Determination of Magnesium In Milk - Principle, Procedure, Result

Determination of magnesium in milk is an analytical test in which total magnesium present in milk is released from milk matrix and then it is quantified by suitable method. Magnesium is an important macro mineral in milk and it is present around 90–150 mg/kg. It has structural role in stabilizing casein micelle and maintaining colloidal stability of milk.

In milk about 65% to 75% magnesium are present in soluble phase as free ions or as complexes and remaining part is bound in colloidal phase with casein proteins and calcium phosphate cluster. Because major portion is associated with proteins and colloids simple water extraction is not sufficient for total magnesium estimation. The magnesium ions has to be liberated from organic environment and then it is measured.

Before measurement milk sample is pre-treated and complex lipid and protein matrix are destroyed and a clear aqueous solution containing dissociated magnesium ions is prepared. Traditional preparation include dry ashing in which sample is combusted in muffle furnace (up to 550°C) and inorganic mineral ash is obtained and it is dissolved in acid. Wet digestion method is also used in which strong acids like nitric acid or perchloric acid with heat are used and organic matter are broken down. In modern lab microwave assisted digestion is used as gold standard in which closed fluoropolymer vessel and high pressure are used and rapid decomposition is done at about 200°C or higher temperature.

After magnesium is freed from milk matrix it is quantified by different techniques as per lab requirement. A classical method is complexometric titration in which EDTA is used for forming stable complex with magnesium and calcium. Indicator like Eriochrome Black T is used and colour change occur from wine red to blue when all free magnesium is bound with EDTA. In automated thermometric titration end point is detected by temperature change because magnesium binding with EDTA is endothermic and heat is absorbed.

For more precision and sensitivity instrumental spectroscopy are preferred. Flame Atomic Absorption Spectrometry (FAAS) is used in which absorption of light by magnesium atoms in flame is measured and releasing agent like lanthanum is added so that milk phosphate do not form refractory compound and interference is reduced. Advanced methods include ICP-OES and ICP-MS in which argon plasma are used and chemical interference are eliminated. ICP-MS is considered as reference technique in many regulatory framework (AOAC ISO) and it gives high sensitivity and high throughput result.

Principle

Principle for determination of magnesium in milk is based on releasing the magnesium from milk complex organic environment and then it is measured by chemical or instrumental method. In milk magnesium are present in soluble phase as ions and also it is tightly bound with casein micelles and colloidal calcium phosphate. Because simple aqueous extraction is not sufficient the protein and lipid matrix has to be destroyed and bound magnesium is released into solution. This is achieved by complete mineralization by dry ashing in muffle furnace at high temperature or by wet digestion using strong acids like nitric acid with hydrogen peroxide and heat or microwave pressure. In some method chemical coagulation by trichloroacetic acid (TCA) is used and proteins are precipitated and magnesium is released into clear serum and it is filtered or centrifuged for analysis.

After magnesium is liberated into stable homogeneous aqueous solution it is quantified by complexometric titration method. In this method EDTA is added gradually and it binds with magnesium. Indicator like Eriochrome Black T is used and colour change occur from wine red to blue and end point is indicated. In thermometric titration the end point is detected by temperature change during reaction and magnesium can be distinguished from calcium because calcium complexation with EDTA releases heat (exothermic) whereas magnesium complexation absorbs heat (endothermic).

For higher sensitivity and accuracy instrumental spectroscopy are used for quantification of extracted magnesium. Flame Atomic Absorption Spectrometry (FAAS) is used in which absorption of light by magnesium atoms in flame is measured. Releasing agent like lanthanum is added so that phosphates present in milk do not bind magnesium and interference is prevented. Inductively coupled plasma methods (ICP-OES and ICP-MS) are also used as gold standard in mineral analysis. The acid digested milk solution is introduced into hot argon plasma and magnesium is atomized and ionized and it is quantified by characteristic light emission (ICP-OES) or by mass to charge ratio (ICP-MS) and chemical interference from milk matrix are minimized.

