A Comparison of Two Microwave Digestion Systems for Analyzing Biological Tissues for As, Cd, Pb, and Hg, by ICP–MS/MS

The primary goal of this study was to evaluate two microwave digestion systems for the acid decomposition of biological tissues: (1) a single reaction chamber (SRC) system and (2) a rotor-based, closed vessel microwave (RBCVM) system. Four key toxic trace elements, arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg), were determined in seven certified reference materials (CRMs), and one other RM by inductively coupled plasma–tandem mass spectrometry (ICP-MS/MS). Results for both digestion systems were within ±10% for As, Cd, and Pb, and within ±20% for Hg compared to certified or reference values and were deemed “fit-for-purpose.” Measurement repeatability was largely comparable between the two systems. The conclusion of the study was that the performance of the two digestion systems was deemed equivalent for As, Cd, Pb, and Hg in the eight materials evaluated.

High-quality data for trace element analysis of biological tissues is a foundation of modern toxicology, medicine, and environmental remediation studies. Many analytical techniques require the sample to be digested or solubilized before being introduced into instrumentation. Digestion is the process by which the sample matrix is decomposed or oxidized, in turn solubilizing the sample and freeing the analytes bound to the matrix into a form compatible with the analytical technique of choice. Therefore, robust digestion techniques are vital to method accuracy, repeatability, reproducibility, and the quality of the analytical data reported.

Rotor based, closed vessel microwave (RBCVM) digestion is now well established as a highly effective method for many different matrices and is widely used for preparing biological tissues for trace element analysis (1). Compared with a classical open vessel hotplate or hot block approach, rotor based, closed vessel microwave digestion reduces contamination errors, volatile element losses, reduces overall digestion times, and increases completeness of digestion (2-6). Controlling and monitoring the vessel temperature and pressure are critical reaction parameters for ensuring digestion completeness. Some microwave digestion systems use contactless monitoring of the vessel temperature to ensure even heating and thus efficient digestion. For example, the CEM Microwave Assisted Reaction System (MARS) 6 system utilizes a floor-mounted sensor to determine the temperature of each vessel sequentially. Sequential monitoring of the temperature allows for estimating the overall batch temperature, which is used to control the amount of microwave power applied to the batch. However, there is some concern about analytical batches containing different matrices because of varying reactivity (7).

In response to these concerns, a microwave digestion system that utilizes a single chamber instead of a turnstile has been promoted. Single reaction chamber (SRC) microwave digestion systems lower the samples into a pressurized chamber and measures the temperature within the chamber. The chamber typically contains a mix of nitric acid and water, which when pressurized absorbs the microwave power and applies the temperature change to the vessels. According to the manufacturer, the chamber system allows for greater control of the batch temperature than the sequential monitoring approach of the RBCVM. The manufacturer claims that this system can double sample throughput, since it does not require batching, and offers complete digestion of “the most difficult samples (8).”

Garitta and associates found that digestion completeness of bovine liver was greater using a RBCVM compared to the SRC at the same digestion temperature (9). This difference was attributed to variation in nitric acid regeneration. Additionally, they found no significant differences in the recovery of the selected analytes. To our knowledge, there have been no studies that have compared these two digestion systems for single batch analyses using multiple biological matrices.

The present study was designed to compare and evaluate a RBCVM and SRC system for multiple biological matrices. Eight biological reference materials (RMs), including five standard reference materials (SRMs), two other CRMs, and a keratin RM from New York State (NYS), were used to assess the completeness of digestion as well as accuracy and precision for four key toxic elements that are ranked among the top 10 on the Agency for Toxic Substances and Disease Registry (ATSDR) Substance Priority List: #1 arsenic (As), #7 cadmium (Cd), #2 lead (Pb), and #3 mercury (Hg) (10). In addition to public health concerns, these elements have some unique analytical challenges. For example, As is subject to the carbon enhancement effect in inductively coupled plasma-mass spectrometry (ICP-MS) such that completeness of digestion and residual carbon could be a factor (11). Pb reportedly complexes with sulfates and silicates, which can lead to poor recoveries if not completely extracted during the digestion (12). However, Hg is a particularly challenging element to determine in biological matrices due to its tendency to be reduced to Hg⁰, which is volatile and can lead to losses, and due to its tendency to adhere to plastic components (12). Therefore, these four key elements were chosen for their unique analytical challenges along with their public health and environmental significance (13).

