Trace-level multiple-element analysis through inductively coupled plasma–mass spectrometry (ICP-MS) provides suitable support for biopharmaceutical upstream process development and batch quality control. To set up a platform method for trace-level multiple elements in industrial cell culture media, scans of all relevant elements of fed-batch cell harvests from various mammalian cell lines were initially obtained and the distinct presence of major elements was observed. After an initial scan, 14 target elements were sorted into two analysis modes based on their specific concentration levels in the harvests. Following this sorting process, multiple-element standard curves with particulate ranges of each element were constructed. Results using various mammalian cell line harvests validated this method of trace-element analysis and provided a theoretical basis for metabolic pathways in distinct cell lines, based on differentiating elemental uptake patterns. Moreover, this multiple-element analysis demonstrated the efficiency of using multiple-element standards for quick result turnover. These results show the capability of ICP-MS to monitor multiple trace elements with high sensitivity in various industrial cell culture media.

	The importance of inductively coupled plasma–mass spectrometry (ICP-MS) for trace-level elemental quantification has been affirmed through increasing interest and research within the field of environmental science and human clinical studies. The analysis of trace-level elements has expanded significantly into the pharmaceutical industry because of the growth of biologics in clinical trials and as commercial therapies, in many cases addressing previously unmet medical needs. These therapeutic proteins are large molecules of significant complexity because of protein secondary and tertiary structure as well as post-translational modifications. The complexity arises, in part, as the proteins are produced by mammalian cells during growth over many days in proprietary growth media. The media contain nutrients for optimal growth and influence expression of the target protein. Of particular interest, several trace elements (such as Mn

) have been reported to improve glycosylation profiles of final products in Chinese hamster ovary (CHO) cells, by acting as a cofactor in a mechanism with β-1,4-galactosyltransferase (1). Sensitive instrumentation such as ICP-MS could be of great benefit in analyzing trace-level elements in spent media originating from different industrial cell lines. This technique naturally leads to facilitating cell culture process optimization by ensuring that critical product quality attributes, such as glycoforms, meet the standard of innovator drugs.

	Based on reported studies about trace-element concentrations in human serum, a minimum sample volume of 500 µL is required for each sample (2,3). In some publications, the sample preparation approach is to use a digestion (4). The primary advantage of using the digestion method is to achieve complete decomposition of the matrix. However, the digestion procedure can introduce random contamination and volatile elements that interfere with the interpretation of results. In this work, we eschew the digestion process and implement a “dilute-and-shoot” method instead. Based on the characteristics of drug substance samples, appropriate dilutions for each sample are determined and applied. This approach provides the advantage of a minimum sample preparation volume requirement, which may be as low as 100 µL. With the dilute-and-shoot approach, sample preparation time is significantly reduced and possible sources of contamination are minimized. Moreover, assay cost is significantly less than the digestion method because it avoids microwave digestion during sample preparation.

	It is noted that this method can result in buildup of sample residues on the MS nebulizer and sample introduction line, requiring frequent cleaning of both the nebulizer and pump tubing. Moreover, a standard minimum dilution limit must to be set to ensure the nebulizer and sample introduction lines are not saturated by protein matrices. After considering results obtained from internal work regarding analysis of trace-element concentration in CHO cell culture media, the minimum dilution of each sample preparation was defined as 1:100 in this study.

	The analytical approach used for this experiment was validated by performing an accuracy check and a precision spike study. The method was then performed to assay a wide range of concentrations of the following elements of interest: Mg, Cr, Mn, Fe, Co, Ni, Cu, As, Se, Mo, Sb, Ba, Tl, and Pb. These elements originated from two common industrial cell lines, designated in this study as cell line 1 and cell line 2.

	The entire study was performed using an Agilent 7700 quadrupole ICP-MS system (Agilent Technologies). The instrument parameters are summarized in Table I. The 14 elements were analyzed using ICP-MS in two separate modes. Most of the elements of interest were analyzed using standard mode. However, for Fe analysis, He mode with a collision cell was used to eliminate intense plasma- and polyatomic-based interferences such as 

	Standards and samples were prepared with high purity water that had a resistivity of 18.2 MΩ. Trace metal–grade nitric acid (Fisher Chemical) and ICP-MS trace-metal standards (Inorganic Ventures) were used in all standard preparations. To facilitate the monitoring of instrument parameters and the stability of target elements during the analysis, an internal standard mixture consisting of Li, Sc, Y, In, and Bi was added to each sample at a 2.5-µg/L level. The data are presented in Figure 1.

	For initial scanning, semiquantitative (SemiQuant) mode was used with ICP-MS (5). For full quantitative (Full-Quant) mode based on distinct levels of each element, specific standard curve levels were designed as presented in Table II.

