Preparation of Food Samples Using Homogenization and Microwave-Assisted Wet Acid Digestion for Multi-Element Determination with ICP-MS

Elemental analysis is an analytical process for determining the elemental composition of various samples. It can be used to control the intake of metals into human bodies (especially heavy metals¹) since their high concentrations may cause unwanted health problems. Heavy metals are also one of the main environmental contaminants, therefore, control of their presence in the environment is necessary². Moreover, elemental analysis can be employed to determine the geographical origin of food products³ and to control the quality of food and water resources⁴. In addition, it is used for the determination of micro- and macronutrients in soils⁵ and to gain insights into geological processes throughout history by examining the chemical composition of minerals and sediments⁶. Studies have also been made to determine the presence of rare metals in electrical and electronic waste for further metal regeneration⁷, to evaluate the success of drug treatments⁸, and to verify the elemental composition of metal implants⁹.

Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) are commonly used techniques for the elemental analysis of various samples¹⁰. They allow simultaneous determination of multiple elements with limits of detection (LOD) and limits of quantification (LOQ) as low as ng/L. In general, ICP-MS has lower LOD values¹¹ and a wider linear concentration range compared to ICP-OES¹². Other techniques to determine elemental composition are microwave-induced plasma optical emission spectrometry¹³ and several variants of atomic absorption spectroscopy (AAS), including flame atomic absorption spectroscopy, electrothermal atomic absorption spectroscopy², cold vapor atomic absorption spectroscopy, and hydride generation atomic absorption spectroscopy¹⁴. Furthermore, elemental determination with low LOD and LOQ is possible with different electroanalytical methods, especially with anodic stripping voltammetry^15,16. Of course, there are other methods to determine the elemental composition of samples, but they are not as frequently employed as the above-mentioned methods.

Direct elemental determination of solid samples is feasible using laser-induced breakdown spectroscopy and X-ray fluorescence¹⁷. However, for elemental determination with ICP-MS, ICP-OES, and AAS it is necessary to convert solid samples into a liquid state. For this purpose, acid digestion is performed using acids and auxiliary reagents (in most cases H₂O₂). Acid digestion is carried out at elevated temperature and pressure, converting the organic part of the sample into gaseous products and converting the metal elements into water-soluble salts, thus dissolving them in the solution¹⁸.

There are two main types of acid digestion, open vessel digestion and closed vessel digestion. Open vessel digestion is cost-effective¹⁴ but has limitations, such as the maximum digestion temperature, which coincides with the boiling temperature of acids at atmospheric pressure. The sample can be heated on hot plates, heating blocks, water baths, sands baths², and by microwaves¹⁹. By heating the sample in that manner, much of the generated heat is lost to the surroundings²⁰, which extends the digestion time¹⁴. Other disadvantages of open vessel digestion include greater chemical consumption, the greater possibility of contamination from the surrounding environment, and possible loss of analytes due to the formation of volatile components and their evaporation from the reaction mixture²¹.

Closed vessel systems are more convenient for the digestion of organic and inorganic samples compared to open vessel systems. Closed vessel systems utilize a variety of energy sources to heat the samples, such as conduction heating and microwaves²². Digestion methods which use microwaves include microwave-induced combustion²³, single reaction chamber systems²⁴, and commonly used microwave-assisted wet acid digestion (MAWD)^25,26. MAWD allows digestion at higher operating temperatures, ranging between 220 °C and 260 °C and maximum pressures up to 200 bar, depending on the instrument's working conditions²⁷.

The efficiency and rate of MAWD depend on several factors, including the chemical composition of the samples, the maximum temperature, the temperature gradient, the pressure in the reaction vessel, the amount of acids added, and the concentration of acids used²⁸. In MAWD, complete acid digestion can be achieved in a few minutes due to the elevated reaction conditions compared to longer-lasting digestions in open vessel systems. Lower volumes and concentrations of acids are required in MAWD, which is in line with current green chemistry guidelines²⁹. In MAWD, a smaller amount of sample compared to open vessel digestion is needed to perform acid digestion, usually up to 500 mg of sample is sufficient^30,31,32. Larger sample quantities may be digested, but they require a larger amount of chemicals.

Since the instrument for MAWD automatically controls the reaction conditions and the person does not come in direct contact with the chemicals during heating, MAWD is safer to operate than open vessel digestions. However, the person should always proceed with caution when adding chemicals to the reaction vessels to prevent them from coming into contact with the body and causing harm. Reaction vessels also need to be opened slowly as the pressure is built up inside them during acid digestion.

Although acid digestion is a useful technique for preparing samples for elemental determination, the person performing it should be aware of its possible limitations. Acid digestion may not be suitable for all samples, especially those with complex matrices and those that are highly reactive or could react explosively. Therefore, sample composition should always be evaluated to select the appropriate chemicals and reaction conditions for complete digestion that dissolves all desired elements in the solution. Other concerns that the user must consider, and address are impurities and loss of analytes at every step of sample preparation. Acid digestion must always be performed according to specific rules or using protocols.

The protocol described below provides instructions for the homogenization of food samples in a laboratory-sized mixer, a procedure for cleaning the mixer's components, properly weighing the sample, adding chemicals, performing acid digestion by MAWD, cleaning the reaction vessels after the digestion is complete, preparing the samples for elemental determination, and performing a quantitative multi-element determination with ICP-MS. By following the instructions given below, one should be able to prepare a sample suitable for elemental determination and perform the measurements of digested samples.

