Titanium Dioxide in Food Products: Quantitative Analysis using ICP

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Titanium Dioxide in Food Products: Quantitative Analysis using ICP-MS and Raman Spectroscopy Jin-Hee Lim, Dongryeoul Bae, and Andrew Fong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06571 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Journal of Agricultural and Food Chemistry

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Titanium Dioxide in Food Products: Quantitative Analysis using ICP-MS and Raman

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Spectroscopy

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Jin-Hee Lim*, Dongryeoul Bae, and Andrew Fong

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Office of Regulatory Affairs, Arkansas Laboratory, U.S. Food and Drug Administration, 3900

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NCTR Road, Jefferson, Arkansas 72079, United States

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*Corresponding author

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Tel: +1-870-543-4660

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Fax: +1-870-543-4021

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Email: [email protected] or [email protected]

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ACS Paragon Plus Environment


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Abstract

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Titanium dioxide (TiO2) is commonly used as a color additive in food products. In this study, a

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total of eleven food products such as coffee cream, yogurt snack, hard candy and chewy candy

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that are widely consumed by adults or children were investigated. For characterization of particle

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size, size distribution, crystallinity and concentration of TiO2, particles were first extracted using

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an acid digestion method from food and various analytical techniques were applied. All products

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investigated in this study contained nanosized TiO2 particles (21.3-53.7%) in the anatase phase.

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The particle size of TiO2 was in a range of 26.9-463.2 nm. The concentration of TiO2 in the

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products ranged from 0.015% (150 ppm) to 0.462% (4620 ppm). These values obtained using

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inductively coupled plasma-mass spectrometry (ICP-MS) were considered as the reference and

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were compared with Raman results to evaluate the feasibility of using the Raman method to

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quantitate TiO2 in food products. The Raman method developed in this study proved to

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effectively analyze anatase TiO2 in food products at levels of several hundred ppm or greater.

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Limitations of using the Raman method as a quick screening tool for determination of TiO2 are

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also discussed.

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Keywords: Titanium dioxide, nanoparticles, color additives, ICP-MS, Raman spectroscopy

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Introduction

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Titanium dioxide (TiO2) is widely used in versatile applications to improve the quality and

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palatability of consumer products such as food, personal care products, cosmetics, paints and

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coatings.1-3 With the wide use of TiO2 in various industries, nanosized TiO2 has raised safety

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questions regarding uncontrolled release and potential adverse impact of TiO2 nanoparticles

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(NPs) to the environment and human health.1, 4, 5 The first step in most risk assessment is an

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identification and characterization of nanomaterials. Understanding the physicochemical

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properties of TiO2 such as particle size, size distribution, shape, crystal structure, ionization

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conditions, coating materials, aggregation status, and particle concentration is an important factor

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to be considered as part of risk assessment.1 Although many studies have been conducted for

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developing analytical methods in the food industry, a major challenge in conducting research on

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analytical method development involve techniques to efficiently and effectively identify

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nanoparticles present in complex matrices and to determine the physicochemical properties of

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nanomaterials.6-9

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Many advanced techniques, including electron microscopy, spectroscopy, mass spectroscopy, X-

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ray diffraction (XRD), x-ray fluorescence (XRF), and light scattering, have been applied to

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characterize physicochemical properties of TiO2 in consumer products. Among these techniques,

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size characterization methods have been extensively developed. In recent years, field flow

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fractionation, capillary electrophoresis, and single particle inductively coupled plasma mass

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spectrometry (spICP-MS) which can separate nanomaterials and determine particle size

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simultaneously have emerged as viable methods.10-14 However, using an electron microscope

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with energy dispersive X-ray spectroscopy (EDS) is still the most widely applied method for 3



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determining the size, morphology, and aggregation status analysis of TiO2.13-16 In some cases,

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the selected area electron diffraction (SAED) patterns generated by transmission electron

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microscope (TEM) were used to identify crystalline information.17, 18 For qualitative and

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quantitative trace element analysis, inductively coupled plasma-mass spectrometry (ICP-MS) has

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been commonly used.5, 14 The quality of ICP-MS data for TiO2 analysis is highly dependent on

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sample preparation. Digesting the sample to a clear solution is essential for accurate analysis of

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materials using ICP-MS. Samples such as TiO2, SiO2 or carbon nanotubes, which are very stable

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in acids, require additional efforts for complete digestion. TiO2 is stable in nitric acid (HNO3),

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and slowly ionized in hydrofluoric (HF) acid.19, 20 Using a mixture of concentrated HNO3 and HF

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for sample digestion, more accurate and reliable results were obtained.11, 20-23 However, HF is an

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extremely dangerous chemical and requires diligence and utmost caution when used.