	Samples from two typical industrial cell lines were chosen for this trace-element study, cell line 1 (mammalian) and cell line 2 (human). Both cell lines typically have a two- to three-week bioreactor process before harvest. A spike study was performed with trace-level elements on log phase (day 3) to stationary phase (day 13) samples from both cell lines. To investigate the contribution of matrix effects, trace-element standard mixture was spiked into each sample at a 1-µg/L level. Bioreactor sample aliquots were centrifuged at 14,000 rpm (20,817 RCF) for 5 min to precipitate any cell debris before assay. Samples were serially diluted twice at a factor of 1:10 with 2% nitric acid in high purity water, for a total dilution of 1:100. For each sample, a further 1:4000 dilution was performed using the same diluent to capture highly abundant elements in cell media (for example, Mg, Fe, and Cu). After preparation, samples were centrifuged at 3500 rpm (1301 RCF) for 5 min before analysis. 

	Semiquantitative-mode measurement with ICP-MS was performed initially, with full scanning of all possible elements in the samples. Notably, results show that more obvious concentration changes of Mg, Cu, Se, and Ba were found from day 3 and day 13 samples. Based on these initial data, a list of 14 elements was selected, which included the critical elements listed above (Mg, Cu, Se, Ba) as well as others considered to be of interest, but in reduced amounts (Cr, Mn, Fe, Co, Ni, As, Mo, Sb, Tl, and Pb). 

	The calibration ranges investigated for each element are summarized in Table II. Given the significantly higher abundance of 

Mg in mammalian cell media observed in previous experimental results, both calibration ranges for these elements began at a level of 0.25 μg/L, while the range for the other ultratrace elements began at a level of 0.05 μg/L. Calibration curves for all 14 elements were constructed with the calibration blank as zero and a six- to eight-point calibration series. Results for each isotope, calibration range, and linearity are also reported in Table II. The 

 values for all 14 elements were ≥0.9998. With the exception of Fe, all the elements of interest were analyzed using standard mode. He mode (with a collision cell) was performed to analyze Fe. 

Se consistently presented higher polyatomic interference with other elements and had corresponding higher counts than estimated (6).

	Table III presents the instrumental background equivalent concentration (BEC), method limit of detection (LOD), and method limit of quantification (LOQ) for all 14 elements in this study. BEC was measured directly by ICP-MS during the run, method LOD was calculated as 3.3 times the standard deviation of triplicate analyses of the calibration blank, and the method LOQ was calculated as 10 times the standard deviation of the triplicate analyses of the calibration blank (7). To summarize, the instrument BEC for all elements was within the range of 0.006–0.359 μg/L, and the method LOD was demonstrated to be in the range between 0.002–0.086 μg/L.

	The accuracy and precision of this method was confirmed by triplicate standard checks and sample spike studies. Table II illustrates the check standard recovery (%) for all investigated elements based on ICP-MS standard mode and He mode, respectively. The range of recovery percentage (94.6–105.4%) demonstrated acceptable accuracy for all calibrants. Upstream samples obtained from 2-L bioreactors containing cells from cell line 1 and cell line 2 were used in this study to investigate matrix effects. Early phase (day 3) and late phase (day 13) samples were chosen to compare elemental uptake by the cells during specific growth phases. The multiple standard solution (1 μg/L) was spiked into each sample before assay to measure the related recovery. 

	Table IV summarizes the results of all spike recoveries (%). The spike recovery values were within the range of 65–125%, indicating the capability of this method to recover successfully in the presence of matrix. Based on specific concentration levels in cell culture media, some abundant elements such as 

Fe were analyzed using a 1:4000 dilution. Elements such as Co and Cu were analyzed by larger dilutions within cell line 2 samples and smaller dilutions in cell line 1 samples.

	Compiled results for investigated elements in each cell line media are displayed in Figure 2. For a better comparison, 

Cu were grouped together and all other elements were combined. Among all the elements, 

Se concentrations were significantly decreased with process time in both cell lines. Specifically, 

Ni had much higher concentrations in cell line 1 media than in cell line 2 media and the concentration of 

Se was much higher in cell line 2 media than in cell line 1 media (in day 3). On the contrary, 

Ba concentrations were increased from log phase to stationary phase in both cell lines. In addition, the 

Mg concentration had an upward trend in cell line 1 media but exhibited a downward trend in cell line 2 media. Similarly, a slight increase of 

Cu in cell line 1 media was evident, but a significant decrease in cell line 2 media was observed from the results.