1. Sample homogenization

1. Using a clean ceramic knife, manually cut the food samples (broccoli, mushrooms, sausages, and noodles) into smaller pieces to speed up the drying process. Prepare enough samples for a minimum of 6 replicates of the acid digestion (ensure that the minimum mass of the dried samples is 1500 mg).
   NOTE: Increasing the surface area of the sample exposes a larger portion of the sample to the heated surrounding air, increasing the rate of evaporation of the water.
2. Place the sample in a 250 mL glass beaker and dry it at 105 °C to a constant weight using a dryer.
3. Remove the glass beaker with the sample from the dryer and insert it in the desiccator.
4. Allow the sample to cool down to room temperature.
   NOTE: Samples must be weighed at a constant temperature to ensure that the weight accurately reflects the mass. Temperature variations can affect the volume and density of the samples measured.
5. Open the desiccator and transfer the glass beaker with the sample on the analytical balance. Measure the weight of the glass beaker with the sample.
6. After the weighing is completed, put the sample back into the dryer.
   NOTE: If the sample shrank significantly during drying, one could transfer it to a smaller glass beaker using a plastic spatula for more convenient weighing.
7. Repeat the process as described in steps 1.3-1.6 until a constant weight of the sample is achieved.
8. Place the dried heterogeneous sample into the mixer beaker (see Table of Materials), ensuring it does not exceed the maximum volume of the mixer beaker.
9. Insert the mixer beaker into the mixer and close the guard door (Figure 1).
10. Press the start button to activate the blades for grinding and mixing the sample.
11. Perform the grinding until the sample turns into a fine powder or a homogenous paste. To achieve such a product, repeat the grinding process several times.
12. When the sample is homogenized, switch off the mixer, open the guard door, and remove the mixer beaker.
13. Remove the homogenized sample from the mixer beaker and transfer it to a clean 50 mL glass beaker using a clean plastic spatula (Figure 2).
    NOTE: If the sample is too hard and could potentially damage the mixer's components, such as the blades and mixer beaker, it can be homogenized by other means, such as crushing it in mortars. Mixers are usually unsuitable for homogenizing hard materials, frozen samples, or easily flammable samples, which could damage the mixer's components. The use of organic solvents in the mixer is discouraged.
    ​CAUTION: Use safety gear and ensure that the mixer's door is adequately closed as the mixer's blades rotate at high speeds.

2. Mixer cleaning

1. Add ultrapure water (see Table of Materials) to the mark of the empty mixer beaker.
2. Insert the mixer beaker into the mixer and perform the standard mixing procedure.
3. Take the beaker out of the mixer and pour out the wastewater. If necessary, repeat the process with ultrapure water several times until the water remains clean even after mixing.
4. Remove the contaminated blades and diaphragm seal from the mixer and clean them thoroughly with ultrapure water.
   NOTE: Use neutral detergents to improve cleaning efficiency, especially when dealing with samples with a high fat content, as fat easily adheres to the surface of the laboratory inventory.
   CAUTION: Wear appropriate protective equipment, such as gloves, when removing and cleaning the blades to reduce the risk of potential injuries from their sharp edges.
5. Dry the cleaned components in the dryer at 105 °C and reinsert them into the mixer.
   ​NOTE: Ensure that the mixer's components are completely dry before reinstalling them in the mixer, to prevent carry-over of the water to the following sample.

3. Sample weighing

1. Remove the cover lid from the 100 mL trifluoromethoxyl-polytetrafluoroethylene TFM-PTFE reaction vessel³³.
2. Place the open reaction vessel on the analytical balance and ensure that the balance is leveled and zeroed before each measurement (Figure 3).
   NOTE: Weighing must be performed at room temperature. Avoid areas where severe temperature fluctuations and air flow could affect the measured weight. Ensure that the weighing area is clean and free of any contaminants.
3. Transfer the homogenized sample into the reaction vessel using a plastic spatula and weigh 250 mg of the sample. Do not weigh the sample below the minimum weight limit of the analytical balance.
4. Once the weighing is complete, place the cover lid on the reaction vessel to protect the sample from contamination.
   NOTE: Exceeding the weight limit of the digestion procedure can result in incomplete digestion. Handle the sample and reaction vessels with care to avoid any external contamination.

