Rapid Determination of Melamine in Liquid Milk and Milk Powder by HPLC on the Acclaim Mixed-Mode WCX-1 Column with UV Detection

Significance of the Topic

Melamine adulteration in milk and dairy products poses severe health risks, as demonstrated by infant toxicity cases. Routine monitoring of melamine in both liquid milk and milk powder is essential for food safety and regulatory compliance. A rapid, cost-effective analytical method supports high-throughput screening in manufacturing and quality control laboratories.

Objectives and Study Overview

This study aimed to develop and validate a streamlined HPLC-UV method for the quantification of melamine in liquid milk and milk powder. The approach leverages a mixed-mode weak cation-exchange/acquity column to achieve rapid separation without extensive sample cleanup.

Methodology and Instrumentation

Sample Preparation

• Milk Powder: Weigh ~1 g powder, add 10 mL water, vortex, ultrasonicate 30 min, add 1 mL 3% acetic acid, chill 30 min, centrifuge ≥10 000 rpm for 15 min, dilute to 10 mL, filter 0.2 μm.
• Liquid Milk: Add 1 mL 3% acetic acid to 10 mL sample, chill 30 min, follow powder protocol thereafter.

Instrumentation

• Dionex UltiMate 3000 HPLC system with VWD-UV detector (240 nm) and optional PDA detector.
• Column: Acclaim Mixed-Mode WCX-1 (4.6 × 250 mm, 5 μm) with guard cartridge (4.3 × 10 mm).
• Mobile Phase: 10 mM ammonium acetate/10 mM acetic acid buffer (pH 4.3) – acetonitrile (80:20 v/v).
• Flow Rate: 1.0 mL/min, Column Temperature: 30 °C, Injection Volume: 20 μL.
• Calibration: seven standards from 0.05 to 40 μg/mL prepared from a 1000 μg/mL stock.

Main Results and Discussion

The method achieved baseline separation of melamine with a retention time around 5.2 min and a total runtime under 10 min. Key performance metrics:

• Linearity: 0.05 to 40 μg/mL, r=0.9999 (y=0.9120·c).
• Reproducibility: RSD 0.037% (retention time) and 0.472% (peak area) over ten injections of 1 μg/mL.
• Detection Limit (MDL): 0.015 μg/mL.
• Recovery: 88–90% in spiked milk powder (40 mg/kg) and liquid milk (4 mg/L).

Comparison with a traditional RP-ion-pair method using an Acclaim 120 C18 column showed equivalent results, validating the simplified sample prep and mixed-mode separation. Use of a PDA detector confirmed peak identity and minimized false positives.

Benefits and Practical Applications

The proposed protocol offers:

• Minimal sample preparation without SPE or evaporation.
• High throughput with <10 min analysis time.
• Compatibility with UV and MS detection.
• Robust performance across diverse dairy matrices.

This makes it suitable for routine quality control in dairy processing and regulatory laboratories.

Future Trends and Potential Applications

Advances may include integration with tandem MS detection for enhanced specificity, development of automated on-line sample cleanup, miniaturization via UHPLC, and extension to other food and feed matrices. Emerging mixed-mode stationary phases could further improve selectivity for ionic contaminants.

Conclusion

A simple, rapid, and reliable HPLC-UV method using an Acclaim Mixed-Mode WCX-1 column has been established for melamine determination in liquid milk and milk powder. The procedure requires minimal cleanup, yields high sensitivity and reproducibility, and is readily adaptable to high-throughput screening.

References

1. Filigenzi MS, Tor ER, Poppenga RH, Aston LA, Puschner B. Determination of melamine in muscle tissue by LC–MS/MS. Rapid Commun Mass Spectrom. 2007;21(24):4027–4032.
2. Ding T, Xu J, Li J, Shen C, Wu B, Chen H, Li S. Determination of melamine in plant protein powders by HPLC–DAD and LC–ESI–MS/MS. Se Pu. 2008;26(1):6–9.
3. Kim B, Perkins LB, Bushway RJ, Nesbit S, Fan T, Sheridan R, Greene V. Melamine in pet food by EIA, HPLC–DAD, and UPLC–MS/MS. J AOAC Int. 2008;91(2):408–413.
4. Wang L, Henday SM, Liu X, Tracy M, Schnute WC. Simultaneous determination of melamine and cyanuric acid by LC–MS on mixed-mode WAX-1. Dionex Corp LPN 1991-01. 2007.
5. GB/T 22388-2008. Determination of melamine in raw milk and dairy products. 2008.
6. GB/T 22400-2008. Rapid determination of melamine in raw milk by HPLC. 2008.
7. Dionex Corp. Product Manual: Acclaim Mixed-Mode WCX-1 Column.