Effects of Wet-Blending on Detection of Melamine ... - ACS Publications

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Effects of Wet-Blending on Detection of Melamine in Spray-Dried Lactose Betsy Jean Yakes, Marti M. Bergana, Peter F. Scholl, Magdi M. Mossoba, Sanjeewa R Karunathilaka, Luke K Ackerman, Jason D. Holton, Boyan Gao, and Jeffrey C Moore J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00834 • Publication Date (Web): 24 May 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Effects of Wet-Blending on Detection of Melamine in Spray-Dried

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Lactose

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Betsy Jean Yakes‡, Marti M. Bergana§, †, Peter F. Scholl‡, Magdi M. Mossoba‡, Sanjeewa R.

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Karunathilaka‡, Luke K. Ackerman‡, Jason D. Holton§, Boyan Gao+ and Jeffrey C. Moore Î, *

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‡

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Regulatory Science, 5001 Campus Drive, College Park, MD 20740 USA

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§

U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of

Abbott Nutrition, Division of Abbott Laboratories, Research and Development, 3300 Stelzer

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Road, Columbus, OH 43219 USA

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+

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USA

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Î

U.S. Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD 20852 USA

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†

Current Address: Consultant, U.S. Pharmacopeial Convention

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742

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Corresponding Author

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*Phone: +1 301 816 8288. Fax: +1 301 816 8157. E-mail: [email protected]

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ABSTRACT

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During the development of rapid screening methods to detect economic adulteration, spray-dried

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milk powders prepared by dissolving melamine in liquid milk exhibited an unexpected loss of

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characteristic melamine features in the NIR and Raman spectra. To further characterize this

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“wet-blending” phenomenon, spray-dried melamine and lactose samples were produced as a

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simplified model and investigated by near-infrared spectroscopy, Raman spectroscopy, 1H NMR,

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and DART-FTMS. In contrast to dry-blended samples, characteristic melamine bands in NIR

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and Raman spectra disappeared or shifted in wet-blended lactose-melamine samples. Subtle

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shifts in melamine 1H NMR spectra between wet- and dry-blended samples indicated differences

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in melamine H-bonding status. Qualitative DART-FTMS analysis of powders detected a greater

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relative abundance of lactose-melamine condensation product ions in the wet-blended samples,

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which supported a hypothesis that wet-blending facilitates early Maillard reactions in spray-dried

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samples. Collectively, these data indicated the formation of weak, H-bonded complexes and

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labile, early Maillard reaction products between lactose and melamine contributes to spectral

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differences observed between wet- and dry-blended milk powder samples. These results have

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implications for future evaluations of adulterated powders and emphasize the important role of

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sample preparation methods on adulterant detection.

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Keywords. Adulteration, DART-FTMS, H-bonding, lactose, Maillard reaction, melamine, milk

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powder, NMR, NIR, Raman spectroscopy, wet-blending

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INTRODUCTION

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The U.S. Pharmacopeia (USP) has been leading a collaborative research project to develop

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reference standards and analytical methods for the targeted and non-targeted detection of

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economically-motivated adulteration in foods. As a first model, skim and nonfat dry milk

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powders (SMP and NFDM, respectively; collectively called MPs) were recently evaluated using

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both targeted and non-targeted techniques.1 Non-targeted methodologies are of interest due to

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their potential utility for high-throughput, in-situ screening and ability to flag a food matrix as

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adulterated with new or unknown compounds that might be missed by targeted methods. Non-

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targeted procedures that offer these advantages include near infrared (NIR), mid-infrared (MIR),

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and Raman spectroscopies, in conjunction with chemometrics.2 However, one challenge of

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applying these vibrational spectroscopies is that highly variable and complex food matrices can

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degrade method performance.

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To evaluate the effect of matrix complexity on the efficiency and accuracy of detection while

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also investigating the potential to detect MP adulteration at different points in the supply chain,

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two classes of melamine-spiked MP samples were prepared and studied in our previous work.1

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The first class had melamine mixed with MP through physical mixing (i.e., dry-blending (DB-

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MP)), while the second was prepared by spray-drying melamine dissolved in liquid milk (i.e.,

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wet-blending (WB-MP)). Interestingly, the WB-MP samples did not exhibit the NIR first

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overtone, primary amine stretching vibration (6812 cm-1) expected for melamine. Instead, this

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band was replaced by new bands that were broader and frequency-shifted, and these observations

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were attributed to wet-blending (WB). As such, targeting the prominent 6812 cm-1 band, as

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commonly performed,3-5 could no longer be considered appropriate for the detection of 3 ACS Paragon Plus Environment


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melamine from all adulteration routes. The implications from the discovery of this WB effect are

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clear: other nitrogen contaminants may also present similar spectral challenges and would

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thereby be less likely to be detected by targeted spectroscopic methods.