4. Acid addition

1. Pour approximately 40.0 mL of 68 wt% HNO₃ and 5.0 mL of 30 wt% H₂O₂ into separate 50 mL glass beakers, respectively.
   NOTE: Chemicals must be of high purity with trace metal impurities of less than 1.0 µg/L (ppb), ideally in the ng/L (ppt) range. Trace metal impurities affect the accuracy and repeatability of elemental determination.
2. Place the reaction vessels in a fume hood, open the cover lids, and add the below mentioned volumes of 68 wt% HNO₃ and 30 wt% H₂O₂ with 1 mL or 5 mL automatic pipettes, according to the following specifications:
   1. Broccoli, mushrooms, sausages, and noodles; for 250 mg of sample add 5.0 mL 68 wt% HNO₃ and 1.0 mL 30 wt% H₂O₂. Prepare three replicates for every sample.
   2. To determine the accuracy of the method (in terms of recovery, Rec), use the procedure described in step 4.2.1 and add 37.5 µL of 100 mg/L ICP multi-element standard solution (see Table of Materials) into the reaction vessels using a 200 µL automatic pipette. For every sample, prepare three replicates.
      NOTE: The volume of 37.5 µL was selected as it corresponds to an increase of 15.0 µg/L for the spiked solutions of samples compared with the concentration in the non-spiked solutions of samples. Moreover, the increase in concentration for the spiked solution of samples corresponds to the final concentration that is still in the linear concentration range for every analyte measured.
   3. Prepare a sample blank using the same volume of 68 wt% HNO₃ and 30 wt% H₂O₂ as used for the digestion of food samples in step 4.2.1. For a sample blank, do not add the sample to the reaction vessels.
      CAUTION: HNO₃ used for digestion is corrosive and produces fumes. For this reason, acid addition must be performed in a fume hood. Standard laboratory protective equipment must be employed (gloves, safety goggles, and laboratory coat). If there is contact with acid, the affected area should be immediately rinsed under the stream of cold water, and medical help should be sought.
3. Place the cover lid on reaction vessels and allow samples to react with the added 68 wt% HNO₃ and 30 wt% H₂O₂ for 2-3 min.
4. Screw the thread cover on the vessel and tighten the cover lids.
5. Shake the reaction vessel using light hand movements to fully incorporate the samples into chemicals.
   NOTE: Do not leave the specimens on the walls or lids of the reaction vessels, as there is a possibility that they will not be completely digested.

5. Microwave-assisted wet acid digestion

1. Turn on the microwave system (see Table of Materials) for acid digestion by pressing the start button (Figure 4).
2. Open the microwave oven door and remove the rack.
3. Distribute the closed reaction vessels symmetrically in the rack to ensure even irradiation of the samples by microwaves.
4. Insert the rack into the microwave chamber and mount it on a holder (Figure 5).
5. Close the microwave oven door.
6. Set a suitable digestion program on the microwave oven touch screen using a pen shaped tool. Choose an appropriate temperature gradient, the final temperature, and the number of samples to be digested. The recommended digestion program for different food samples is listed below:
   1. Broccoli, mushrooms, sausages, and noodles: 10 min increase to 160 °C, 10 min increase to 200 °C, 15 min at 200 °C, maximum power 900 W.
7. Start the digestion program and monitor the change in reaction conditions on the screen. Stop the digestion process if the temperature does not increase according to the prescribed program.
   NOTE: During digestion, sudden temperature spikes may be seen on the microwave oven screen. They occur when the samples react exothermically with the chemicals. The microwave system will automatically regulate the temperature by adjusting the output power.
8. Wait until microwave-assisted digestion is completed and the temperature of the sample decreases.
9. Open the microwave oven door and remove the rack from the microwave oven chamber. Close the door and switch off the instrument.
10. Remove the reaction vessels from the rack and wait for them to cool down to room temperature.
11. Slowly open the lid covers manually to release the gases formed during acid digestion. Turn the reaction vessels in the direction of the fume hood (Figure 6).
12. Completely remove the cover and rinse the cover and the walls of the reaction vessel with a small amount of ultrapure water.
13. Quantitatively transfer the contents of the reaction vessel into a clean 25 mL glass volumetric flask through a glass funnel by repeated rinsing of the cover and reaction vessel with ultrapure water.
14. Dilute the sample with ultrapure water to the mark of the volumetric flask. Close the volumetric flask with a stopper and mix the content of the volumetric flask.
    NOTE: Further dilution of the digested samples with ultrapure water should be performed as they should contain less than 5% (V/V) of residual acid³⁴ and less than 2 g/L of dissolved elements, also referred to as total dissolved solids³⁵.
15. Take a 20 mL plastic syringe and connect it with a polyamide syringe filter (25 mm diameter, 0.20 µm pore size). Fill the plastic syringe with the diluted sample and filter its content into a 50 mL plastic centrifuge tube by applying pressure. Use a new plastic syringe and syringe filter for every sample to avoid any cross-contamination.
    NOTE: Samples need to be filtered to remove any insoluble materials or solid particles that may remain undigested after MAWD. These particles may interfere with elemental determination measurements by clogging the instrument components. When filtering the samples, ensure to discard the first couple of drops. Use hydrophilic filters (made of polyamide) for aqueous solutions. Hydrophobic filters (PTFE) are not suitable for the filtration of aqueous solutions as they require a higher applied pressure, which could result in membrane rupture³⁶.
16. Close the 50 mL plastic centrifuge tube with a screw cap and put the sample in the refrigerator until the measurements.
    NOTE: Digested samples are stored in the refrigerator at lower temperatures to preserve them and extend their storage time.

6. Reaction vessel cleaning

1. After the digested samples were transferred to 50 mL volumetric flasks, add 5.0 mL of 68 wt% HNO₃ and 5.0 mL of ultrapure water with 5 mL automatic pipettes into the reaction vessels.
2. Close the reaction vessels with the cover lids and insert them into the rack. Transfer the rack to the microwave oven chamber.
3. Apply the following microwave program: 15 min increase to 160 °C, 10 min increase to 180 °C, maximum power 900 W.
4. Monitor reaction conditions during heating. After the heating is completed, let the reaction vessels cool down.
5. Open the microwave oven, remove the reaction vessels from the rack, and slowly open them in the fume hood.
6. Discard the content of the reaction vessels into plastic waste containers.
7. Rinse the reaction vessels with ultrapure water to remove any excess material or chemicals.
8. Dry the reaction vessels in the dryer at 105 °C before the next use.
   ​NOTE: The same microwave procedure (time, power, temperature gradient, and volume of chemicals) used for the acid digestion of samples may be used for cleaning the reaction vessels. Alternatively, the reaction vessels can be cleaned without the microwave system by submerging them in concentrated HNO₃ or HCl for several hours and rinsing them with ultrapure water.