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Due to difficulties of ionizing TiO2 in ICP-MS analysis, researchers developed new approaches

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for TiO2 quantitation.23-25 One of the more recently developed methods for quantitation of food

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additives or toxic materials in food involved Raman spectroscopic methods.24-29 There are

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several research articles describing quantitative analysis of food products using Raman.26, 30-32

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Tan et al.32 described a single-drop Raman imaging technique for the analysis of trace

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contaminants in milk and Weng et al.31 reported quantitative analysis of ediphenphos residue in

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rice. Another group24, 25, 33 reported the quantitation of TiO2 in table sugar using Raman

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spectroscopy and also developed the flavonoid-assisted microextraction method for TiO2

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detection. The published methods showed a simple and rapid quantitative Raman method with

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standard TiO2 powders; however, those methods have limitations when applied to complex food

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matrices containing multiple organic and inorganic compounds. In addition, more studies are 4


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necessary to develop extraction methods to reduce or eliminate strong fluorescence effects

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caused by various food ingredients or to improve recovery.

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The aim of this work was to characterize TiO2 nanoparticles in consumer products such as coffee

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cream, yogurt snack, hard candy, chewy candy which are widely consumed by adults or children,

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and to evaluate the feasibility of Raman method to be used as a quick and simple technique for

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the quantitative determination of TiO2 in complex matrices. The TiO2 concentration determined

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by ICP-MS was used as a reference value to evaluate the accuracy of the Raman technique. The

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limitations of Raman spectroscopy associated with quantitation of total TiO2 in commercial

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products are presented.

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Materials and Methods

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Titanium - Ti (1000 and 10,000 mg/kg) and Scandium - Sc (1000 mg/kg) single-element ICP-

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MS standard solutions were acquired from Ultra Scientific (Metuchen, NJ), Ricca Chemical Co.

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(Arlington, TX), and Spex CertiPrep Group (Metuchen, NJ). TiO2 powders were purchased from

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the national institute of standards and technology (NIST 1898; Gaithersburg, MD), US Research

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Nanomaterials, Inc. (30-50 nm, Rutile, Houston, TX), nanoComposix Inc. (25 nm, 80%

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anatase/Rutile, San Diego, CA), Pronto Foods (Food & Pharmaceutical grade; Chicago, IL), and

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MakingCosmetics Inc. (Snoqualmie, WA). Nitric acid (HNO3, Optima 67-70%), hydrofluoric

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acid (HF, Optima 41-51%), hydrogen peroxide (H2O2, Optima 30-32%) ethanol (C2H5OH,

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denatured), and acetone [(CH3)2CO, ACS grade] were purchased from Fisher Scientific (Houston,

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TX). Aluminum oxide (Al2O3, >98%) was obtained from Sigma Aldrich (St. Louis, MO). Silicon

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oxide (SiO2, 99.9%, 20-60 nm) was purchased from SkySpring Nanomaterials, Inc. (Houston,

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TX). Type I ultra-pure water (18MΩ-cm) was available through a Direct-Q 3UV water 5



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purification system (EMD Millipore, Billerica, MA). The commercial products that claimed to

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contain TiO2, color additives, E171 white coloring, or artificial color were randomly obtained

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from various retailers, where E numbers are codes for specific substances used as food additives

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in Europe.34, 35 A total of eleven products, including coffee cream, cookie, yogurt snack, hard

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candy and chewy candy were tested and reported in this study. Each product was given a unique

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identification name such as RS01-RS11. Product information is listed in Table 1.

