Determining the total fat content in food and feed samples is a routine procedure for quality control labs in the food industry and third-party contract labs. Traditional methods require that laboratories adopt matrix-specific approaches, leading to higher costs and extended turnaround times.

Based on simultaneous hydrolysis and extraction, the Milestone ETHOS X approach offers a robust and reliable alternative. This method delivers accurate and precise results while reducing the fat analysis cost per sample and turnaround time. In this report, the Milestone ETHOS X method is demonstrated as a viable and modern alternative to conventional official procedures that may no longer meet the demands of today’s laboratories.

Image Credit: Stock-Asso/Shutterstock.com

Introduction

The Nutrition Facts Label has become a fundamental feature of packaged foods and beverages. The U.S. Food and Drug Administration (FDA) regularly updates its regulations on food labeling requirements.¹

Fat content is one of the most important parameters of food labeling. Determining total fat, saturated fat, monounsaturated fat, and trans-fat content in food samples is essential for complying with these requirements. Total fat content, in particular, holds significant importance, as it influences the proper dosage of expensive ingredients and adherence to nutritional labeling regulations.

Numerous official methods have been developed for calculating total fat content, each tailored to specific matrix types. This variety of methods complicates analysis in high-throughput laboratories, resulting in elevated costs and increasing the risk of sample re-processing.

The Milestone ETHOS X approach utilizes a simultaneous microwave-assisted hydrolysis and extraction process designed to significantly reduce both solvent consumption and processing time.

This microwaved-assisted approach enables the usage of a single method for any matrix. In this study, the external contract laboratory Bureau Veritas India tested the Milestone ETHOS X to evaluate its performance compared to official methodologies.²

Experimental equipment

• Milestone ETHOS X 
• SR-15 eT extraction rotor 
• RAR-15 evaporation rotor 
• Aluminum caps 
• Vacuum system with condensation module 
• Analytical balance (with direct interface to ETHOS X terminal) 
• Conventional methods were performed with the Soxhlet extraction apparatus or the Mojonnier flask tube with associated glassware

Figure 1. Milestone’s ETHOS X with SR-15 extraction rotor (left) and RAR-15 evaporation rotor (right). Image Credit: Milestone S.r.l.

Solvents and reagents

Solvents and reagents for conventional procedures were purchased in the grade indicated in official methods. The ETHOS X method utilized sulfuric acid (25%) and Cyclohexane ACS reagent grade.

Samples

A list of food was selected, as reported in Table 1.

Table 1. Food sample selections. Source: Milestone S.r.l.

The samples were homogenized before the weighing step to ensure a representative aliquot. No drying step was applied to the sample, which has been used as-is.

Procedure

Conventional methods:

Confectionery products

FSSAI Manual for sugar and confectionary:2015

Ammonia hydrolysis followed by triple solvent extraction using the Mojonnier apparatus.

Dairy products

Rose-Gottlieb method - IS 1479: part 2:1961

Ammonia hydrolysis followed by triple solvent extraction using the Mojonnier apparatus.

Meat products

FSSAI Manual for Meat and Fish products:2017(2.1)

Hydrochloric acid hydrolysis is followed by Soxhlet solvent extraction (petroleum ether).

Ready-to-eat products

IS 12711: 1989

Soxhlet solvent extraction (petroleum ether).

Spices products

IS 1797:2017

Soxhlet solvent extraction (diethyl ether).

ETHOS X procedure:

Approximately 1 to 3 g of the sample was weighed directly into the SR-15 eT extraction vessels. Then, 10 mL of sulfuric acid (25%) and 25 mL of cyclohexane were added, and the final mass was recorded. Magnetic stirring bars were placed in each vessel, and the SR-15 eT was properly assembled. The microwave program is detailed in Table 2. 

Table 2. ETHOS X microwave hydrolysis and extraction program. Source: Milestone S.r.l.

After the program, the SR-15 eT vessels were opened, and aliquots of the organic phase were transferred into aluminum caps for weighing. The aluminum caps were reweighed after the solvent was quickly evaporated using the RAR-15 evaporation rotor.