7. Multi-element determination with ICP-MS

1. Take the 50 mL plastic centrifuge tubes containing the digested samples from the refrigerator and allow them to warm to room temperature.
2. Dilute the samples by a factor of 10 to decrease the concentration of acid in the digested sample and to decrease the concentration of the component of the sample matrix. Using an automatic pipette, transfer 2.50 mL of the sample into a 25 mL glass volumetric flask and then fill it up to the mark with ultrapure water.
3. Transfer the diluted samples into the 15 mL plastic tubes and place them into the appropriate positions in the autosampler.
4. Prepare the ICP-MS instrument (see Table of Materials) for the measurements:
   1. Switch on the ventilation and the chiller that supplies the ICP-MS with cooling water and cools its components.
   2. Use the compatible software to ensure that the rinsing solution (1 wt% HNO₃) flows continuously from the autosampler to the ICP-MS without pulsating.
   3. Open Ar (99.999% purity) and He (99.999% purity) gas cylinders to supply the ICP-MS with both gasses. Check the gas flow in the software and adjust it if necessary.
      NOTE: Use collision cell with He gas when spectral interferences are expected due to the formation of polyatomic ions (e.g., ⁴⁰Ar¹⁶O⁺ interfering with ⁵⁶Fe⁺)³⁷.
   4. Start up the plasma and calibrate the instrument using the tuning solution (see Table of Materials).
   5. Once the instrument is calibrated (torch position, gain voltage, lens voltage, mass/resolution, pulse/analog (P/A) calibration, database (DB) calibration, and validation), select the desired measurement method and perform the measurements.
5. When working with unknown samples, perform a semi-quantitative determination to obtain information on what elements are present in the sample and their approximate concentration.
   NOTE: It is advisable to additionally dilute the samples for the semi-quantitative determination as the detectors have a limit of the concentration of elements they can detect at once. Lower sample concentrations can extend the lifetime of the instrument components.
6. After obtaining the data on the approximate concentrations of the elements in the samples, create a method for the quantitative elemental determination in the software. Select the operating conditions of the ICP-MS (Table 1) and select the desired elements (in the present case Cu, Fe, Mn, and Zn). Determine the number and concentrations of solutions of the standard required to create a calibration curve (sometimes referred to as an analytical curve or working curve) (Table 1).
   NOTE: Prepare at least six different concentrations as calibration points for the calibration curve.
7. Prepare solutions of standard for the calibration curve. Using automatic pipettes, pipette the required volume of 100 mg/L multi-element standard solutions into 25 mL glass volumetric flasks, to prepare solutions of standards with the following concentrations: 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 10.0 µg/L, 20.0 µg/L, 30.0 µg/L, 40.0 µg/L, and 50.0 µg/L. Fill the flasks to the mark with 1 wt% HNO₃. Additionally, prepare a calibration blank using the 1 wt% HNO₃ solution.
8. Transfer the prepared solutions of standard and samples into the 15 mL plastic tubes, place them into the autosampler, and start the instrument following the procedure described in step 7.4.
9. Perform the quantitative measurement of the selected elements using the calibration curve methodology.
10. Once the measurements are completed, turn off the plasma, close the Ar and He gas supplies, switch off the ICP-MS chiller, and turn-off the ventilation system.

Homogenization
All samples were dried to a constant mass with the laboratory dryer to eliminate any moisture. Transferring the sample to a desiccator allowed it to cool to room temperature without binding moisture from the surrounding environment. The food samples were then homogenized using the laboratory mixer to obtain a fine powder. The resulting homogenized particles were uniform in size and evenly distributed, ensuring that subsamples (samples drawn from a larger sample) used for acid digestion were representative. The samples were easily removable from the mixer beaker with the help of a plastic spatula, except for the dried meat sample, which was more challenging to remove due to its higher fat content. Higher fat content made the sample partially adhere to the glass walls of the mixer beaker. The comparison of fresh, dried, and homogenized samples is given in Figure 2.

The instrument's components had to be cleaned multiple times with ultrapure water to eliminate all food particles that remained in the mixer.

It is essential to ensure that the weighed mass of the sample does not exceed the maximum value allowed in the reaction vessels. Weighing was carried out using an analytical balance at a constant temperature, and a plastic spatula was used to avoid contamination with metals that may arise from metal spatulas.

Acid digestion
All samples used in the protocol were food samples containing various amounts of carbohydrates, proteins, and fats. HNO_3, in combination with H₂O_2, is suitable for the digestion of these molecules, and other chemicals are not required. The chemicals were treated in a fume hood since HNO₃ forms fumes. After adding the chemicals into the TFM-PTFE reaction vessels, cover lids were mounted on the top of the reaction vessels and were sealed well to avoid possible contamination and loss of analyte. The reaction vessels were symmetrically distributed in the rack to ensure uniform microwave irradiation inside the microwave system.