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TiO2 Particle Extraction from Food Products

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Sample products selected in this study contain various food ingredients or organic compounds.

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To remove the complex matrices, TiO2 containing products were digested in HNO3.

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Approximately 100-200 mg of each sample and 2 mL of nitric acid were heated at 180 ˚C for 3

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min using a Mars 6 microwave digestion system (CEM, Matthews, NC). Some samples such as

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candy, which has a very low concentration of TiO2 and contain various food color additives,

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needed an additional pre-extraction procedure. Approximately 1 g of candy samples was first

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dissolved in a 5 mL of mixture solution containing H2O2/HNO3 (10:0.1, v/v) at 80 ˚C. After the

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solid food was dissolved, the samples were centrifuged at 7000 × g for 10 min and the precipitate

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only was acid digested in 2 mL of nitric acid at 180 ˚C for 3 min. Digested solutions were then

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transferred into 15 mL tubes and centrifuged at 7000 × g for 10 min. After centrifugation,

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extracted particles were rinsed with ultra-pure water and acetone three times. For further analysis,

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the extracted particles were dispersed in ethanol and stored at room temperature.

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X-Ray Diffraction (XRD)

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The crystalline structure of TiO2 was characterized using an AXS XRD D2 Phaser (Bruker,

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Madison, WI) operated at 30 kV and 10 mA at 24 ˚C. Particles dispersed in ethanol were placed

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onto the zero diffraction silicon sample holder purchased from MTI Corp. (Richmond, CA).

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Dried samples were loaded into the instrument and XRD patterns were collected from 20-80˚ (2θ)

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using a Lynxzeye decector (Bruker) and Cu Kα (λ=1.541 Å) radiation. The data was analyzed

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using EVA 4.6 software.

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Transmission Electron Microscopy (TEM)

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A 2100 TEM (JEOL, Peabody, MA) operated at acceleration voltage of 200 kV was used for

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particle size characterization. Elemental analysis data was collected using an Oxford X-Max 80T

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EDS system. The isolated TiO2 particles were dispersed in ethanol and sonicated using ultrasonic

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bath for 10 min. A small amount (5-10 µL) of sample was placed on a carbon coated Cu grid

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(300-mesh) and dried overnight. The TiO2 particle size was statistically determined using ImageJ

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(NIH, Bethesda, MD).

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Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

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To determine Ti concentration, sample products were first digested in an acid matrix using a

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MARS-Xpress microwave digester (CEM) (maximum power: 1600 W). Approximately 100 mg

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of sample products, 3 mL of HNO3, and 1 mL of HF were transferred into a digestion vessel.

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Samples were digested at 210 ˚C for 20 min. After microwave digestion, the sample solutions

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were diluted to 25 mL with ultra-pure water. A second dilution step was performed by diluting

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0.1-1 mL of diluent to 50 mL with ultra-pure water and internal standard (Sc) added. The

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samples were analyzed using an 8800 ICP-QQQ with a PFA inert kit and nebulizer (Agilent 7



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Technologies, Santa Clara, CA). The concentrations of Ti were determined in single mode (m/z

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47) with Helium (He) collision cell.23 Initial and continuing calibration verification standards,

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reagent and sample blanks were prepared and analyzed along with the samples. A weight

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fraction of Ti was calculated from the molecular weight of TiO2 (Ti=60%) and was used to

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determine percent (%) concentration of TiO2 in food products. All samples for ICP-MS were

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analyzed in triplicate.

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Raman Spectroscopy

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Raman scattering measurements were performed using a LabRam HR Raman spectrometer

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equipped with a 633 nm excitation laser and a 10X objective (Horiba Scientific, Edison, NJ). The

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spatial resolution and laser spot diameter were approximately 1.5 µm and 3.0 µm, respectively.

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The Raman spectra were obtained with a 100 µm slit aperture with a 5 s acquisition time.