Requirements

Requirements are as follows–

Sample preparation and matrix transformation–

Milk sample is warmed (38°C–40°C) and it is mixed properly for uniform distribution of fat globules and trace minerals.
Trichloroacetic acid (TCA) is used for protein coagulation in titration method and proteins and fats are precipitated then it is filtered or centrifuged and clear milk serum is obtained.
Dry ashing is done by carbonization and heating in muffle furnace (540°C–550°C) and organic matrix is destroyed and mineral ash is obtained and it is dissolved in nitric acid or hydrochloric acid.

Wet digestion is done by heating with conc. acids like HNO3 HCl and H2O2 on hot plate or digester block and organic matter are broken down.
Microwave assisted digestion (MAD) is used by nitric acid and hydrogen peroxide in closed fluoropolymer vessel and high pressure is applied and digestion is done around 200°C.

Analytical quantification techniques–

Complexometric titration (EDTA) is used and milk serum is buffered to pH 10 by ammonia/ammonium chloride buffer and Eriochrome Black T (EBT) is used as indicator. It is titrated with standard EDTA till colour changes from wine red to clear blue.

Thermometric titration is done by automated titration using Na4EDTA and end point is detected by enthalpy change (calcium is exothermic and magnesium is endothermic). Acetylacetone is added in milk serum for separating calcium and magnesium end point.
Flame Atomic Absorption Spectrometry (FAAS) is used and magnesium is measured using hollow cathode lamp at 285.2 nm. Releasing agent like 1% lanthanum chloride is added so that phosphate do not bind magnesium and interference is prevented.
ICP-OES is used and magnesium is measured in argon plasma and wavelength like 279.553 nm or 285.213 nm are used. Internal standard (Yttrium or Scandium or Strontium) are used and ionization buffer like cesium chloride is used for matrix effect due to potassium and sodium.

ICP-MS is used for high sensitivity and magnesium (24Mg) is quantified and collision/reaction cell (CRC) with helium gas is required for removing polyatomic interference (like doubly charged calcium).

Quality control and regulatory standard–

Certified reference materials (CRMs) are analysed along with sample like NIST SRM 1549 (Nonfat Milk Powder) or NIST SRM 1849a (Infant/Adult Nutritional Formula) for checking accuracy.
Method performance requirement as per AOAC SMPR 2014.004 are followed and LOQ should be ≤ 1 mg/100 g and recovery should be 90%–110% and RSD (precision) should be ≤ 5%.

Safety and equipment requirement–

PPE are used and fume hood is required and acid resistant lab coat chemical splash goggles face shield and heavy duty neoprene gloves or heat resistant gloves are worn.
Caution is required during handling conc. HNO3 and HCl and during using pressurized microwave digestion vessel and it is allowed to cool before opening because hot corrosive vapour can cause hazard.

Procedure

Procedure are as follows–

Phase 1 (sample preparation and matrix destruction)–

Liquid milk is warmed (38°C–40°C) and it is mixed gently so that fat globules are distributed evenly. Milk powder are mixed properly or it is reconstituted in deionized water.

Trichloroacetic acid (TCA) is added for coagulation of proteins and fats (mainly for titration method). The mixture is filtered or centrifuged and clear serum containing magnesium ions is obtained.
Dry ashing is done by pre ashing on hot plate and fats and sugars are removed then it is kept in muffle furnace (up to 550°C) for 12–24 hours. White mineral ash is obtained and it is dissolved in strong acid like nitric acid or hydrochloric acid.
Wet digestion is done by heating sample on hot plate with strong acids like hydrochloric acid or nitric acid till clear digested solution is formed.

Microwave assisted digestion is done by taking milk sample with nitric acid and hydrogen peroxide in closed pressurized fluoropolymer vessel and it is heated in microwave up to 200°C or higher and organic matter is destroyed rapidly.

Phase 2 (analysis and quantification)–

For complexometric titration buffer is added to maintain pH 10 and indicator like Eriochrome Black T is added. The sample is titrated with EDTA till colour changes from wine red to blue completely and end point is noted.
Thermometric titration is done by automated EDTA titration and temperature change is measured by probe and magnesium binding shows endothermic change. Acetylacetone is added for separating calcium and magnesium end point.