During acid digestion, the door of the microwave system was closed, and the door could not be opened until the end of the protocol. The entire process of acid digestion can be monitored on the screen of the device, showing the temperature change with time (Figure 7).

After acid digestion was completed and the solutions of the digested samples had cooled to room temperature, the reaction vessels were opened in the fume hood. They were opened as slowly as possible. If the pressure is released too quickly, even small droplets of the reaction mixture may escape, resulting in loss of analyte. When the reaction vessels were opened, a yellow or yellow-orange gas was released (Figure 8). The coloration of the fumes can be attributed to NO₂, which forms orange fumes at higher temperatures. The pressure increase in the reaction vessels was due to the oxidation of food samples with HNO₃, resulting in the formation of gases such as CO₂, H₂O, NO, etc. After the reaction vessels were degassed, a light-yellow or colorless solution of the digested sample remained in the reaction vessel, indicating that total acid digestion by MAWD had been achieved. This was further confirmed by the absence of visible particles left in the solution.

The final step of sample preparation involved diluting the digested samples with ultrapure water to reduce the residual acidity (RA). High RA values interfere with the measurements by increasing the background signal. Dilution also decreases the concentration of metal ions in the liquid sample²⁶. When transferring the solution of digested samples into volumetric flasks, the components of the reaction vessel were thoroughly rinsed with ultrapure water to completely transfer the analyte. One problem which occurs is that small drops of ultrapure water, which may contain the analyte of interest, adhere to the walls of the reaction vessels. After dilution with ultrapure water to the 25 mL mark, all samples became colorless. The final solutions of digested samples contained water-soluble salts, as the metal elements present in the sample reacted with HNO₃ to form highly soluble nitrates. Elemental analysis techniques can determine the metal ions that form water-soluble salts. When filtering the diluted solutions, it is important to discard the first few drops to ensure that any particles or contaminants are removed. After filtration, the solutions were tightly sealed to prevent any leakage and then stored in the refrigerator.

The main limitation of the procedure of acid digestion is sample throughput. The MAWD system can digest only a limited number of samples at a time. Additionally, each digestion and subsequent sample preparation step can take several hours to complete. Furthermore, reaction vessel cleaning is also time-consuming, but it is crucial to minimize the risk of cross-contamination between samples.

Multi-element determination with ICP-MS
For each element, a calibration curve was constructed. They were obtained by plotting the intensity as a function of analyte concentrations (Figure 9). The linear concentration ranges for all measured elements were in the range from 1.0 µg/L to 50.0 µg/L.

The LOD and LOQ for every element were calculated using Equation 1 and Equation 2, respectively. In both equations, s_blank represents the standard deviation of the several measurements of calibration blank (10 replicates)^38,39, while b₁ represents the slope of the calibration curve.

     (1)

     (2)

The obtained LODs were 0.5 ng/L, 2.8 ng/L, 2.8 ng/L, and 3.2 ng/L for Mn, Cu, Fe, and Zn, respectively. The obtained LOQs were 1.6 ng/L, 9.2 ng/L, 9.5 ng/L, and 10.8 ng/L for Mn, Cu, Fe, and Zn, respectively.

Six replicate digestions of every sample were performed. Three replicate digestions of every sample were performed without spiking the sample with solutions of standard, and three replicate digestions were performed with the addition of a solution of a known amount of analyte standard to test the accuracy (spike recovery test⁴⁰) and precision of the entire methodology. For accuracy determination before the digestion procedure, 37.5 µL of 100 mg/L ICP multi-element standard solution was pipetted into the reaction vessels containing the sample, which resulted in an increase of concentration of 15.0 µg/L in spiked samples that were diluted by a factor of 10. This also corresponded to an increase of 15.0 µg per gram of sample for every measured metal ion. The accuracy and precision were determined using Rec and relative standard deviation (RSD), respectively.

The accuracy of an analytical method can be assessed by the spike recovery test. For this purpose, a solution of a known amount of analyte standard is added to the sample, which is then digested under the same reaction conditions as samples that are not spiked⁴¹. The Rec is calculated using Equation 3, where γ_i is the measured concentration of the spiked samples after digestion, while γ_t represents the determined concentration of the unspiked sample by considering the increase of the added solution of analyte standard. The γ_i and γ_t are averages of the three replicates. The analytical method is deemed to be accurate when Rec is in the range of 80.00%-120.00%⁴².

      (3)

The precision of an analytical method is evaluated with RSD. It describes the closeness of agreement between independent results, which were obtained through several replicate measurements. RSD is calculated using Equation 4, where s_m represents the standard deviation of the replicate measurements for the concentration determination, while represents the average value of the determined concentrations. The analytical method is deemed precise if the RSD value is lower than 20.00%⁴³.

     (4)

All samples were diluted with ultrapure water by a factor of 10 before the ICP-MS measurements (for the first set of measurements). The dilution decreased the concentration of the matrix components introduced into the analyzer. Moreover, by diluting the sample, the RA decreases. A high RA might compromise plasma ionization efficiency or result in matrix interference issues. If the concentration of the analytes after the first set of measurements is lower than the LOQ, the dilution factor should be lower than 10. The quantification of the metal ions was carried out using a calibration curve. The values of the calculated results should have the same precision (the same number of significant figures) as the solution of the standard employed for the calibration. The content of metal ions in the sample was expressed as µg per gram of weight (µg/g). This was achieved by multiplying the measured mass concentration of the analyzed sample with the dilution factor to obtain the concentration in the original digested sample. This mass concentration was then multiplied by the volume of the digested sample (25 mL) and then divided by the initial weighed mass of the homogenized sample (the initial weighted mass is the mass of the sample that was weighted into the reaction vessel for the MAWD). All values are reported as an average of three replicates.

