Effects of Adulteration Technique on the NIR Detection of Melamine in Milk Powder
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1 SUPPLEMENTAL FIGURES AND TABLES Effects of Adulteration Technique on the NIR Detection of Melamine in Milk Powder Peter F. Scholl,*, Marti Mamula Bergana,, Betsy Jean Yakes, Zhuohong Xie, Steven Zbylut **, Gerard Downey, Magdi Mossoba, Joseph Jablonski 2, Robert Magaletta ç, Stephen E. Holroyd çç, Martin Buehler Î, Jianwei Qin ø, William Hurst, Joseph H. LaPointe^, Dean Roberts ÎÎ, Carol Zrybko ç, Andrew Mackey ç, Jason D. Holton, Greg A. Israelson, Anitra Payne œ, Moon S. Kim ø, Kuanglin Chao ø, Jeffrey C. Moore * 1
2 Effects of Adulteration Technique on the NIR Detection of Melamine in Milk Powder SUPPLEMENTAL FIGURES AND TABLES Supplemental Table S1: Impedance Spectroscopy Data Sample Description SMP (non-spiked) USP Sample Code Melamine (% w/w) t g * [h] f [Hz] τ [h] n (fitting parameter) S W W WB melamine/smp W W4A Time to reach 20 mhz W4C W Supplemental Table S2: XRD Acquisition Sampling Rates Sample Description USP Sample Melamine Rate Code (% w/w) (⁰/min) melamine A DB A027 + S085 melamine/smp WB melamine/smp W W W
3 Supplemental Figure 1a. NIR Reflectance Spectra of WB and DB SMP Samples (Lab N2). (Green) non-spiked pilot-scale SMP (W0-1); (red) 0.033, 0.089, 0.30, and 0.39% WB melamine in SMP (W2-1; W3-1; W4A-1; W4C-1); (blue) 0.012, 0.046, 0.098, 1.2% DB melamine in SMP (A027 + W0-1); (black) melamine (A027). Reflectance spectra SNV normalized and offset for clarity. An expanded view from nm and the 2 nd derivative spectra are provided in Supplemental Figures 1b-c. 8 3
4 Supplemental Figure 1b-c. NIR Reflectance Spectra of WB and DB SMP Samples (Lab N2). (B) Expanded view ( nm). (C) 2 nd derivative with gap size of 7 points. (Green) non-spiked pilotscale SMP (W0-1); (red) 0.033, 0.089, 0.30, and 0.39% WB melamine in SMP (W2-1; W3-1; W4A-1; W4C-1); (blue) 0.012, 0.046, 0.098, 1.2% DB melamine in SMP (A027 + W0-1). 15 4
5 Absorbance Units DB Loadings WB Loadings Reagent Melamine Spectrum PC2 PC Supplemental Figure 2. PCA Loading Plots of DB and WB Melamine in SMP (Lab N3). PCA loading plots and reagent melamine spectrum are scaled and offset for clarity. Spectra were mean center value processed, without applying SNV and 1 st derivative smoothing, to highlight similarity between the spectrum of crystalline melamine and the PC1 loading plot in dry-blended samples. Samples: DB SMP (A027 + W0-1) at 0.089, 0.39, 0.50, 1.0%; WB Set-1 SMPs at 0.033, 0.089, 0.30, 0.39% Wavelength (nm) 37 5
6 Supplemental Figure 3. SIMCA Classification Class Distances (Lab N3). SIMCA class distance increased with the amount of added melamine. 45 6
7 Supplemental Figure 4. Raman Spectra of Wet- and Dry-blended Melamine in SMP (Lab R1). (blue) Raw spectra of 0.39% WB melamine in SMP (W4C-1); (red) 0.39% DB melamine in SMP (A027+ W0-1); (grey) non-spiked SMP (W0-1). The narrow wavenumber range indicated in the green boxed area was presented in Figure 5a. 52 7
8 Supplemental Figure 5. Ratio Raman Imaging Spectra for WB and DB Melamine in SMP. Powder samples were put in 47 mm diameter Petri dishes. A point-scan Raman chemical imaging system with a 785 nm laser was used to acquire hyperspectral images. The system scanned a mm 2 area for each sample using a CCD exposure time of 0.1 s and a step size of 0.25 mm for both X and Y directions, resulting in a hypercube (10,000 spectra each with 1024 bands). A dark current image acquired with the laser off and a cap covering the probe was subtracted from the original sample image. A polynomial curve-fitting method was used to correct the fluorescence background. Mean spectra were obtained by averaging 10,000 fluorescence corrected spectra for each sample. A spectral ratio method was used to remove the SMP background and boost melamine Raman signals. The Raman peak positions for the 673 cm 1 melamine peak in the reference spectrum for (red) DB 0.33%/0%, (blue) WB 0.33%/0%, and (purple) WB 0.33%/0.089% are 673, 684, and 681 cm 1, respectively. 8
9 WB DB Melamine Supplemental Figure 6. 1 H NMR Spectra of Wet- and Dry-blended Melamine in SMP. (Green) WB 0.39% melamine in SMP (W4C-1); (red) DB 0.39% melamine in SMP (A027 + W0-1); (blue) reagent melamine. Solvent (99.9% DMSO-d 6 ); DMSO (2.5 ppm), H 2 O (3.3 ppm). 73 9
10 Supplemental Figure 7. XRD ( ⁰ 2θ) of DB Melamine in SMP. (Blue circles) reagent melamine (A027); DB melamine in non-spiked SMP (A027 +S085) (orange squares) 10%; (grey triangles) 5%; (purple dotted line) 1%; (red line) 0.5%; (green dashed line) non-spiked SMP (S085)
11 Supplemental Figure 8. DART-FTMS Spectra of WB and DB Melamine in SMP. (A) WB (0.39% melamine), chronogram averaged spectra ( min), 100% = 1.90E8. (B) WB (0 % melamine), min, 6.58E7. (C) DB (0% melamine), min, 1.55E8. (D) DB (1.0% melamine), min, 4.25E7. Retention times refer to Figure 8. (HMF+H) +, C 6 H 7 O 3, experimental mass error (EME, ppm). (MEL+H) +, C 3 H 7 N 6, EME ppm. (LAC+NH 4 -H 2 O) +, C 12 H 24 O 10 N, EME ppm. (LAC+MEL-2H 2 O+H) +, C 15 H 25 O 9 N 6, EME ppm. (LAC+MEL-H 2 O+H) +, C 15 H 27 O 10 N 6, EME ppm. (LAC+MEL+H) +, C 15 H 29 O 11 N 6, EME ppm. Lactose (LAC), melamine (MEL), hydroxymethylfurfural (HMF), lactose+melamine addition product (LAC+MEL)
12 Supplemental Figure 9. Expanded DART-FTMS Spectra (m/z ) of WB and DB Melamine in SMP. (A) WB (0.39% melamine), chronogram averaged spectra ( min), 100% = 2.39E7. (B) WB (0% melamine), min, 7.96E6. (C) DB (0% melamine), min, 2.37E6. (D) DB (1% melamine), min, 1.09E7. Retention times refer to Figure 8. (LAC+HMF-2H 2 O+H) +, C 18 H 25 O 12, EME ppm. (LAC+HMF-H 2 O+H) +, C 18 H 27 O 13, EME ppm. (LAC+HMF+H) +, C 18 H 29 O 14, EME ppm. Lactose (LAC), melamine (MEL), hydroxymethylfurfural (HMF), lactose+melamine adduct (LAC+MEL), lactose+hydroxymethylfurfural addition product (LAC+HMF). 12
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