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LabSpec 6.4 software was used for Raman data acquisition and analysis. The TiO2 calibration

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curve was developed with six independent TiO2 working standards with 0.07-2.3wt%. The

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calibration standard powders were prepared by mixing anatase TiO2 particles (food and

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pharmaceutical grade) with Al2O3. In the case of food samples, particles were extracted by acid

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digestion described previously. Dried particles were mixed with Al2O3, where the amount of

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Al2O3 was dependent on the original amount of each sample digested (100-200mg). In the case

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of RS06, multiple samples were digested to prepare one Raman sample due to low TiO2

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concentration and the extracted TiO2 was concentrated almost 20-fold while mixed with Al2O3.

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The mass of extracted samples and Al2O3 were recorded. Well mixed standards or samples were

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placed onto a glass slide and pressed with another slide in order to make a flat surface. A flat

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surface facilitated focusing and moving the stage quickly. To improve the accuracy of 8


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quantitative analysis, more than fifteen discrete locations in each sample were selected under the

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Raman microscope by controlling x-y axis and scanned in the range of 50-800 cm-1. All

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experiments were performed in triplicate with independent samples and average concentration

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reported.

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Results and Discussion

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A total eleven food products were selected and procured for this study. A list of products with

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sample identification number, product type, and TiO2 label information are provided in Table 1.

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Ten of the eleven products investigated clearly listed the presence of TiO2 as ‘Titanium Dioxide’

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with color, color additives, or E171. However, the RS03 product label just indicated the presence

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of color additive as ‘Color added’. The color of the eleven products was diverse and many other

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color additives were listed along with TiO2 on their ingredient list.

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Crystallinity, Shape, Particle Size and Size Distribution Analysis

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To isolate TiO2 particles from the complex matrices, the acid digestion method was adopted in

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this study. The TiO2 recovery after the extraction procedures was tested before the method was

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applied to sample products. Between 91.4-96.3% of TiO2 spiked in chewy candy, sucrose, and

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water samples were extracted after the acid digestion and centrifugation procedures (n=6). This

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test confirmed that the extraction efficiency was higher than 90%. In addition, the pure particles

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and the extracted particles were characterized to prove that the method did not alter the size,

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morphology and crystallinity of TiO2 particles. The XRD patterns of TiO2 before and after

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extraction presented only anatase. In this study, it was difficult to demonstrate statistically how

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much the acid effect on particle size and morphology of TiO2 because of the broad size 9



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distribution of TiO2 and the characteristic of TiO2 easily agglomerate/aggregate in water.

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However, the preliminary results proved that this extraction method can be applied to

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characterize size and concentration of TiO2 in food products using various analytical instruments

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below.

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To isolate TiO2 particles from the complex matrices, all food products were acid digested leaving

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TiO2 as an insoluble precipitate. Following digestion, the white TiO2 particles were collected

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after centrifugation. The crystallinity, shape, particles size and size distribution were

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characterized using TEM and XRD. This method allowed detection of very low amount of

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particles in products such as in RS06. The limit of sensitivity for detecting the other phases of

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TiO2 was evaluated with mixture of anatase and rutile TiO2 powders. The XRD results proved

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that the instrument was sufficient to detect approximately 1% rutile in 99% anatase TiO2. As

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shown in Figure 1, TiO2 particles extracted from the food products (RS01-RS11) were in the

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anatase phase. No other types of TiO2 structures such as rutile or brookite were detected. The

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peak positions were consistent with the standard diffraction pattern of anatase TiO2 (JCPDS no.

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21-1272).22 Diffraction peaks were found at 2θ = 25.3°, 36.9°, 37.8°, 38.5°, and 48.1°, which can

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be indexed to the (101), (103), (004), (112), and (200) planes. Some impurities were found at

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28.7° in RS07, but the peak did not correspond to any peaks of rutile phase at 2θ = 27.5°, 36.3°,

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41.3°, 54.4°, 62.8° (JCPDS no. 21-1276). Figure 2 showed TEM images and size distribution

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histograms. The extracted nanoparticles were mostly spherical in shape, but some particles were

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rod-shaped. In addition, as expected, many particles were aggregated and size ranged from

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several hundred nanometers to several micrometers. The size of individual TiO2 particles was

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calculated using ImageJ software and more than 350 particles of each sample were measured. As