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Based on the potential effects of elevated temperature and pressure that are used in spray-drying,

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a plausible explanation for these spectral shifts was the development of a different crystalline or

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amorphous form, and this phenomenon has been reported in the literature for other compounds

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(e.g., lactose,6 simvastatin7). Results of XRD, PLM, Raman, and NIR analyses in previous MP

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studies were consistent with such a change only for lactose (i.e., loss in crystallinity features

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upon spray-drying).1 A second explanation considered potential chemical reaction(s) between

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melamine and MP constituents. Quantitative LC-MS/MS and qualitative 1H NMR data did not

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indicate melamine degraded in WB samples. These data collectively provided evidence that the

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WB effect was not predominantly driven by an irreversible chemical modification of melamine.

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In this paper, additional interactions, specifically those that involve the primary amine in

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melamine and a compound naturally found in MP (lactose), are explored. One interaction that

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could have arisen from this system was hydrogen-bonding (i.e., H-bonding), where melamine

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has been shown in literature to H-bond to other molecules and create new structures. These

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structures can range from small to supramolecular, often depending on the mixing method,

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pressure, and temperature, with the chemistry well-described in a number of articles.8-10 Indeed,

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shifts in Raman and IR bands have previously been attributed to melamine H-bonding, notably in

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work by Li et al. that showed both band broadening and shifts for the OH group of ascorbic acid

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and NH groups of melamine in FTIR spectra.11 Mircescu et al. have also performed detailed


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calculations and experiments using Raman spectroscopy that demonstrated how melamine

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spectral bands can be shifted by amine protonation.12

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Maillard reactions between melamine and lactose were also considered herein. Controlling such

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reactions between lactose and amines is important in the food and pharmaceutical industries

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because these determine the organoleptic properties and nutritional quality of foods, as well as

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the stability of drug commodities. As such, these reactions have been well-studied, including

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work on milk protein lactosylation as a function of pasteurization and MP storage conditions.13, 14

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Although lactose serves well as an inactive ingredient in many pharmaceuticals, studies have

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documented unwanted interactions of lactose excipients with drugs that contain amines.15, 16

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Maillard reaction products have been characterized in protein-free model systems containing

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melamine and lactose, using reverse-phase LC-MS/MS.17 Related studies have also evaluated the

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kinetics of reactions between melamine and a variety of aldehydes.18

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Previous efforts to understand the cause(s) of the WB effect in MP were limited due to the

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complexity of the MP matrix.1 As lactose is one of the most prevalent components in MP, from a

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chemical concentration and chemical functional group standpoint,19 a simple melamine-lactose

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model system was developed for this study. This simple model system was used to prepare the

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samples listed in Table 1, and, in order to maintain consistency with our previous MP study,1

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this system was processed using similar spray-drying techniques and at equivalent melamine

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concentrations. NIR, Raman, NMR and DART-FTMS instrumentation were then employed to

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qualitatively analyze this simpler system and characterize spectral changes upon wet-blending.



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This lactose-melamine model gave insight into potential interactions (i.e., H-bonding and/or

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lactose-melamine adducts) that may cause the band disappearance and shifting phenomena.

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MATERIALS AND METHODS

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Samples. Sample information including ID, melamine and lactose concentrations, and

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preparation type are indicated in Table 1. A lactose and melamine solution (0.23 g/mL α-Lactose

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monohydrate, 2.82 mg/mL melamine (both reagent grade from Sigma-Aldrich, St. Louis, MO))

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was prepared by dissolving lactose in water at 52 °C, adding melamine, and holding for 10 min

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before spray-drying. This sample was identified as wet-blended “WB-Lac”. A lactose-only

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control was similarly prepared; this mixture was identified as “SDLactose”. To explore whether

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changes in melamine crystallinity could be contributing to spectral differences, a 4.35 % (w/v)

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aqueous solution of melamine was gently warmed, to aid dissolution, and spray-dried; this

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sample was identified as “SDMelamine”. All spray-dried samples were processed using a Buchi

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B-290 bench-top spray-dryer (New Castle, DE) using an inlet temperature of 220 °C, outlet

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temperature of 123 °C, feed rate of 6 mL/min, and airflow at 357 L/hr. For the dry-blended

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melamine sample, 0.123 g of unprocessed melamine was blended into 10 g of dry SDLactose;

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this sample was identified as dry-blended “DB-Lac”. All samples were stored in a desiccator

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until use.