The reported content of the elements below is given as ± s_m. The content of Cu, Mn, and Zn in the broccoli sample was 5.9 ± 0.5 µg/g, 32.5 ± 2.7 µg/g, and 42.8 ± 0.2 µg/g, respectively. The determined mass concentration of Fe in broccoli samples exceeded the upper limit of the linear concentration range of the calibration curve (i.e., 50.0 µg/L). Thus, the solution of the sample was diluted with ultrapure water by a factor of 2, and the ICP-MS measurement of this solution was performed. The results showed that the broccoli contained 63.0 ± 1.9 µg/g of Fe.

For the mushroom, the content of Zn, Fe, Cu, and Mn was 35.6 ± 1.4 µg/g, 30.4 ± 1.3 µg/g, 18.5 ± 1.0 µg/g, and 5.4 ± 0.3 µg/g, respectively. Sausages contained 42.2 ± 0.9 µg/g of Fe, 25.1 ± 2.6 µg/g of Zn, and 1.0 ± 0.1 µg/g of Cu. The multi-element determination with ICP-MS of the digested solution, which was diluted 10 times, showed that the concentration of Mn was lower than the lower limit of the linear concentration range (i.e., 1.0 µg/L). Thus, the original solution of the sausage sample was diluted only by a factor of 5, and the multi-element determination with ICP-MS was repeated. The content of Mn in sausage samples was determined to be 0.9 ± 0.3 µg/g. Noodles contained 5.4 ± 2.8 µg/g of Zn, 10.3 ± 1.2 µg/g of Fe, 1.6 ± 0.3 µg/g of Cu, and 7.5 ± 0.2 µg/g of Mn.

The Rec for all analytes measured in all four samples was in the range of 80.00%-120.00%, indicating the accuracy of the analytical method. The calculations showed that the analytical method was precise, as the RSD values were below 20.00%, apart from RSD for Zn in noodle samples. The results are reported in Table 2.

Figure 1: Laboratory mixer used for the homogenization of food samples. Please click here to view a larger version of this figure.

Figure 2: Comparison of fresh, dried, and homogenized samples. (A-D) Fresh samples of broccoli, mushrooms, sausage, and noodles. (E-H) dried samples of broccoli, mushrooms, sausage, and noodles. (I-L) homogenized samples of broccoli, mushrooms, sausage, and noodles. Please click here to view a larger version of this figure.

Figure 3: Weighing the sample on an analytical balance. This is performed from above by opening the upper flap. Please click here to view a larger version of this figure.

Figure 4: Microwave system. The microwave system for acid digestion with side touch screen for selecting reaction conditions and monitoring the process of acid digestion. Please click here to view a larger version of this figure.

Figure 5: Components used for microwave-assisted acid digestion. (A) Rack with 14 reaction vessels for acid digestion inside the microwave oven chamber. (B) TFM-PTFE reaction vessels consist of 3 parts. Once the vessels are closed with lids, neither the sample nor gases can escape from or enter the reaction vessels. Please click here to view a larger version of this figure.

Figure 6: The interior of the reaction vessels when opened in the fume hood. (A) The yellow-orange coloration of the fumes is due to NO₂ produced during acid digestion. (B) The yellow coloration of the solution of the digested sample after most of the gases have escaped from the reaction vessel. Please click here to view a larger version of this figure.

Figure 7: Change of temperature with time. A plot showing the temperature change as a function of time during acid digestion with MAWD. T2 stands for the temperature of the reaction mixture inside the reaction vessels. Please click here to view a larger version of this figure.

Figure 8: Opening the reaction vessels under the fume hood, where yellow-orange gases are released. Please click here to view a larger version of this figure.

Figure 9: Example of a calibration curve for Mn. Please click here to view a larger version of this figure.

Figure 10: ICP-MS instrument used for multi-element determination. Please click here to view a larger version of this figure.

Table 1: Operating conditions of the ICP-MS instrument. Please click here to download this Table.

Table 2: Rec and RSD values of broccoli, mushrooms, sausage, and noodles. Please click here to download this Table.

Homogenization
To ensure reproducible results in elemental determination, it is necessary to homogenize samples before acid digestion due to their complex and inhomogeneous structure and composition. Homogenization aims to eliminate constitutional and distributional heterogeneity. Mixing the sample minimizes distributional heterogeneity by evenly redistributing components throughout the sample. Similarly, by bringing the particle size down to a uniform size, constitutional heterogeneity is reduced⁴⁴. The subsamples obtained from the homogenate must contain the same ratio of components as the original sample to be representative⁴⁵.

Homogenization is achieved by applying a force to break down the sample into smaller particles⁴⁶. Samples may be homogenized by cutting, chopping, shearing, crushing, grinding, or mixing. However, the appropriate method must consider the sample's degree of hardness, brittleness, abrasiveness, elasticity, shape, and capacity to adhere to the homogenizer's components method⁴⁷.