Methods

Digestion Systems

Two commercial microwave digestion systems were compared for this study: the CEM Microwave Assisted Reaction System (MARS) 6, which is a RBCVM system and the Milestone SRC ultraWAVE, which is a SRC system. The CEM MARS 6 was fitted with 20-mL perfluoroalkoxyl (PFA) XPress vessels and was equipped with iWAVE technology. The Milestone SRL ultraWAVE system was fitted with 15-mL quartz vessels, as glass vessels were contaminated with Hg and Pb. Vessels for both systems were acid washed with 2% (v/v) HNO₃ for 24 h between digestion batches.

Each microwave digestion system program was independently optimized to digest 250 mg (251.8 ± 2.7 mg) of sample with 3.0 mL of concentrated HNO₃ and 0.5 mL of concentrated hydrochloric acid (HCl). The resulting digestion method specifications are listed in Table 1. Digestion batches included one aliquot of each reference material (RM) and a digestion blank. Digestion batches were analyzed in triplicate for three independent digestion batches per system. After digestion, samples were volumetrically diluted to 10 mL with double deionized water. The study design can also be found in Figure 1.

Equipment

A Labconco freeze drier was used prior to digestion to remove any residual water from the RMs. The five step, 10-h program used was previously validated in our laboratory.

An Agilent 8800 ICP-MS/MS (Agilent Technologies) equipped with an octopole reaction system (ORS) was used for analysis. Oxygen gas was used with on-mass detection to address potential interferences from tungsten oxide (WO) at mass-to-charge (m/z) 202 on ²⁰²Hg and MoO at m/z 111 on ¹¹¹Cd, and to achieve a +16 mass transition (to measure the metal oxide) for ⁷⁵As, which was monitored at m/z 91. Although not required for interference correction, O₂ gas mode was also used on-mass detection to monitor ^{Σ(206+207+208)}Pb for ICP-MS/MS efficiency. Two internal standards were also monitored: ¹⁰³Rh for As and Cd; ¹⁹³Ir for Hg and Pb. Ir was also monitored at a +16 mass transition. The method detection limit was calculated according to the International Union of Pure and Applied Chemistry (IUPAC) harmonized guidelines for a single laboratory validation of analytical methods (14) and was based on analyzing digested National Institute of Standards and Technology (NIST) SRM 8435 Whole Milk Powder, which has very low endogenous concentrations of the target analytes.

Reagents, Standards, and Solutions

Single element stock solutions traceable to the NIST) were obtained from High Purity Standards and used for the duration of this study. Standard solutions were prepared and stored in 100-mL Nalgene polypropylene volumetric flasks (Thermo Fisher Scientific). All HNO₃ used in this work was double distilled in-house from reagent grade acid using a duoPUR Sub-Boiling Distillation System (Milestone Inc.). Veritas Double Distilled HCl (GFS Chemicals Inc.) was used for digestion and rinse. Double deionized (>18 MΩ·cm) water (DDI) was produced using a NANOpure Diamond UV/UF water system (Barnstead International).

Instrumental rinse solutions were prepared and stored in acid-washed 2-L fluorinated ethylene propylene containers (GFS Chemicals Inc.) and contained 30% (v/v) HNO₃, 5% (v/v) HCl, 1,000 µg/LAu (Inorganic Ventures), and 0.005% (v/v) Triton X-100 (Sigma Aldrich Co.). Diluent solutions were prepared and stored in acid-washed 2-L fluorinated ethylene propylene containers (GFS Chemicals Inc.) and contained 2% (v/v) HNO₃, 1,000 µg/L Au (Inorganic Ventures), and 0.005% (v/v) Triton X-100 (Sigma Aldrich Co.), and 0.5 µg/L Rh, Ir, and Y as internal standards (single element stocks, High Purity Standards).