	The calibration results shown in Table II were linear over the measured concentration ranges for each element with 

 values of no less than 0.9998. Based on the percentage of check standard recovery, we can conclude that this analytical method is able to analyze all elements of interest without significant mass interferences. Noticeably, the spike recovery (%) of 

Fe shown in Table IV was lower than other elements in both cell lines. He mode with a collision cell was performed in Fe analysis with the intent to eliminate 

 and other polyatomic interferences. However, there are other potential sources of interference including adjacent elements such as Mg and Cu. These potential interferences may have contributed to the low spike recovery for Fe. Furthermore, high-density polyethylene (HDPE) centrifuge tubes that contained samples may introduce leachable trace elements during a typical storage period (8). Another possible reason for low spike recovery of 

Fe could be high salt and carbon effects. As observed in the semiquantitative-mode results, significantly high amounts of 

S were observed in both cell lines. The high concentrations of those elements increase noise levels in the measurement environment, which presents increased difficulty for discernment of the 

Fe signal. To reduce potential matrix effects in cell culture media of future studies, 0.01% Triton X-100 or 0.01% NaCl solution can be added to the diluent.

Mg concentration increased from log phase to stationary phase in cell line 1. On the contrary, 

Mg concentration in cell line 2 decreased from log phase to stationary phase. This difference may result from metabolic pathways unique to each cell line, which produces differing Mg uptake in facilitation of their metabolism. In addition, 

Se concentration in both cell lines was decreased in relation to cell growth, whereas much more significant uptake of 

Se was observed in cell line 2. Previous publications about Se (9) have reported an effect of Se on cell cycle and apoptosis. Therefore, a higher requirement of Se in cell line 2 may have a larger effect on cell viability and longevity, which correlates quite closely with product titer.

	We have demonstrated that a dilute-and-shoot method combined with quadrupole ICP-MS is suitable to accurately analyze Mg, Cr, Mn, Fe, Co, Ni, Cu, As, Se, Mo, Sb, Ba, Tl, and Pb in two industrial cell lines using low sample volumes. The minimal required volume may be as low as 100 μL for each sample prep, which is significant for industrial researchers because it preserves precious sample. This method demonstrated the capabilities of ICP-MS and its functionality of analyzing trace-level elements simultaneously in two typical industrial cell lines. This assay has the potential to significantly accelerate the optimization of upstream processes in the biopharmaceutical industry and increase analytical testing efficiency.

	This research was supported by the Process Development and Quality Control laboratories at Cook Pharmica LLC. 

		W.O. Moellmer, T.G. Miller, S. Wilbur, and E. Soffey, 

		G. Kapadnis, “Analysis of Extractable and Leachable Metals in Plastic Materials of Construction as Per USP <661.1> Acid Extraction Procedure Using The Agilent 5110 VDV ICP-OES,” Agilent Technologies, 2016.

Wei Hong, Ilyas Holmes, Todd Stone, Robert Henry, Victor Vinci

 are with Cook Pharmica LLC, in Bloomington, Indiana. Direct correspondence to: 

Articles

This article demonstrates that a dilute-and-shoot method combined with quadrupole ICP-MS is suitable to accurately analyze trace elements in two industrial cell lines using low sample volumes.

ICP-MS Analysis of Multiple Trace Elements in Industrial Cell Lines

This article focuses on the use of a cell-based inductively coupled plasma–mass spectrometry (ICP-MS) approach that utilizes ion–molecule chemistry to reduce many of the traditional spectral interferences seen in the analysis of high-purity hydrochloric acid used in manufacturing integrated circuits and semiconductor devices. In addition, a solid-state radio frequency (RF) generator allows the use of lower powers to reduce formation of argon-based polyatomic interferences while still maintaining strong ionization conditions. The combination of controlled gas-phase reaction chemistry with the unique RF generator results in the elimination or significant reduction of many of the problematic chloride-, water-, and argon-based polyatomic spectral interferences, allowing the determination of a suite of 43 ultratrace elements in semiconductor-grade hydrochloric acid by following the validated analytical protocols described in the Semiconductor Equipment Manufacturers International (SEMI) Tier C guidelines.

	The semiconductor industry provides one of the most demanding application areas for inductively coupled plasma–mass spectrometry (ICP-MS) with regard to the detection capability requirements. Consumer demand for smaller electronic devices and more compact integrated circuits has resulted not only in the need for lower trace metal contamination levels on the surface of silicon wafers, but also in the high-purity chemicals and gases used in various stages of the semiconductor manufacturing process. To reduce costs and increase yield, chip manufacturers are making larger diameter wafers with ever smaller chip components. This trend, which is being driven by initiatives like the International Technology Roadmap for Semiconductors (ITRS) (1), is setting the course for the next generation of semiconductor devices and has resulted in requirements for lower and lower trace-element contamination levels in all semiconductor-related materials. Whereas 20 years ago the Semiconductor Equipment Manufacturers International (SEMI) organization deemed that 10-ppb (µg/L) purity levels were adequate for many of the process chemicals, today Tier C levels of 100 ppt (ng/L) are typical-and for some of the more critical chemicals, 10-ppt (ng/L) levels are required (2).

