Sensitive and precise quantitation of melamine in milk


Using the SCIEX Triple Quad 3500 system to meet regulatory guidelines set by the Food Safety and Standards Authority of India (FSSAI)

Binumol T1 , Rakesh Reddy1 , M Chandrasekar1 , Sabarinathan1 , Manoj Pillai1 and Jianru Stahl-Zeng2
1SCIEX, India; 2SCIEX, Germany

Abstract


This technical note demonstrates a fast and simple method for the analysis of melamine in milk, which achieved a limit of quantification (LOQ) of 2.5 ng/mL (Figure 1). The sensitivity of the method using the SCIEX Triple Quad 3500 system enabled analysis at levels significantly less than the FSSAI maximum residue limit (MRL) of 150 ng/mL. Quality control (QC) samples (n=6) showed an accuracy range of 88–98% and precision (%CV) of <6% across the spiking range of 2.5–225 ng/mL.

Introduction


Milk naturally has high nutritional value but is prone to adulteration by the addition of substances to change its nutritional profile. Melamine (1,3,5-triazine-2,4,6-triamine) is a nitrogen-rich compound that has been added to milk to artificially inflate its protein content. 1,2 High concentrations of melamine can cause tissue injury, such as acute kidney failure, urolithiasis, bladder cancer or death.

Many analytical methods have been developed for the quantitation of melamine to help ensure food safety and detect potential adulteration2 . In this technical note, a simple, robust, selective and sensitive LC-MS/MS method was developed to quantify melamine in milk.

Figure 1. Extracted ion chromatogram (XIC) of 2.5 ng/mL melamine in milk. Overlaid quantifier and qualifier transitions are shown. 

Key features of the method to quantify melamine in milk
 

  • A fast and sensitive LC-MS/MS method was developed on the SCIEX Triple Quad 3500 system for the analysis of melamine, utilizing a simple sample extraction with a 5-minute runtime

  • The LOQ achieved was 2.5 ng/mL and was less than the 150 ng/mL MRL set by the FSSAI

  • Excellent precision and accuracy were observed, with %CV values <6% for QC samples (n=6) and accuracies between 94-106% for matrix-matched calibration standards

  • Confident analyte detection and quantification in the milk matrix were achieved using 2 selective MRM transitions for melamine

Methods


Sample preparation. The melamine standard was purchased from Toronto Research Chemicals (Toronto, Canada). Milk samples were purchased from the local market in Gurgaon, India and stored at room temperature. Prior to extraction, the melamine standard was spiked into the milk to prepare matrix-matched calibration standards (2.5–400 ng/mL) and QC samples (2.5, 5, 10, 75, 150 and 225 ng/mL).

The matrix-matched calibration standards, QC spikes and real-world milk samples were all extracted using the same procedure. First, 1 mL of milk was added into a 15 mL tube with 3 mL of 3% acetic acid in ethyl acetate. The solution was vortexed for 10 minutes and then centrifuged at 4500 rpm for 10 minutes. The organic layer was collected and evaporated under a stream of N2 gas. Samples were reconstituted with 1 mL of diluent (1:1 (v/v), acetonitrile/water with 0.1% formic acid), vortexed, transferred to a 2 mL centrifuge tube and centrifuged at 13,000 rpm for 10 minutes. Finally, the supernatant was transferred to an autosampler vial for instrumental analysis.

Chromatography. LC separation was performed using a Nexera XR LC instrument and a Phenomenex Luna 3 µm PFP (2) 100 Å (2.0 × 150 mm) column. Mobile phase A was water with 0.1% formic acid by volume and 5mM ammonium acetate. Mobile phase B was 95:5 (v/v) acetonitrile/water with 5mM ammonium acetate and 0.1% formic acid by volume. The gradient ramped from 5% to 95% mobile phase B. The gradient conditions used are shown in Table 1. A flow rate of 0.3 mL/min and an injection volume of 10 µL were used.

Table 1. Gradient conditions for the analysis of melamine.

Mass spectrometry. Samples were analyzed on the SCIEX Triple Quad 3500 system operated in multiple reaction monitoring (MRM) mode. The Turbo V ion source was used with electrospray ionization (ESI) in positive polarity. Two optimized MRM transitions were monitored, m/z 127 > 85.0 for the quantifier and m/z 127 > 68.2 for the qualifier.

Data processing. Data were processed using SCIEX OS software, version 1.7.

Method sensitivity, accuracy and precision


The matrix-matched calibration curve showed excellent linearity with correlation coefficients (r) ≥0.99 for both the quantifier and qualifier transitions. A linear regression and weighing factor of 1/x2 were applied (Figure 2). The method LOQ was 2.5 ng/mL, demonstrating excellent sensitivity (Figure 1 and Table 2). Further, the calibration standards yielded accuracies between 94% and 106% across all concentration levels (Table 2). The matrix-spiked QC samples showed excellent accuracy (88–98%) and precision with %CV values <6% across all spiking levels (Table 3). Overall, these results met the FSSAI MRL guidelines (Figure 3) and demonstrated the ability of the SCIEX Triple Quad 3500 system to achieve the sensitive, precise and accurate quantitation of melamine in milk samples using a simplified sample preparation procedure. 

Figure 2. Matrix-matched calibration curve for melamine. Calibration levels ranged from 2.5 to 400 ng/mL with r > 0.99 for both quantifier and qualifier transitions.

Table 2. Accuracy (%) and ion ratio of matrix-matched calibration standards. Melamine was spiked into milk samples prior to extraction.

Figure 3. Representative XIC for melamine spiked into the milk sample at the FSSAI MRL of 150 ng/mL. Ion ratio lines show the ± 20% tolerance.

Sample analysis. Various milk samples were collected from the local market of Gurgaon, India and analyzed for the presence of melamine. All samples were detected but were below the current method LOQ, deeming these samples safe for consumption in accordance with the FSSAI MRL.

Table 3. Precision (%CV) of QC samples for melamine in milk (n=6). Melamine was spiked into the milk samples prior to extraction. The %CV achieved was <6% at each spiking level.

Conclusion
 

  • A fast and sensitive LC-MS/MS method for the quantitation of melamine in milk samples in a 5-minute runtime using the SCIEX Triple Quad 3500 system

  • A simple extraction method followed by LC separation and MS/MS detection enabled the quantitation of melamine in milk samples at concentrations as low as 2.5 ng/mL

  • Acceptable accuracies (88%-98%) and precision <6 %CV were observed for QC spikes. The matrix-matched calibration yielded linearity with an r >0.99 for both quantifier and qualifier transitions.

  • Method performance and LOQs were sufficient to meet regulatory requirements set by the FSSAI

References
 

  1. Jalili J. (2017) A review paper on melamine in milk and milk products. Dairy and Vet. Sci. J. 1(4), 555566.

  2. Tyan Y.-U., Yang M.-H., Jong, S.-B., Wang, C.-K., Shiea, J.. (2009) Melamine contamination. Anal. Bioanal. Chem. 395(3), 729-735.

  3. LC-MS/MS analysis of emerging food contaminants. SCIEX technical note, RUO-MKT-02-12454-A.
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