cell analysis?

Single cell -omics is a growing field of research, since cellular heterogeneity is what triggers the beginning of many diseases. Among single cell -omics techniques (such as genomics, trancriptomics, and proteomics), metallomics is also playing an important role, since metals are key regulators of cell functions. In addition, the monitoring of the differential uptake of drugs containing metals by individual cells might explain their effectiveness. The evaluation of the intracellular content of different elements in individual cells can be performed by ICP-MS using the previously established concept of single particle ICP-MS, considering some critical points (a cell is more sensitive to the analysis process than a particle). Through the years, we have been evaluating the suitability of different ICP-MS based strategies for single cell analysis and I would say we have found the most successful combination for the sensitive and sequential determination (with triple quadrupole-based mass analyzers) of single elements in single cells.

You’ve recently worked with selenized yeast as a reference standard. Why did you choose this standard, and what are the challenges involved in using these cells as a reference standard?

Different approaches are available for individual introduction into ICP-MS. To address the suitability of every system, different authors use different strategies. Reference materials are generally used to “validate” analytical results, but it is uncommon to have reference standards specifically for “cell-based” materials. We realized that selenium enriched yeast certified reference material, or SELM-1, was a yeast material that was certified in total Se content. While talking to microbiologists at our university, they noted that yeast cells were robust enough to be intact over time, and if preserved in the correct way, they should be alive. Thus, we started a multiple-techniques study on the suitability of the reference material (for example, using confocal microcopy of labeled cells, flow cytometry, and ICP-MS analysis). We also found it necessary to address the differential Se content intra- and extracellularly.

Are there any specific sample preparation and analysis conditions that are required to be successful in this analysis?

Indeed, we realized the importance of matrix effects when using ICP-MS. It seems strange that often something so well-known for so many years is not considered carefully when conducting this type of analysis. The paper we published establishes that the reference material can be used as such for sc-ICP-MS if carefully washed a few times and then dried before analysis. If this procedure is not followed, the accuracy of the results might be compromised when using external calibration.

Are there any sample cell types that are easier or more difficult to analyze, whether you learned this through testing or presume it from past experiences?

Yes. In our experience (our first paper is from 2017), we have researched yeast cells (

,and human cells (A2780, A2780cis, OV-CAR3, A549, MCF-7, MDA-MB 231, STR-747, HepG2 corresponding to ovarian, lung, breast, and liver cancer cells). The first two models (yeast and bacteria) are much more resistant to the nebulization and transport into the plasma so the chance for leaking of the intracellular content into the sample matrix is minimized. With human models, it is much more challenging due to the delicate composition of the cell membrane that can be easily disrupted (3). In some cases, we fix the cells to minimize disruption, but this can be risky if constitutive elements need to be analyzed.

How does this work differ from what has been previously done by yourself or others?

Spectroscopy spoke with Montes-Bayón of the University of Oviedo about her work with single cell ICP-MS to study the uptake and apoptotic status nanoplatinum treated cells.

ICP-OES and ICP-MS Techniques for Today's Spectroscopists

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Study of Heavy Metals and Methyl-Mercury in Fungi in Markets of Madrid

Feature: Toward Normalization of Quantitative Single Cell ICP-MS Experiments