The ETHOS X easyCONTROL software monitors and records all the steps and weights required for the calculation. Thanks to the software's capabilities and direct interface with the analytical balance, total fat values are provided at the end of the run. The data can be stored, and customized reports can be generated.

Results and discussion

In this study, the total fat content of various food samples was analyzed by applying the ETHOS X simultaneous hydrolysis and extraction method and comparing the results with those obtained through the conventional approach.

Table 3 presents the data collected. A comprehensive evaluation grid of parameters was applied to each food category, with tests conducted for both methods.

Each result obtained was statistically analyzed to compare the validation parameters, with the comparison summarized in Table 4.

The total fat content measured using the ETHOS X consistently aligned with the values obtained through the traditional approach for all tested food categories. The new approach demonstrates excellent reproducibility, evidenced by minimal relative standard deviations across 10 repetitions.

Table 3. Results comparison of conventional and ETHOS X. Source: Milestone S.r.l.

Table 4. Results for Confectionary, comparison on values (number replicates = 10). Source: Milestone S.r.l.

Working range

Using an analytical balance, a typical working range varies from 0.1 to 100% of total fat content, depending on the sample mass.

The operational range of the ETHOS X method for total fat determination is influenced by both the sample mass and the balance’s capacity, as it is calculated based on the minimum mass able to be weighed by the analytical balance. A higher sample mass is associated with a lower detectable fat content value.

Conclusions

This study examined the total fat determination of a wide range of common grocery items typically available on the market. 

The ETHOS X method produced results consistent with those of conventional methods. The external contract lab deemed all statistical parameters necessary for validating the method satisfactory. The ETHOS X method’s innovative ability to perform simultaneous hydrolysis and extraction in the same run enables unparalleled turnaround time, minimal solvent consumption, and an optimized workflow.

Likewise, the 15 positions available on the SR and RAR rotors deliver the highest throughput on the market. Following gravimetric evaluation, the total fat residue can also be utilized for fatty acid methyl ester (FAME) analysis, as outlined in the relevant application reports.

The ETHOS X's unique capabilities effectively address contemporary food laboratories' demands in terms of productivity, user-friendliness, cost-efficiency, and turnaround time.

References and further reading:

1. FDA (2019). Food Labeling & Nutrition. (online) U.S. Food and Drug Administration. Available at: https://www.fda.gov/food/food-labeling-nutrition. 
2. Lorenzi, S. (2025). Fat Determination with ETHOS X microwave extraction system - Milestone - Helping Chemists. (online) Milestonesrl.com. Available at: https://www.milestonesrl.com/products/microwave-extraction/ethos-x-for-fat-determination (Accessed 3 Feb. 2025).

About Milestone S.r.l.

Milestone was established in 1988 as the first specialized laboratory instrument manufacturer focusing on advanced microwave technology for sample preparation. Today we hold over 50 patents and have over 20,000 global users spanning across government, academic, contract, and manufacturing industries. Milestone’s headquartered is in Italy with offices in the Unites States, China, Japan, and Korea. The R&D and manufacturing sites are in Germany (MLS) and Switzerland (MWS), along with the offices for Austrian, German and Swiss markets. We have grown as advanced microwave technology manufacturer operating worldwide through a network of over 100 exclusive distributors, all providing our customers premium application and service support.

The key to Milestone’s technological leadership lies in bringing together individuals from diverse scientific and engineering disciplines to solve real world problems with innovative microwave instrumentation. The excellent support of the Application Team through all these years has highly contributed to the success of our laboratory instruments and our company. All our products are manufactured, assembled and checked by Milestone Production Department according to ISO procedures, to ensure they fully satisfy the needs of our customers.

Milestone has UNI EN ISO 9001:2015 certification representing international standards for quality management in TÜV Rheinland - a global, multi-accredited organization accepted by the majority of the world.

This information has been sourced, reviewed and adapted from materials provided by Milestone S.r.l..

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