Using Microwave Digestion and FAST Flame Sample Automation to Analyze Micronutrients in Fortified Breakfast Cereal

A breakfast of fortified cereal, fruit and milk is a quick and simple solution to provide a nutritious meal at the beginning of the day. Fortified breakfast cereals are enjoyed across the globe and provide an essential source of nutrition for children.

Consumers opt for fortified products over non-fortified products and expect high-quality cereals. However, in order to produce fortified breakfast cereals efficiently, care should be taken when it comes to formulation and batch-to-batch uniformity.

Continuous analytical measurement of the entire micronutrient content and nutritional additives in the cereal is one way to measure the consistency and quality of cereal products.

Besides this, the ability to analyze samples in a fast and precise way is also critical for well-timed data reporting. Such measures make it possible to make real-time batch modifications and improve continuous process control. Moreover, food manufacturers have to adhere to nutritional labelling guidelines that demand an exact evaluation of micronutrients to comply with regulatory labelling.

Although inductively coupled plasma optical emission spectroscopy (ICP-OES) is often utilized for multi-element analysis, the fast, simple and cost-effective operation of a flame atomic absorption (AA) system presents another option. However, if the latter approach is used for measuring different elements, each sample had to be measured separately for each element which affects the speed benefit of flame AA.

To that end, a fast, high-throughput sample automation system can be employed to address this issue. While samples still need to be analyzed several times, the time taken for each sample is greatly reduced, thereby improving sample throughput in comparison to conventional sample introduction. An automated sample introduction system also improves the accuracy of the analysis while freeing the chemist to focus on other tasks.

This article shows how PerkinElmer’s PinAAcle™ 900 atomic absorption spectrometer combined with a FAST Flame sample automation accessory can be effectively used for studying standard nutritional elements in a wide range of fortified cereals.

Experimental Frame work

All measurements were carried out on PerkinElmer’s PinAAcle 900T atomic absorption spectrometer working in flame mode and coupled to a FAST Flame 2 sample automation accessory. Table 1 shows the preferred elements and instrument parameters for analysis of the cereal samples.

Table 1. PinAAcle 900 Instrument and analytical conditions

Element Cu Fe Mg Mn Zn K Na Ca
Mode Absorption Absorption Absorption Absorption Absorption Emission Emission Absorption
Wavelength (nm) 324.75 248.33 285.21 279.48 213.86 766.49 589.00 422.67
Slit (nm) 0.7 0.2 0.7 0.2 0.7 0.2 0.2 0.7
Acetylene Flow (L/min) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.7
Air Flow (L/min) 10 10 10 10 10 10 10 10
Burner Head Rotation 0 ° 0 ° 0 ° 0 ° 0 ° 45 ° 45 ° 45 °
Acquisition Time (sec) 1 1 1 1 1 1 1 1
Replicates 3 3 3 3 3 3 3 3
Sample Flow Rate (mL/min) 6 6 6 6 6 6 6 6
Intermediate Standard (mg/L) 1 10 1 1 5 400 100 400
Auto-Diluted Calibration Standards (mg/L) 0.05
0.1
0.2
0.5
1
0.5
1
2.5
5
10
0.05
0.1
0.25
0.5
1
0.05 0.1
0.25
0.5
1
0.25
0.5
1
2.5
5
20
40
100
200
400
10
20
50
100
20
40
100
200
400
Calibration Curve Type Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero Non-Linear Through Zero

A high sensitivity nebulizer was utilized with a normal spray chamber and a 10 cm burner head.  External calibrations were carried out using an intermediate standard made in 2% deionized water / HNO3 which was diluted in-line by leveraging the capabilities of the FAST Flame 2 accessory.

La2O3 was introduced to the solutions, diluents, and standards at a concentration of 0.5% by weight to assist with the analysis of calcium (Ca), sodium (Na), potassium (K), and magnesium (Mg).