The sample can be crushed manually with a pestle and mortar or ground in a variety of mills (knife mill, cutting mill, ball mill, mixer mill, etc.), and other forms of homogenizers⁴⁸. Small laboratory-sized mixers with ceramic or metal blades are commonly used for homogenization as they rapidly reduce the particle size by disintegrating and simultaneously mixing the sample. Grinding samples into smaller homogeneous particles increases the specific surface area, which accelerates acid digestion by MAWD.

However, care must be taken to avoid any contamination due to abrasion during grinding. Samples should not be homogenized with blades containing the same metals as the elements to be determined after acid digestion. Consequently, ceramic blades are more frequently used compared to metal blades. Homogenization is often a major contributing factor to cross-contamination, usually from improperly cleaned inventory and instrument components, resulting in systematic errors. After use, each component of the mixer should be thoroughly cleaned and washed with ultrapure water.

Cryogenic grinding is used when homogenizing samples that are more difficult to break up. The sample is frozen using liquid nitrogen (which has -196 °C), making it brittle and easier to homogenize^49,50.

Acid digestion
One of the most frequently used oxidizing acids in acid digestion procedures is HNO₃. It is usually used in combination with a low amount of H₂O₂, which increases the oxidizing power of the acid, thus improving digestion efficiency²⁰. A mixture of these two chemicals is frequently used for the digestion of organic samples, including food samples⁵¹. Digestion of organic samples in MAWD is carried out at elevated pressure and temperatures that exceeds the boiling point (121 °C) of HNO₃ azeotrope at atmospheric pressure²⁷. In MAWD the boiling point of HNO₃ rises to 176 °C as the pressure is increased to 5 atm²⁷. The temperature at which acid digestion is performed in MAWD cannot be achieved in open systems because the HNO₃ would evaporate, reducing the efficiency of acid digestion.

During digestion in closed vessels by MAWD, HNO₃ reacts with the organic sample under harsh reaction conditions, forming gaseous products such as CO₂, H₂O, and NO (Equation 5)^52,53. The benefit of using closed reaction vessels is the lower volume and concentration of the acid required for digestion, as HNO₃ is constantly being regenerated. This regeneration process occurs for as long as O₂ is present in the reaction vessel. The primary source of O₂ is H₂O_2, which is thermally unstable and breaks down into H₂O and O₂ (Equation 6). In the reaction vessel, NO reacts with O₂ to form NO₂ (Equation 7). Formed NO₂ then dissolves in H₂O, resulting in the formation of HNO₃ and HNO₂ (Equation 8). The produced HNO₂ subsequently degrades into H₂O, NO₂, and NO (Equation 9), completing the regeneration mechanism^53,54. The newly formed NO and NO₂ then react by the above-mentioned processes.

     (5)

Where n stands for the number of carbon atoms.

     (6)

     (7)

     (8)

     (9)

When organic samples react with HNO₃, the metals present in their chemical structure will form water-soluble nitrates⁵⁵. Since MAWD aims to convert solids into liquids, the formation of water-soluble salts is desired.

For different samples, different combinations of acids can be employed due to the complexity of the sample composition. Since less easily degradable organic and especially inorganic samples cannot be dissolved solely with HNO₃, other acids such as HCl, HF, HClO₄, and H₂SO₄ are also used^21,56.

Non-oxidizing HCl at elevated temperatures is used for the digestion of salts such as carbonates, phosphates, oxides, borates, sulfides, and fluorides^28,55. When HCl is combined with HNO₃ in a molar ratio of 3:1, aqua regia is formed, which improves the oxidizing abilities compared to HCl and HNO₃ alone due to the formation of nitrosyl chloride (NOCl), Cl_2, and H₂O (Equation 10)⁵⁷. Aqua regia is able to dissolve noble metals such as Au, Pt, Pd²⁸.

     (10)

For the digestion of silicates, HF is frequently used, since it breaks strong bonds between Si and O. When HF interacts with silicate samples (minerals, soil), hexafluorosilicic acid (H₂SiF₆) is formed (Equation 11)^19,58. However, despite HF's ability to digest silicates, it exhibits several drawbacks, including the formation of insoluble fluoride salts⁶, the formation of volatile products with heavy metals¹⁹, and volatile SiF₄²⁷. Moreover, HF cannot be used with glassware and quartz reaction vessels as it dissolves them¹⁸.

     (11)

Reaction vessels for acid digestion
The reaction vessels used in MAWD are designed to withstand high temperatures and elevated pressures during acid digestion. These reaction vessels also exhibit good microwave permeability, allowing microwaves to pass through without being absorbed²⁰. Microwaves passing through the reaction vessels will reach water molecules, which will effectively absorb them since they are polar, consequently heating up the solution containing the sample⁵⁹. Only the liquid phase in the reaction vessels absorbs the microwave radiation while the gaseous phase does not, resulting in a high increase in temperature with a slight increase in pressure¹⁸.

To minimize contamination and analyte loss, reaction vessels are hermetically sealed, preventing any materials from escaping or entering the vessels.

The most commonly used materials for reaction vessels are synthetic polytetrafluoroethylene (PTFE), copolymerized PTFE known as TFM, perfluoroalkoxy alkane (PFA), and quartz^52,60. These materials are chemically inert to most chemicals used in acid digestion, except for quartz vessels, in which HF dissolves. Using only one type of reaction vessel in every experiment is crucial, as using different types of vessels may result in different reaction conditions when subjected to microwave heating. At lower reaction temperatures, PTFE, PFA, and TFM-PTFE reaction vessels are used, while at temperatures above 300 °C, quartz vessels are recommended⁵². This is because polymers deteriorate and decompose at higher temperatures.