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shown in Table 1 and Figure 2, the particles had a broad size distribution, between 26.9 and 10


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463.2 nm. The smallest particle sizes of TiO2 found in products were between 26.9 and 40.3 nm

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and all products contained particles that were less than 100 nm. The mean particle size of each

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product was in the range of 103.5-147.8 nm. The size distribution histogram of each product was

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generated to examine the ratio of nanoparticles in total TiO2 detected in the food products

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(Figure 2). In this study, 21.3-53.7% of TiO2 were found to be nanosized particles and the values

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varied within products without any relationship to product type. A large number of nanosized

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TiO2 particles were found in RS03 (49.8%), RS04 (53.7%) and RS09 (50.4%). Comparison of

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TiO2 particles found in the foods and food grade TiO2 powders proved that size distribution,

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shape and aggregation/agglomeration of TiO2 were very similar (data not presented).

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ICP-MS Analysis

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ICP-MS was chosen as a determinative technique for the quantitative analysis of the food

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products studied for TiO2. The ICP-MS instrument was calibrated for Ti with seven calibration

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solutions ranging in concentration from 10 to 500 µg/L. The calibration resulted in a correlation

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coefficient (r2) with excellent linearity (≥ 0.9999). For the system suitability test, seven

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consecutive injections of a standard that contains mid-level (0.2 mg/L) concentration were

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performed.36 The average concentration of seven consecutive injections and relative standard

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deviation (RSD) were 197.5±1.4 (98.8% recovery) and 0.7%, respectively. Method detection

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limit (MDL) was calculated by multiplying the student's t value (3.143) for the seven replicates

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(n=7) by the standard deviation of the calibration standard at the lowest analyte concentration

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(0.010 mg/L of Ti).23, 36 Limit of quantitation (LOQ) was defined as 10 times the standard

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deviation. MDL and LOQ for ICP-MS analysis were 0.001 mg/L and 0.004 mg/L, respectively.

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Continuing calibration verification (CCV), initial calibration verification (ICV) and spiked 11



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sample recovery (acid digested) were also determined in the validation process. The recovery

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values of CCV and ICV were in the range of 100-107%. The spiked sample recovery was

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102.6%. The concentration of Ti in reagent blank and sample blank was examined at least once a

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batch and the determined concentration was always much lower than MDL. All test results for

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method validation showed good accuracy and precision, within ±10% of nominal values and less

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than 2.0% of RSD.

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After method validation, eleven products containing anatase TiO2 were characterized by ICP-

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MS. The concentration of TiO2 (%) in each product is shown in Table 1. The range of TiO2

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concentrations in the products was between 0.015% and 0.462% (150-4620 mg/kg). As

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mentioned earlier, some of the products such as chewy candy showed multiple colors, which

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means various color additives were used to present a specific color. However, the ICP-MS

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results showed that the amount of TiO2 present in each sample with different colors was very

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close and that the TiO2 was homogeneously distributed regardless of sample product color.

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The U.S. Food and Drug Administration approved the use of TiO2 in 1966 with an allowed limit

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of TiO2 in food at 1% of the overall food weight (US FDA 21CFR73.575).37, 38 The

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concentrations of TiO2 used as color additives in these products were much lower than the 1%

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limit of TiO2.

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Raman Analysis

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We have developed a quantitation method for the determination of TiO2 in commercial food

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products using a Raman spectroscopy. Anatase TiO2 showed a strong peak at 143 cm-1 followed

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by low intensity peaks located at 196, 395, 518 and 638 cm-1, which can be assigned as Eg, Eg,

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B1g, A1g+B1g, Eg modes, respectively.39 For quantitative analysis, six working standard powders 12


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ranging from 0.05-2.36% TiO2 (700 mg/kg to 23600 mg/kg) were prepared and Raman signals

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for each standard were recorded. A calibration curve was constructed from a series of standards

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(Figure 3), where the x-axis is the known concentration (%) and the y-axis is the intensity of

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peaks at 143 cm-1. Values for slope (m) and the y-intercept (b) were determined using Origin Pro

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software. The Raman signal at 143 cm-1 increased in proportion to the particle concentration and