Materials

Eight biological RMs were obtained from various providers: NIST Standard Reference Material (SRM) 8435 Whole Milk Powder; SRM 1486 Bone Meal; SRM 1577c Bovine Liver; SRM 1547 Peach Leaves; and SRM 2976 Mussel Tissue were obtained from the NIST. Additional materials included: CRM DORM-4 Fish Tissue from the National Research Council of Canada (NRCC); BCR 670 Aquatic Plant from the Joint Research Centre, IRMM; and NYS 18-03 Caprine Horn Level 3 that was obtained from the New York State Department of Health (NYSDOH).

Evaluation of Digestion Systems

Digestion completeness was assessed visually after a minimum of 12 h to allow for settling. Completeness of digestion was determined based on the absence of visible particulate matter, and/or a clear digestate. An 800-µL aliquot of digestate was diluted to 4 mL with a diluent solution for analysis by ICP-MS/MS. For NRCC CRM DORM-4 Fish Tissue and NIST SRM 2976 Mussel Tissue, a 1-mL aliquot of digestate was diluted to 50 mL with DDI water and then diluted as described above for analysis.

Eight different RMs were analyzed in this study to assess accuracy and/or agreement with assigned values. Some RMs were designated as CRMs, which includes SRMs from NIST. However, not all assigned values used are designated as “certified.” Good agreement with the certificate of analysis (COA) value was determined based on overlapping expanded uncertainties or within ±20%. The use of multiple RMs allowed for a robust investigation of accuracy and performance due to their homogeneity and in many cases traceability to SI units. Repeatability was assessed as percent relative uncertainty.

Calculations

Standard uncertainty was calculated according to equation 1:

where u_x is standard uncertainty, SD is the standard deviation of the mean concentration, and n is the number of measurements performed. It is important to note that this equation does not include the component of uncertainty from the COA, since not all assigned values for each of the analytes include the uncertainty. Means and standard deviations were calculated for each run and each digest, as shown in Figure 2. The mean standard deviation of the digests was used in equation 1 to determine u_sample and the mean standard deviation of the runs to calculate u_analytical. Propagation of combined uncertainty (u_c) was calculated according to equation 2:

Expanded uncertainty (U) was calculated by multiplying the u_c by a coverage factor (k); 1.96 was selected to represent 95% confidence. Relative uncertainty was calculated using equation 3.

Results and Discussion

Completeness of Digestion

Particulate matter was still visible in the digestates for aquatic plant, fish protein, and mussel tissue CRMs from both microwave systems. In contrast, digested whole milk powder, bone meal, bovine liver, peach leaves, and caprine horn had no visually detectable particulates. Particulate matter that could not be digested with excess time or temperature in nitric and hydrochloric acid was most likely silica or fat residues. Nonetheless, there was no evidence of any clogging of the sample introduction system when analyzing the samples with visible particulates.

There was excellent agreement between assigned and found values for all four analytes suggesting the digestion for fit-for-purpose. Enhancement of the As signal is caused by charge effects with residual carbon and is a well-documented phenomenon. The data here suggests sufficient digestion of the carbon matrix, as As values were derived using an aqueous calibration curve and no significant As signal enhancement was observed.

Agreement with CRM Assigned Values

Table 2 shows the found values and expanded uncertainties for As, Cd, Hg, and Pb in each of the eight RMs for (a) the RBCVM; and (b) the SRC system. Values for As, Cd, and Hg in NIST SRM 8435 Whole Milk Powder were below the established limits of detection (LODs) of 2.7 ng/g, 1.3 ng/g, and 1.2 ng/g, respectively.