In Flame Atomic Absorption Spectrometry (FAAS) the digested solution is aspirated into flame and absorption by magnesium atoms is measured at 285.2 nm. Releasing agent like lanthanum solution is added to prevent chemical interference.
In ICP-OES the sample is nebulized into argon plasma and magnesium concentration is calculated by measuring emitted wavelength of excited ions.
In ICP-MS the acid digested solution is introduced into argon plasma and magnesium is ionized and it is quantified by mass to charge ratio. Collision cell (helium gas) is used for removing polyatomic interference and this method is considered as gold standard (AOAC 2015.06).

Results – Formula for Calculating Magnesium Percentage

Result are as follows–

ICP-OES method–

Australian pasteurized retail milk shows magnesium average value 101.39 µg/g (mg/kg) and range is 97.62 to 110.57 µg/g in different brands. LOQ is 11.56 µg/g and recovery is 116.0%.
Whole milk powder (CRM certified 814 mg/kg) gives 813 mg/kg by dry ashing 794 mg/kg by hot plate wet digestion and 705 mg/kg by microwave digestion.
Nonfat milk powder (CRM certified 1200 mg/kg) gives 1200 mg/kg in FDA validation and about 0.105% to 0.115% depending on digestion method used.

Evaporated milk (unfortified) shows magnesium value about 157 mg/kg in FDA elemental analysis.
General milk sample under EN 13805:2002 protocol shows mean magnesium value 123.5 mg/kg.

FAAS vs EDTA titration–

Cow milk magnesium by FAAS is found 895 to 1219 mg/kg and by EDTA titration it is found lower about 604 to 650 mg/L.

Buffalo milk shows higher magnesium than cow milk and FAAS value is 1227 to 1493 mg/kg and EDTA titration value is 765 to 1350 mg/L.

Automated thermometric titration–

Full cream milk shows average magnesium 13.2 mg/100 ml.
Low fat milk shows average magnesium 14.8 mg/100 ml.
Skim milk (added solids) shows average magnesium 14.5 mg/100 ml.

Formula for calculating magnesium percentage–
Magnesium (%) = (109.24 × w) / W

Where–
w = Weight (g) of magnesium pyrophosphate obtained after ignition of precipitate. It is final weight and accurate weighing is required.
W = Weight (g) of milk sample taken for analysis (example 10 g). The exact initial weight is taken for standardizing the result in percentage.
109.24 = Constant factor and it is derived from molecular weight and stoichiometry of magnesium and pyrophosphate reaction and it is used for converting magnesium pyrophosphate weight into elemental magnesium.

The calculated value gives percentage of magnesium in milk sample. Higher percentage indicates higher magnesium content in sample.

Uses

Uses are as follows–

It is used for nutritional labeling and regulatory compliance and magnesium content is quantified for verifying nutritional claim and proper consumer information and export requirement.

It is used in quality control of milk and dairy products and magnesium level affects physicochemical stability taste and texture. It is part of milk salts balance and it affects heat stability and rennetability during processing.
It is used in product innovation and fortification and precise magnesium analysis is required for fortified products and infant formula and safety standard are maintained.
It is used in cheese manufacturing and monitoring magnesium helps in improving technological property and it gives faster milk clotting firmer curd and increased protein retention in soft cheese.

It is used for controlling bacterial spoilage and magnesium concentration adjustment helps in inhibiting biofilm formation by Bacillus species and it increases heat pasteurization susceptibility and equipment contamination is reduced.
It is used in animal health assessment and feed formulation and magnesium in milk is analysed for detecting deficiency (hypomagnesemia) and diet formulation is guided.
It is used for environmental monitoring and mineral profile of fresh milk acts as bio indicator for agricultural pollution and environmental quality of farming region is evaluated.

Limitations

Limitations are as follows–

Simple water extraction cannot be used because magnesium are bound with casein and colloidal calcium phosphate.
TCA coagulation may not recover complete micellar magnesium and result can be lower than total value.
Dry ashing is time consuming (12–24 hours) and high energy is required and loss of volatile element can occur at high temperature.