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NIR Measurements. Spectra were acquired using an MPA Bruker Optics (Billerica, MA) FT-

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NIR spectrometer equipped with an integrating sphere, diffuse reflection accessory including a

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sample rotating cup, and PbS detector. Spectra were collected at room temperature using 16 cm-1

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resolution in the 10,000 to 4000 cm-1 range at 32 scans. Replicate spectra (N=3) were measured 6 ACS Paragon Plus Environment

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for each test portion in a randomized order. Instrument performance was internally verified

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according to vendor-specific tests with a USP NIR Suitability Reference Standard (Rockville,

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MD). Included in the OPUS software is the Optics Validation Program (OVP), which executes a

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series of performance tests using the standards in an automated filter wheel. This program was

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used to verify that the spectrometer was operating within specifications prior to sample

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acquisition.

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Data were preprocessed and analyzed using PLS_Toolbox software within a Matlab

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computational environment (PLS_Toolbox_7.82, Eigenvector Research Inc., Wenatchee, WA).

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To eliminate scaling effects, standard normal variate (SNV) normalization was applied to the

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derivative spectra, while fourth derivative transformation (Savitzky-Golay algorithm, window

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size = 15 points, fourth order polynomial fit) was employed to eliminate baseline artifacts.

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Raman Spectroscopy. Raman spectra were acquired on a DXRxi Raman Imaging System

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(Thermo Electron North America, Madison, WI) controlled with OMNICxi Raman Imaging

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Software. Automated alignment and calibration procedures were performed prior to sample

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measurement. Instrument parameters were: 780 nm laser at 24 mW, full-range grating (5 cm-1

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nominal resolution (FWHM), 50-3300 cm-1), 10× microscope objective, and 50-µm pinhole

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aperture. Approximately 0.05 g of each powdered sample was placed onto individual, plain glass

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microscope slides (Fisher Scientific, Pittsburgh, PA) for analysis. For melamine samples, spectra

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were obtained with a 0.2 s exposure and 60 scans. For lactose and mixture samples, spectra were

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obtained with a 3 s exposure and 10 scans. For chemical imaging, a 75 × 81 µm2 area was

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defined, and spectra were obtained via an image pixel size of 3 µm and 2 s exposure with 2 scans



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per spectrum. Raman spectral intensities were recorded as counts per second (cps). Data analysis

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was performed with Atlµs in OMNIC for Dispersive Raman (v. 9.2) and GraphPad Prism (v.

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5.02, La Jolla, CA).

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tetramethylsilane ((TMS), Cambridge Isotope Laboratories, Inc., Andover, MA) and vortex

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mixed for ~1 min. Immediately following a 10 min centrifugation at 1417g (Fisher Scientific

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Micro-Centrifuge 59A), the supernatants were transferred to NMR tubes, and the spectra were

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acquired. To maintain similar times between the introduction of solvent and data collection,

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samples were prepared and analyzed sequentially. For reagent melamine, an arbitrary amount of

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melamine was added to DMSO-d6 to allow for evaluation. Proton (1H) NMR spectra were

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collected with a 500 MHz VNMRS spectrometer equipped with a 3 mm PFG-ID probe (Varian,

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Palo Alto, CA), utilizing a pulse width of 90° and a 10 s pre-pulse delay. A sweep width of

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6510.4 Hz and an acquisition time of 2.517 s were employed. Each spectrum (32 scan average)

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was collected at 25 ± 0.1 °C and internally referenced to TMS at 0.0 parts per million (ppm).

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Spectra were processed with MestReNova software (v. 10.0.2, Mestrelab Research, Santiago de

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Compostela, Spain).

H NMR Analysis. Samples (6.0 ± 0.1 mg) were dispersed in 600 µL of DMSO-d6 with

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DART-MS. Powders were fastened to DipIt™ sample introduction tips (melting point

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capillaries, LEAP Technologies, Carrboro, NC) using a thin film of silicone high vacuum grease

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(HVG, Dow Corning Corp., Greensboro, NC). HVG was dissolved (17 mg/mL) in hexane

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(pesticide residue grade, Fluka, Milwaukee, WI) and aged 48 h prior to use to allow SiO2 solids

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to precipitate. Sample capillaries were then dipped into the supernatant, and the solvent was


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evaporated (~15 min) to produce a thin film coating. Three HVG coated sampling capillaries

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were dipped into vials containing each dry, powdered sample, and the capillaries were gently

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tapped against each vial to remove loose particles. Each capillary was subjected to two

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concentrated blasts (

Effects of Wet-Blending on Detection of Melamine ... - ACS Publications

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