Evaluating the efficiency of acid digestion
There are several ways to evaluate the efficiency of acid digestion. The color of the solution can be used to evaluate whether complete or partial digestion of the sample has taken place. Usually, a colorless or slightly yellow coloration of the solution is an indicator of successful digestion, while the darker yellow, orange, green, or brown color of the solution suggests that the digestion process was unsuccessful, meaning that partial digestion took place⁶¹. In some cases, undigested organic or inorganic particulates may be present in the reaction mixture after digestion, requiring filtration before the sample can be introduced into the instrument for elemental determination. Removal of undigested particulates prevents system clogging and plasma instability in the case of ICP-OES and ICP-MS³¹.

The efficiency of acid digestion can also be experimentally evaluated through measurements of residual carbon content (RCC) and RA. RCC represents the amount of organic carbon remaining in the solution that was not converted to CO₂ during digestion⁶². A lower value of RCC is preferred to reduce non-spectral and spectral interferences (e.g., ⁴⁰Ar¹²C⁺) in the elemental determination^63,64. RCC measurements are performed using ICP-OES. The carbon content is determined at an emission wavelength of 193.091 nm^65,66,67. The efficiency of acid digestion is related to the consumption of chemicals. The more acid is consumed, the lower RCC values will be determined²⁵.

The acid is continually consumed during digestion as it reacts with the sample. In most cases, a small amount of acid remains unreacted. The amount of RA can be determined by titration with NaOH¹⁰ or KOH^25,54. Lower values of RA are preferred, as the higher acid concentration in the final digested solution can increase the background signal in analytical techniques such as ICP-MS²⁵ and ICP-OES⁶⁸. Higher RA values may also indicate the use of lower initial acid concentration for digestion⁶⁹.

Multi-element determination with ICP-MS
ICP-MS consists of several components. The peristaltic pump pumps the sample solution from the autosampler to the nebulizer. The liquid sample is then converted into an aerosol by the nebulizer by mixing it with Ar gas. Subsequently, the spray chamber filters aerosol droplets, thus allowing the introduction of the finest aerosol droplet fraction into the plasma⁷⁰. Ar plasma is generated and maintained within the torch by the radiofrequency coil, resulting in temperatures of approximately 10,000 K⁷⁰. The aerosol is atomized and ionized in the Ar plasma. Ions then continue through the interface into the high vacuum region. Ion optics guide the ions through the collision cell, where the stream of He gas collides with the monoatomic ions of the analytes and polyatomic ions. Since polyatomic ions are larger than analytes of the same nominal mass, they collide more frequently with He, lose more kinetic energy, and are thus efficiently removed⁷¹. In the next step, ions reach the mass analyzer (in the present case, quadrupole). In the mass analyzer, the ions are separated based on their mass-to-charge ratio (m/z)⁷². Following the separation by m/z, the ions reach the detector (in the present case, an electron multiplier) (Figure 10).

Critical steps and limitations
There are several critical steps and some limitations within the protocol. Ensuring the samples are entirely dry before continuing the process and avoiding contamination are crucial steps in homogenization. To prevent contamination, effort must be taken to keep all glassware clean during the whole process⁷³, as it may affect the accuracy of the analysis. In the event of contamination, the sample must be discarded, and the preparation process repeated, which can be time-consuming. When applying this protocol to other samples not described in this protocol, complete digestion might not be achieved, as some samples may require higher temperatures and different chemicals to completely dissolve all metals present in the sample. For acid digestion, high purity chemicals are needed, which can be expensive. Using high purity chemicals helps minimize interferences, ensuring improved reliability, accuracy, and precision of measurements performed by ICP-MS. The sample preparation process is time-consuming and has a low sample throughput as the preparation can last several days (drying, homogenization, acid digestion), limiting the number of samples that can be prepared in one day.

When performing multi-element determination with ICP-MS, spectral interferences (polyatomic and isobaric) can be encountered. Polyatomic interferences, which usually occur in plasma, combine at least two isotopes, while isobaric interferences represent isotopes of other elements with the same m/z as measured analytes⁷⁴. Eliminating these interferences (e.g., with a collision cell) is important. In addition to spectral interferences, the results are also affected by non-spectroscopic interferences consisting of sample introduction into the ICP-MS instrument, aerosol droplet size distribution, plasma stability, ion transport through the interface etc.⁷⁵.

The protocol described herein has the potential for other applications apart from that for the food samples. With slight modifications in the homogenization and acid digestion steps, it could be adapted for the preparation of inorganic samples, soil⁷⁶, electronic waste²⁸, etc. The adjustments can involve using different chemicals, varying their volumes, and altering the digestion temperature to suit different sample types. Moreover, as technology and methodologies evolve, further improvements and automation could be incorporated into the protocol, increasing its efficiency, and reducing the overall sample preparation time.

In summary, this protocol demonstrates the homogenization of food samples in a laboratory mixer, microwave-assisted wet acid digestion using a mixture of 68 wt% HNO₃ and 30 wt% H₂O₂ at an elevated temperature and pressure, and elemental determination with ICP-MS. The protocol can be used to train personnel in preparing samples for elemental determination as the protocol provides step-by-step instructions and explains the theory behind homogenization, acid digestion, and elemental determination.