	Tier C refers to third-generation guidelines generated by the SEMI North American Process Chemicals Committee and published in its 

) (3). The guidelines are intended to describe the quality required to produce integrated circuits whose critical dimensions lie in the range of 0.09–0.2 µm. Hydrochloric acid (HCl) is currently in the 

 at the Tier C guideline level, meaning that the maximum contaminant level of 18 elemental impurities should each be less than 100 ppt (ng/L) in the process chemical. On completion and approval of the validated analytical method described in the guideline, it will meet the SEMI Grade 4 Standard (4).

	Traditionally, ICP-MS has been the technique of choice for ultratrace-element determinations in many of the high-purity chemicals used in the semiconductor and electronics industries. However, to get to the next required purity level of 10 ppt (ng/L) and beyond, conventional ICP-MS has shown that it does not have the detection capability for the full suite of trace metals-even with the use of spectral background reduction techniques like cool or cold plasma conditions and collision-cell technology. To achieve the next required analytical levels, a cell-based ICP-MS approach capable of using pure, highly reactive gases to produce targeted, controlled reactions to eliminate argon-, matrix-, and solvent-based spectral interferences is required. When combined with optimized plasma conditions, detection limits can be dramatically lowered for many of the semiconductor elements. 

	It is well-recognized that the ability to control ion-molecule chemistry in cell-based ICP-MS is essential when using the highly reactive gases required for maximum removal of atomic and polyatomic spectral interferences to meet the SEMI Tier C levels and beyond (5,6). For example, consider the analysis of hydrochloric acid, which requires the removal of the chloride-based interferences such as oxygen-chloride (

) on arsenic at mass 75. In addition, other prominent interferences include argon-oxide (

) on iron at mass 56 and chlorine-hydroxide (

 52. Pure ammonia is the most effective gas for removing 

 while allowing for low-parts-per-trillion (nanograms-per-liter) determinations of vanadium, iron, and chromium (7), limited only by levels of contamination. Oxygen has proven most effective for the determination of arsenic in hydrochloric acid by reacting with 

 91, where it is measured interference-free, away from the 

	In addition, the use of cold plasma conditions has been very effective at reducing the number of argon ions formed in the plasma, which minimizes the formation of argon-based species such as 

 and facilitates the determination of elements like 

 (8). Even though cold plasma has been used for many years, it has several limitations, including the ability to deal with real-world matrices such as concentrated mineral acids, susceptibility to matrix suppression effects, and the inability to switch rapidly between cold and normal plasma conditions during a multielement analysis. 

	However, advances in solid-state free-running radio frequency (RF) generators have enhanced the capability of ICP-MS to analyze real-world samples using cold and normal plasma conditions (9). The combination of pure, highly reactive gases in a controlled reaction environment with cold plasma conditions provides the most efficient reduction of polyatomic interferences, allowing ultratrace levels to be accurately measured. Table I shows chloride-, water-, and argon-based spectral interferences, which can potentially interfere with a suite of analyte masses during the analysis of hydrochloric acid, together with the optimum reaction gas and plasma conditions. 

	The goal of this investigation was to meet or exceed the SEMI Tier C guideline levels for the analysis of semiconductor-grade hydrochloric acid. This guideline states that spike additions made at 50% of the proposed specification level (100 ppt in 37% HCl) must have recoveries within 75–125% of the expected value. To evaluate the long-term stability, a 10-h run was performed monitoring a solution of 20% HCl spiked with 50 ppt of all elements.

	In semiconductor plants, high-purity HCl (35–38%) is most commonly used as an etchant in the chip fabrication process. One of the main requirements is that it must contain extremely low levels of elemental impurities so the etching process does not deposit elemental contaminants on the surface of the wafer or chip. To analyze high-purity HCl by ICP-MS, it is typically diluted twofold with ultrapure deionized water before aspiration into the instrument. To eliminate potential contamination from dilution, ultrapure 20% HCl (Tamapure-AA-10, Moses Lake Industries) was used in this work and analyzed directly without dilution. Calibration standards (10, 20, and 40 ng/L) were made from 10-mg/L multielement stock solutions via serial dilution, with the final standards being prepared in 20% HCl. Spikes of 10 and 25 ppt in the 20% HCl were used to evaluate recovery. Although the 25-ppt spike meets the SEMI Tier C guidelines, the 10-ppt spike was also measured to evaluate the analytical methodology with an eye toward future SEMI requirements.

	All analyses were performed on a NexION 2000 S ICP-MS system (10) (PerkinElmer, Inc.) using the instrumental parameters shown in Table II.