Values for As found using the RBCVM and the SRC system were all in good agreement with the COA values across seven RMs and were within 3% of each another indicating excellent agreement between the two systems. There was good agreement between both systems for Cd and the COA values for six of the materials. Relative differences between the two techniques for Cd values ranged from 0.03% in the plant CRM to 6% in the bone RM, again indicating good agreement between the two systems. There was good agreement between both systems for Hg for three out of the five RMs with assigned values. Differences for Hg results for both digestion systems ranged from 20% for bone to 2% in the mussel tissue SRM. Notably, Hg differences between digestion systems exceeded 10% for the bone meal and fish protein RMs, and values were significantly higher for both systems compared to the COA value. For Pb, found values for both systems were within 10% of each another, ranging from 0.1% in fish to 7% in bovine liver. There was also good agreement between found values and the COA values for both systems and for all eight RMs.

The reasonable agreement between the two systems, and with the respective COA values implies overall fitness-for-purpose for both with little bias between the systems for the four analytes measured. Overall, both the RBCVM and SRC resulted in acid digests that performed well by ICP-MS/MS for the four analytes and the various matrices investigated.

Repeatability

Differences in the relative uncertainty for As, Cd and Pb between the two systems were less than 2%, implying excellent agreement between the two. For As, Cd, and Pb, relative uncertainties were less than 5% confirming that the systems were operating under conditions of repeatability for these elements. Relative uncertainties for mercury were somewhat larger for both systems by comparison, ranging from 5% to 16%. However, there was no significant difference between the two systems for Hg.

Other Practical Considerations

Contamination

After rinsing with DDI, the standard disposable glass vessels for the SRC system had double the background concentration for Hg and more than 20 times background concentration for lead compared to the RBCVM system vessels. Therefore, we used the reusable quartz vessels with the SRC system, which had comparable background levels to the RBCVM vessels. Results of the digestion blanks analyzed indicated no contamination (< LOD) for As, Cd, Hg, and Pb in the SRC quartz vessels. Only one RBCVM vessel had a detectable Cd digestion blank of 3 ng/g, indicating some exogenous contamination during the digestion. However, this did not impact overall Cd performance.

Maintenance

The SRC system requires somewhat more “hands on” maintenance compared to the RBCVM system. Cleaning and lubrication of all stainless-steel parts and Teflon components are required after each digestion batch in the SRC as nitric acid is highly corrosive. This process takes much longer than the cleaning of the RBCVM system, which requires wiping down of the system interior using DDI after each use. Additionally, some degradation of the stainless steel of the SRC was noted (even with the recommended daily maintenance) which may increase the risk of contamination for elements such as Cr and Mn. Both systems require ventilation, however the SRC system also requires a supply of nitrogen gas to pressurize the chamber that adds additional maintenance, cost, and potential contamination.

Both system configurations used in this study require cleaning of the reusable vessels after each use (24-h acid leaching with 2% (v/v) HNO₃) for high quality, low-level trace element studies. The quartz vessels of the SRC required more initial and periodic “deep” cleaning cycles, running a digestion program with just nitric acid present, compared to the PFA vessels of the RBCVM. For example, the SRC quartz vessels had noticeable white deposits after digesting (Figure 3) that could not be removed with acid washing nor by sonicating after one to two uses. There were no noticeable deposits observed on the RBCVM vessels during the study.

Conclusions

In this study, the RBCVM and SRC microwave systems performed similarly when digesting various biological matrices. The performance for As, Cd, Hg, and Pb in biological matrices was comparable between the two systems including accuracy-based evaluation against the COA data for the four analytes. Repeatability for both systems was similar for the four elements: As, Cd, Pb, and Hg. Other considerations including the laboratory’s internal processes and needs may play a role in evaluating microwave-assisted digestion systems. Future studies are needed to investigate additional trace elements and different matrices.

Disclaimer

Use of trade names and references to specific manufacturers does not imply an endorsement by the New York State Department of Health.

Acknowledgments

This work was supported in part by grant funding from the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH) under Award Number U2CES026542 (Parsons).

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