With a combination of a high-speed autosampler, switching valve and peristaltic pump, the FAST Flame 2 accessory gives rapid sample turnaround with short signal stabilization times, instant rinse-out, and no sample-to-sample memory effect. It quickly fills a sample loop through vacuum and switches to inject as the autosampler moves to the subsequent sample.  This not only prevents the time delay related to peristaltic pumping or self-aspiration, but also prevents the lengthy rinse-in and rinse-out times because of autosampler movement and flushing, thereby shortening complete sample-to-sample analytical times to 15 seconds.

Since the FAST Flame 2 accessory mechanically pumps the sample during variability due to differences in sample viscosity, tubing length, and dissolved solids are removed.  Pumping the sample also maintains sample flow stability for longer periods.

With this in-line dilution capability, analysts can make an intermediate standard and allow the FAST Flame 2 accessory to automatically create all in-line calibration standards as needed.

The system can even be configured to detect quality control over-range samples and use the in-line dilution capability to automatically re-run a sample falling beyond the calibration range at an increased dilution factor so the concentration falls within the calibration range.  The result is precise measurement as well as an efficient quality control check.

In order to realize precise analysis of the cereal samples, they must be turned into an instrument-ready solution. Here, the use of open-vessel digestion using a simple heating block and nitric acid can prove effective, but could leave behind undigested matter which would further need centrifugation or filtration before being introduced into the instrument.

All of this may lead to reduced recovery and poor precision. In contrast, closed-vessel microwave digestion provides complete sample digestion, prevents the necessity for additional steps, ensures the highest element recovery, and provides increased safety and higher throughput.

Table 2 shows a range of breakfast cereal samples that were analyzed. Using a PerkinElmer Titan MPS™ microwave sample preparation system, these samples, along with NIST™ SRM 3233 (Fortified Breakfast Cereal standard reference material) were prepared both spiked and unspiked. Titan MPS is a sample digestion oven that uses a special vessel and system design and provides ease of use with improved safety and throughput.

With pressure control through a reference vessel and non-contact temperature control for individual vessel, the Titan MPS system ensures precise digestion and zero sample contamination, irrespective of the type of sample. Table 3 details the microwave digestion technique.

Table 2. Cereal types analyzed and corresponding data labels

Cereal Type Data Label
Multi Grain G
Oat O
Rice R
Corn C
Wheat W1, W2

Table 3. Titan MPS system digestion method

Method Step Target Temp (°C) Pressure Limit (bar) Ramp Time (min) Hold Time (min) Power Limit (%)
1 140 35 10 2 60
2 195 35 3 25 100
3 50 35 1 20 0

To each vessel, about 10 mL of concentrated nitric acid and 1 g of crushed cereal were added. All spiking was carried out before sample digestion, with spike concentrations chosen based on the reported SRM values.

Results and Discussion

For individual elements, calibration curves were produced from an intermediate standard using the in-line dilution capabilities of the FAST Flame 2 accessory, creating the final standards in real-time. Table 4 shows the calibration results.

Table 4. Calibration results

Element Correlation Coefficient ICV Concentration (mg/L) Measured ICV (mg/L) ICV (% Recovery)
Cu 0.99997 0.500 0.494 98.8
Fe 0.99998 5.00 5.06 101
Mg 0.99996 0.500 0.456 91.2
Mn 0.99999 0.500 0.511 102
Zn 0.99990 2.50 2.54 102
K 0.99936 200 208 104
Na 0.99962 50.0 48.6 97.2
Ca 0.99999 200 207 104

The exceptional correlation for the calibration standards shows the value of the standard dilution and automatic in-line sample capabilities of the FAST Flame 2 accessory.

The autonomous calibration verification recoveries corroborate the validity of the calibration and accuracy of the standards created with the dilution system. The results for the analyses of NIST™ SRM 3233 Fortified Breakfast Cereal are shown in Table 5.