	Analyses were accomplished with a combination of reaction (with NH

) and standard modes (no cell gas), using either normal plasma (1600 W) or cold plasma (600 W) conditions for the 43-analyte multielement method. To maximize the effectiveness of reaction mode, 100% ammonia and 100% oxygen were used in two different ways: Ammonia effectively removed interferences at the analytical mass, and oxygen was used to shift the analyte to a new analytical mass. The latter approach was found to be most effective for the measurement of arsenic and selenium. Table sI (

) displays the method parameters for all 43 analytes.

Interference Reduction Strategies Using Reaction Mode

	It is important to emphasize that the effects of interferences were removed in reaction mode in two different ways:

		removing the interference at the analytical mass, and 

		shifting the analyte to a new mass away from the interference. 

	An example of removing the interference at the analytical mass is the removal of the 

 at mass 51. Although ammonia reacts rapidly with 

 to remove it, the reaction is driven to completion by using 100% ammonia in the cell. In addition, because the reaction cell incorporates a quadrupole, the RF-only parameter (RPq) acts as a low mass filter to control the chemistry and prevent the formation of new interferences, which is critical when using highly reactive gases. This is particularly important in removing the 

 interference since one of the intermediate products is 

 at mass 51. However, because the RPq parameter serves as a low mass filter, it can be set to make 

 unstable in the cell, which prevents the formation of 

	This functionality is demonstrated in Figure 1, which shows the optimization of the RPq parameter for 1 μg/L V in 10% HCl. As the RPq parameter increases to 0.7, the background decreases sharply, which represents where 

Cl+ is no longer stable in the cell (blue plot). As a result, 

 is ejected from the cell, meaning that the reaction to 

can no longer occur, resulting in interference-free analysis of V

	The second method of dealing with interferences is using reaction chemistry in mass-shift mode where the analyte reacts with the reaction gas moving it to a new analytical mass. An example of this approach is the analysis of arsenic (As), which reacts rapidly with oxygen to form 

 is measured free of interferences. Figure 2 shows the conversion of 

 decreases (blue plot), while the signal for 

 increases (red plot), demonstrating complete conversion. 

 at mass 91 is that the presence of zirconium (

) in a sample may produce a false positive reading. However, because 

 reacts rapidly with oxygen (rate constant ≈ 10

 forms, allowing for interference-free measurement of 

 even if Zr is present at the same concentration as As, as shown in Figure 3. In this figure, mass 91 is monitored as a function of oxygen flow for a 1-μg/L Zr standard (blue) and a mixed standard containing 1 μg/L Zr + 1 μg/L As.

	To assess the effectiveness of interference removal, method detection limits (MDLs) and background equivalent concentrations (BECs) were determined in 20% HCl using a 1-s integration time. The MDLs (analyte signal and background noise) were calculated by multiplying the standard deviation of the blank (10 analyses) by three, and the BECs (analyte signal and background signal) were determined by measuring the background concentrations of the blank at each analyte mass. A spike recovery test was carried out based on SEMI Tier C guidelines, but using a lower spike level of 10 ppt (ng/L), Table sII (

) shows both the MDLs and BECs for all elements, as well as the 10-ppt (ng/L) spike recoveries. The majority of MDLs and BECs are less than 1 ng/L, demonstrating the effectiveness of using both controlled reaction chemistry and cold plasma conditions. All spike recoveries fall within 10% of the true values, showing the accuracy of low-level measurements.

	Calibration curves were established with 10-, 20-, and 40-ng/L (ppt) standards using the method of standard additions. All curves had regression values of >0.999, demonstrating both the linearity of the analysis and the ability to accurately measure at low concentrations. Figure s1 (

) shows three calibration curves, which highlight the effectiveness of the interference reduction: calibration curves for 

 were acquired using ammonia as the reaction gas and cold plasma conditions, demonstrating the ability to remove significant plasma-based interferences (

 was acquired with ammonia and normal plasma conditions, exemplifying how effectively a significant chloride interference (

) can be removed. The accuracy of low-level measurements was then verified by measuring the recoveries of 10-ng/L (ppt) spiked additions in 20% HCl, which are all within 10% of the true value.

	With the ability to remove interferences and quantitative accuracy established, the stability of the methodology was evaluated by measuring a 50-ng/L (ppt) spike in 20% HCl over 10 h of continuous aspiration. The results shown in Figure 4 demonstrate the stability for all 43 elements when using a mixed-mode method consisting of standard (no cell gas) and reaction (NH

) modes with both normal and cold plasma conditions. These data show that for every element the drift of the signal is less than 10% from the initial reading, while the RSDs over the 10 h are less than 3% for all elements.

