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

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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

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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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1

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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 in Spray-Dried

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