Table 5. NIST™ SRM 3233 Fortified Breakfast Cereal recovery values

Element In-line Dilution Factor Certified SRM Concentration (mg/kg) Measured SRM Concentration (mg/kg) % Certified Value Recovery
Cu 1 3.97 4.26 107
Fe 5 766 751 98.0
Mg 40 1093 1142 105
Mn 1 33.1 30.9 93.4
Zn 10 628 587 93.5
K 3 3060 3278 107
Na 5 6830 7249 106
Ca 20 36910 37870 103

All of the elements read within 10% of the certified values, which confirm the precision of the methodology. A number of different dilution factors were utilized for the various elements, which were conducted in-line with no operator intervention.

Once the precision of the methodology was ascertained, the cereal samples were analyzed. Figure 1 shows the results, demonstrating several trends. In all samples, K and Na were present at the highest concentrations, while Mn and Cu were present at the lowest concentrations.

It was observed that Wheat Cereal 1 (W1) included considerably less Cu, Na, Mn, and Fe when compared to all the other samples, indicating that this cereal is more natural and less fortified than others.

On the contrary, Oat Cereal (O) is at or close to the top for all elements, which indicates that it is one of the most fortified cereals. Ca, Zn, and K, which fall into distinct levels that are equally divided among all the samples, indicate varied levels of fortification.

Results from analysis of seven cereals

Figure 1. Results from analysis of seven cereals

Given the broad range of elements amongst the samples, it is not possible to always apply the same dilution factor to all the samples for the same element. The dilution factors automatically determined and carried out in-line with the FAST Flame 2 accessory are shown in Table 6.

Table 6. In-line dilution factors

Sample Cu Fe Mg Mn Zn K Na Ca
MG 1 5 40 1 10 3 4 6
O 1 5 40 1 10 3 4 8
R 1 5 40 1 10 3 4 8
C 1 5 40 1 10 3 5 6
W1 1 5 40 1 10 3 4 8
W2 1 5 40 1 10 3 4 10

In order to evaluate any potential matrix effects from the wide range of samples, all samples were spiked with all elements at the levels illustrated in Table 7. Figure 2 shows the resulting spike recoveries.

It was seen that the recoveries of all sample method spikes are within 10% of the measured values for all elements and did not need per-sample matrix matching, thus highlighting the value and effectiveness of using the Titan MPS system to digest the samples in a safe and complete manner. The range of cereals displayed spike recoveries within 10%, which further confirmed the robustness of the instrument and sample preparation methods.

Table 7. Pre-Digestion Spike Levels (all units in mg/kg)

Sample Cu Fe Mg Mn Zn K Na Ca
MG 28.9 578 578 28.9 578 2634 5268 10536
O 32.3 646 646 32.3 646 2995 5989 11979
R 29.6 592 592 29.6 592 2815 5631 11261
C 29.6 593 593 29.6 593 2828 5656 11312
W1 26.5 530 530 26.5 530 2865 5729 11459
W2 28.8 576 576 28.8 576 2847 5693 11387

Spike recoveries for all elements for all samples

Figure 2. Spike recoveries for all elements for all samples

The quantified concentrations of most of the elements in the samples were sufficiently different and fell beyond the calibration curve. However, the in-line dilution capability of the FAST Flame 2 accessory enabled dilution of these samples in real-time, so that the absorbance fell within the calibration curve, resulting in precise analyses.

The FAST Flame 2 reacts to the over-range samples and automatically dilutes the samples reliably and accurately with no user intervention, thus saving time and removing further sample handling and extensive re-preparation steps.

Conclusion

This article has shown how the PinAAcle 900 AA spectrometer effectively and reliably analyzes breakfast cereal samples for various elements such as Mg, Cu, Fe, Mn, K, Zn, Ca, and Na across a broad concentration range.

The combination of PinAAcle 900 and FAST Flame 2 sample automation accessory reduces manual errors when conducting dilutions and making calibration standards, offers long-term stability, and improves throughput for the laboratory.

The application of the Titan MPS system for sample digestion prevented sample and matrix issues and allowed the use of external standards without the need for specific analytical parameters or matrix matching. When studying smaller batches of samples, the same analyses can also be carried out without the use of the FAST Flame 2 accessory.

This information has been sourced, reviewed and adapted from materials provided by PerkinElmer.

For more information on this source, please visit PerkinElmer.

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