	Tables sI and sII as well as Figure s1 are available online in a supplementary information section. Please visit 

www.spectroscopyonline.com/supplementary-information-optimized-icp-ms-analysis-elemental-impurities-semiconductor-grade-hydroch

	This study has demonstrated the ability of the methodology used in this investigation to perform the elemental analysis of 20% HCl at SEMI Tier C guideline levels and beyond, without the need for dilution. The combination of fast switching between hot and cold plasma together with reaction cell technology using 100% ammonia and oxygen led to the elimination of spectral interferences on many of these elements. This capability allows for sub-part-per-trillion detection limits, excellent spike recoveries and accurate quantitation at the 100-ppt Tier C guideline level while offering the kind of long-term stability required by a high-throughput semiconductor production laboratory. In addition, the data show that quantitation at the SEMI Tier D guideline level (proposed Grade 5) of 10 ppt can be achieved.

		International Technology Roadmap for Semiconductors (ITRS 2.0), Semiconductor Industry Association (2015), 

http://www.semiconductors.org/main/2015_international_technology_roadmap_for_semiconductors_itrs/

		Semiconductor Equipment and Materials International (SEMI) (Organization, Milpitas, California, 

SEMI Book of Standards (BOSS): Process Chemica

http://ams.semi.org/ebusiness/standards/semistandard.aspx?volumeid=13)

		SEMI C27-0708 - Specifications and Guidelines for Hydrochloric Acid, 

http://ams.semi.org/ebusiness/standards/SEMIStandardDetail.aspx?ProductID=211&DownloadID=30

		T.S. Cheung, C. Wong, and H.R. Badiei, Technical Note, PerkinElmer Inc., Woodbridge, Ontario, Canada.

		NexION 2000 ICP-MS System: Product Description, 

http://www.perkinelmer.com/lab-solutions/resources/docs/BRO-NexION-2000-ICP-MS-012730_01.pdf

 are with PerkinElmer, Inc., in Shelton, Connecticut. Direct correspondence to: 

A closer look at the use of a cell-based ICP-MS approach that utilizes ion–molecule chemistry to reduce many of the traditional spectral interferences seen in the analysis of high-purity hydrochloric acid used in manufacturing integrated circuits and semiconductor devices

Optimized ICP-MS Analysis of Elemental Impurities in Semiconductor-Grade Hydrochloric Acid

) nanoparticles (NPs) are widely used in industry, manufactured goods, and consumer products, such as food additives. However, there are several significant challenges associated with the measurement of silicon by inductively coupled plasma–mass spectrometry (ICP-MS). First, silicon has a moderately high first ionization potential (8.152 eV), which means it is less than 70% ionized in a typical argon plasma, so the sensitivity is significantly lower than for a fully ionized element. In addition, background polyatomic ions affect all three silicon isotopes, 

Si, with the most intense interferences (

Si isotope. Finally, silicon-based materials are widely used in manufactured goods and industrial materials ranging from glass, alloys, and air and water filters to organic compounds such as silicone tubing, sealants, adhesives, lubricants, and coatings. Consequently, maintaining a low background for silicon in the analytical laboratory can be highly problematic. When characterizing silica in nanoparticle form, the analysis is especially difficult. In this article, we describe a method to measure ultrafine silica nanoparticles using ICP–tandem MS (ICP-MS/MS) to control the elemental and polyatomic ion backgrounds. The method provides high sensitivity to ensure accurate analysis at the small (~50 nm) particle sizes of interest in environmental and biological applications.

	Nanomaterials are increasingly used in industrial processes, manufactured goods, medicines, and as ingredients in consumer products such as cosmetics, sunscreen, and food. The measurement of nanoparticles (NPs) is a subject of interest because the fate of NPs in the environment and the potential for toxic effects once absorbed into biological systems are not yet well understood.

	Some of the most common types of NPs are difficult to measure accurately using conventional single-quadrupole inductively coupled plasma–mass spectrometry (ICP-MS). Such instruments often cannot achieve the combination of high sensitivity and low background that is required to allow the signal generated from a small particle to be distinguished from the background. Much of the work reported thus far has concentrated on particles that contain elements such as zirconium, silver, and gold (1,2), which are relatively easy to measure by ICP-MS. However, many of the natural and engineered NPs of interest are composed of elements that are much more difficult to measure by ICP-MS-for example, iron, sulfur, titanium, and silicon.