Wet digestion require conc. acids (nitric acid perchloric acid) and these are corrosive and hazardous and safety handling with fume hood is needed.
Microwave digestion is limited by small sample size and cooling time and limited vessel processing capacity and in high fat sample risk of explosion is present if not controlled.
In EDTA titration pH must be maintained around 10 and if pH goes below 9 magnesium–EDTA complex becomes unstable and end point is not proper.

EDTA binds calcium also so mutual interference occur and masking agent or specific indicator or thermometric separation is required.
In FAAS phosphate interference occur and magnesium pyrophosphate is formed and low reading is obtained if lanthanum releasing agent is not added.
FAAS is low throughput because one or few elements are analysed at a time and it is slow.

ICP-OES and ICP-MS are costly and maintenance cost is high due to argon gas requirement.
In ICP-OES matrix effect occur due to potassium and sodium and magnesium signal can be suppressed or enhanced if internal standard is not used.
In ICP-MS polyatomic interference occur and carbon or calcium complex can mimic magnesium isotope so collision cell or dilution is required.

References

Alfa Chemistry. (n.d.). Comprehensive protocol for total hardness determination in tap water using EDTA titration.
American Dairy Products Institute. (2023). Analytical method #008: Total ash and alkalinity of ash.
AOAC International. (2014). AOAC SMPR 2014.004 Standard method performance requirements for minerals and trace elements in infant formula and adult/pediatric nutritional formula.

Ben-Ishay, N., Oknin, H., Steinberg, D., Berkovich, Z., Reifen, R., & Shemesh, M. (2017). Enrichment of milk with magnesium provides healthier and safer dairy products. npj Biofilms and Microbiomes, 3, Article 22. https://doi.org/10.1038/s41522-017-0032-3
Brown, S. D., & McCurdy, W. H. (2005). Chemistry 120: Experiment 2 EDTA titration of calcium.
Food Safety and Standards Authority of India. (2024). Manual of methods of analysis: Trace elements (Method No. FSSAI 09.003:2024).

Kira, C. S., & Maio, F. D. (2004). Comparison of partial digestion procedures for determination of Ca, Cr, Cu, Fe, K, Mg, Mn, Na, P, and Zn in milk by inductively coupled plasma-optical emission spectrometry. Journal of AOAC International, 87(1), 151-156. https://doi.org/10.1093/jaoac/87.1.151
Metrohm. (n.d.). Automated determination of calcium and magnesium in milk with thermometric titration (Application Bulletin 342/1e & 342/2e).
Mindak, W. R., & Dolan, S. P. (2010). 4.4 Inductively coupled plasma-atomic emission spectrometric determination of elements in food using microwave assisted digestion. In Elemental analysis manual for food and related products. U.S. Food and Drug Administration.

Nguyen, D. D., Solah, V., Daubney, S., & Jani, S. (2024). Determination of Ca, P, K, Na, and Mg in Australian retail pasteurised milk using inductively coupled plasma atomic emission spectroscopy (ICP OES). Journal of Analytical Methods in Chemistry, 2024, 4417607. https://doi.org/10.1155/2024/4417607
Pacquette, L. H., Thompson, J. J., Malaviole, I., Zywicki, R., Woltjes, F., Ding, Y., Mittal, A., Ikeuchi, Y., Sadipiralla, B., Kimura, S., Veltman, H., & Miura, A. (2018). Minerals and trace elements in milk, milk products, infant formula, and adult/pediatric nutritional formula, ICP-MS method: Collaborative study, AOAC final action 2015.06, ISO/DIS 21424, IDF 243. Journal of AOAC International, 101(2), 536-561. https://doi.org/10.5740/jaoacint.17-0318
Seely, O. (2026). Complexometric calcium determination (Experiment). Chemistry LibreTexts. California State University, Dominguez Hills.

Shelke, M. E., & Sheikh, R. R. (2020). Relative study for the determination of calcium and magnesium in milk samples by using complexometric EDTA titration and FAAS. International Journal of Chemical and Physical Sciences, 9(2), 1-7.
Truman State University. (2008). Determination of Mg by titration with EDTA. CHEM 222 Lab Manual.
University of Canterbury. (n.d.). Determination of total calcium and magnesium ion concentration.

Determination of Magnesium In Milk – Principle, Procedure, Result