	Nanostructured synthetic amorphous silica (SAS) is one of the most commonly used nanomaterials. It is an authorized food additive (E-551) and is also found in a wide variety of industrial and consumer products from polymers, composites, paints, and textiles to toothpastes, detergents, and cosmetics. Silica NPs also have great potential for a variety of other uses, including silica aerogels as thermal and electrical insulators, and imaging and therapeutic applications in medicine (3). Reliable analytical methods are needed to allow monitoring of compliance with upcoming regulations (4) and for the assessment of nano-safety (5). However, Si measurement by ICP-MS is not easy because of the combination of relatively low sensitivity and high spectral background. Silicon is only about 70% ionized and the major isotope (

Si – 92.23% abundance) is interfered by the intense background polyatomic ions CO

. These limitations mean that conventional single-quadrupole ICP-MS is unable to detect silica particles with diameters smaller than about 200 nm (6). This capability is not sufficient for characterization of the nanoscale particle sizes of interest in emerging applications and for regulatory requirements; the accepted definition of a nanomaterial is one with major dimensions of 100 nm or less (7).

 can be resolved using a reactive cell gas such as hydrogen (H

) in the collision–reaction cell (CRC) of an ICP-MS system. However, ion–molecule reaction chemistry can only be controlled effectively using a tandem mass spectrometer, where an additional mass filter is situated before the CRC of the ICP-MS system. This extra mass filter enables MS/MS operation, where nontarget ions are prevented from entering the cell. This approach ensures that reaction processes and the resulting reaction product ions are consistent, even when the sample composition is complex or variable. The general layout and performance capability of a triple-quadrupole configuration of tandem ICP-MS is described in the literature (8).

 NPs were measured by single-particle ICP-MS (spICP-MS), using MS/MS mode and H

 reaction gas on a triple-quadrupole ICP-MS system. The instrument software included an optional single nanoparticle application module for method development, signal processing, and data interpretation. The practical applications capabilities of the instrument for the measurement of a range of SiO

	An Agilent 8900 ICP-QQQ system (model #100, Advanced Applications Configuration) was used for the analysis of silica nanoparticles. The instrument was equipped with a glass concentric nebulizer, a quartz spray chamber, a quartz torch with a 1-mm injector, and standard nickel sampling and skimmer cones. Samples were introduced with a standard peristaltic pump and pump tubing (1.02 mm i.d.). The configuration of the instrument used has a novel argon gas delivery path that uses special inert materials to minimize contamination of the gas supply through the instrument, reducing the elemental background for common contaminant elements Si and S. 

Si was measured in continuous fast time-resolved analysis (fast TRA) mode, using a dwell time of 0.1 ms (100 μs) per point with no settling time between measurements. The 

Si signal was measured on-mass in MS/MS mode, with both quadrupoles (Q1 and Q2) set to 

 28. Hydrogen cell gas was used to resolve the on-mass polyatomic interferences, N

 and CO. The main operating conditions of the instrument are shown in Table sI online; please visit 

www.spectroscopyonline.com/supplementary-information-accurate-measurement-ultrafine-silica-nanoparticles-using-icp-msms

Reference Materials and Sample Preparation 

 NP reference materials with nominal diameters of 50, 60, 100, and 200 nm determined by transmission electron microscopy (TEM) were purchased from nanoComposix. The reference materials were diluted with deionized (DI) water to give particle concentrations of 40, 40, 100, and 1000 ng/L (ppt) for the 50-, 60-, 100-, and 200-nm particles, respectively. The solutions were sonicated for 5 min before analysis to ensure sample homogeneity. To allow the elemental response to be calculated, a Si ionic standard of 5 μg/L (ppb) was prepared in DI water. MS/MS with H

 cell gas should be effective in removing the CO

 polyatomic overlap. To check this interference removal capability, a SiO

 NP solution containing a matrix of 1% ethanol was also prepared.

	NPs measured using TRA with ICP-MS give a narrow peak or signal plume lasting around 0.5 ms for each particle that passes through the plasma; the peak signal area of each particle plume is proportional to the particle mass. The signals measured in DI water (without particles) and in solutions containing SiO

 particles with diameters of 50, 60, and 100 nm are shown in Figure 1. Fast TRA mode allows multiple measurements to be made during the signal peak from each individual particle. The shape and duration of the ion plume from each NP can be measured, and the total ion count can be converted to a particle mass and therefore diameter (assuming spherical particles). The Single Nanoparticle Application module of the ICP-MS MassHunter software includes the calculations to convert signal intensity to particle diameter, along with other key parameters, such as minimum detectable particle size and background equivalent diameter. Because of the high sensitivity, low elemental background, and effective interference removal of the ICP-MS instrument used, the smallest (50-nm) SiO

 particles provided clear signal peaks (Figure 1b) that were easily distinguished above the low background signal seen in DI water (Figure 1a). As expected, the 60-nm and 100-nm particle solutions gave proportionally higher signal peaks as shown in Figures 1c and 1d, respectively.

	Frequency distributions were calculated automatically by the instrument software from the signals measured for the four SiO

 nanoparticle solutions shown in Figure 1; the frequency distribution plots are shown in Figure 2. The particle signals were clearly separated from the background caused by the dissolved  ionic component, although the background and particle signals were partially overlapped for the 50-nm particles (shown in Figure 2b). From these results, we can estimate that the practical detection limit for the particle diameter was below 50 nm. The background equivalent diameter (BED) calculated by the instrument software from the 50-nm particle solution was 22 nm.

	The particle size distribution results for the different SiO

 NP solutions are shown in Figure 3. The plots confirm that all the SiO

 particle sizes could be determined accurately, including the 50 nm NPs. The high sensitivity and low background of triple-quadrupole ICP-MS combined with the effective removal of polyatomic interferences in MS/MS mode with H

 cell gas, enabled the smaller NP sizes to be clearly distinguished from the background signal. All the particle size plots demonstrated a Gaussian distribution, which agrees with the information provided by the supplier of the NP solutions. The triple-quadrupole ICP-MS results obtained for median, mode, and mean particle sizes also agreed well with the reference sizes obtained by TEM (Table I). Also, the measured particle concentrations agreed well with the theoretical particle concentrations, although the concentration result for the 50-nm solution was slightly high (49 ng/L compared to an expected value of 40 ng/L) because of the overlap with the background signal.

	Figure 4 shows the mean signal intensity measured by spICP-MS/MS plotted against the reference particle diameter determined by TEM. The logarithmic plot has a slope of 2.84, which is in good agreement with the theoretical cube relationship between diameter and mass indicated in equation 1: 

	Equation 1 is used to calculate particle mass (

Resolving Carbon Interference Using MS/MS and H

	Real samples of interest-such as biological materials, food matrices, polymers, pharmaceutical ingredients, and organic solvents-may contain a high level of carbon, which causes a 

Si. Triple-quadrupole ICP-MS in MS/MS mode with H

 cell gas can eliminate this interference effectively. A mixed solution of the 100-nm and 200-nm SiO

 NPs was prepared in a sample containing 1% ethanol. The particle size distribution for this sample is shown in Figure 5. Despite the high concentration of carbon, the size distribution for each group of particle sizes was consistent with the result obtained by TEM, and the different particle sizes were separated with excellent resolution. This result suggests that the spICP-MS/MS technique can resolve the carbon interference successfully, allowing accurate determination of particle sizes for SiO

 nanoparticles in natural sample matrices.

	Triple-quadrupole ICP-MS operating in MS/MS mode with H

 cell gas was used for the successful determination of SiO

 nanoparticles at sizes smaller than 100 nm. The triple-quadrupole ICP-MS method delivered exceptionally low background signals and high sensitivity, allowing the smallest (50-nm) SiO

 particles to be distinguished from the background signal. MS/MS operation gave effective control of the H

 reaction chemistry, eliminating polyatomic interferences from N

 and CO, even in the presence of a high level of carbon matrix. Dedicated software was used to calculate the particle sizes, providing accurate results for individual NP reference materials and mixed solutions. The spICP-MS/MS method provided fast analysis times, excellent detection limits for particle size and concentration, and accurate results for SiO

		J. Tuoriniemi, G. Cornelis, and M. Hassellöv, 

		C. Degueldre, P.-Y. Favarger, and C. Bitea, 

		D. Napierska, L.C.J. Thomassen, D. Lison, J.A. Martens, and P.H. Hoet, 

		L. Calzolai, D. Gilliland, and F. Rossi, 

		S. Dekkers, A.G. Oomen, E.A. Bleeker, R.J. Vandebriel, C. Micheletti, J. Cabellos, G. Janer, N. Fuentes, S. Vázquez-Campos, T. Borges, and M.J. Silva, 

		M.D. Montaño, B.J Majestic, Å.K. Jämting, P. Westerhoff, and J.F. Ranville, 

		International Organization for Standardization, 

ISO TS 80004-1:2010: Nanotechnologies - Vocabulary - Part 1: Core terms,

		L. Balcaen, E. Bolea-Fernandez, M. Resano, and F. Vanhaecke, 

 is with Agilent Technologies in Stockport, UK. 

 is with Agilent Technologies in Tokyo, Japan. 

 is with Agilent Technologies in Bellevue, Washington. Direct correspondence to: 

A method to measure ultrafine silica nanoparticles using ICP-MS/MS to control the elemental and polyatomic ion backgrounds is described here.

Accurate Measurement of Ultrafine Silica Nanoparticles Using ICP-MS/MS

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Special Issues-09-01-2017

ICP-MS Analysis of Multiple Trace Elements in Industrial Cell Lines

Optimized ICP-MS Analysis of Elemental Impurities in Semiconductor-Grade Hydrochloric Acid

Accurate Measurement of Ultrafine Silica Nanoparticles Using ICP-MS/MS

Vol 32 No s9 Spectroscopy September 2017 Applications of ICP & ICP-MS for Today's Spectroscopists Supplement PDF