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the calibration curve showed a good linear correlation (r2=0.9982). The calibration curve was

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verified by measuring twelve independent standard powders with various concentrations (0.3-2.0%

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TiO2). The percent recovery values (%RV) for twelve calibration verification samples was

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between 85.4 and 108.8%, and average recovery was 96.1±7.1%, exhibiting good reproducibility

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by this quantitative Raman determinative method. In addition, to evaluate the quality of Raman

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working standard, three independent samples with approximately 1% TiO2 (10000 mg/Kg) were

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also analyzed by ICP-MS (Table 2). The recoveries for ICP-MS and Raman analyses were

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calculated as follows:

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%RV1= ([TiO2]ICP-MS / [TiO2]known value) × 100

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%RV2= ([TiO2]Raman / [TiO2]ICP-MS) × 100

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where [TiO2]ICP-MS are the TiO2 concentration determined by ICP-MS, [TiO2]Raman are the TiO2

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concentration determined by Raman, and [TiO2]known value is the theoretical values. Correlation

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between the ICP-MS and Raman techniques was determined by calculating the difference value

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(DV). %DV were defined as

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%DV= ([TiO2]Raman - [TiO2]ICP-MS) / (([TiO2]Raman + [TiO2]ICP-MS)/2) × 100

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The mean %RV for ICP-MS and Raman ranged from 91.9-106.3% and the %DV for the

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standard samples between ICP-MS and Raman methods were found to be less than 10% (Table

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2). These results indicate that powder working standards can be applied to quantitative

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determination of TiO2 particles using Raman.

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To calculate MDL and LOQ for Raman analysis, seven individual measurements were carried

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out using the lowest calibration standard (0.05%). The calculated MDL and LOQ were 0.006%

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(60 mg/kg) and 0.02% (200 mg/kg), respectively. LOQ is the lowest concentration reportable for

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Raman analysis, but does not imply that only products containing more than 0.02% can be

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analyzed. In this study, samples were concentrated while the particles were isolated and mixed

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with Al2O3 matrix. In some cases, the final TiO2 concentration in Al2O3 was 10 times higher than

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the original concentration. For example, the original concentration of yogurt snack sample (RS06)

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has 0.013% TiO2 (130 mg/kg) in products (lower than LOQ). However, TiO2 were detectable by

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Raman after concentration from the extraction process. The %RV for this sample was 86.7% as

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shown in Table 1. To test extraction efficiency, five independent spiked samples were prepared

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and analyzed by Raman. Test results are summarized in Table 3. Recoveries of each spiked

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sample ranged between 107.2% and 117.0%, which are higher than expected (average recovery:

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112.9%).

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After the Raman method validation, quantitative analyses of RS01-RS11 samples were

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conducted and results are summarized in Table 1. For efficient TiO2 extraction, nitric acid was

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used to digest food and other ingredients, excluding TiO2. Recovery (%RV2) results demonstrate

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that almost all food compounds were successfully digested and extracted TiO2 particles were

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well distributed in Al2O3. Because original concentration of TiO2 information in food products is 14


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not available on label (manufacturers are not required to label the final concentration of TiO2 in

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food products), Raman results were only compared with ICP-MS data. The %RV2 ranged from

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86.7-116.0%. The %DV was between -14.3 and 14.8%. These data demonstrate that the Raman

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technique was able to quantitate % concentration of TiO2 particles in food products (RS01-RS11)

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as shown by %DV under ±20% for each concentration level analyzed. In addition, this Raman

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technique can be utilized as an alternative method to quantitate metal oxide compounds such as

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TiO2 in food.

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To achieve better quantitative results using Raman spectroscopy, one should consider several

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factors which can affect the Raman analysis: mixing with Al2O3 matrix; fluorescence effect;

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crystallinity of TiO2; and other ingredients remaining after acid extraction. As shown in TEM

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image (Figure 2 and Table 1), some extracted particles aggregated and particle size varied from

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nanometer to micrometer. Particle aggregation in the matrix can cause signal variation. For a

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successful quantitative analysis, the particles were first milled and redispersed in Al2O3 matrix

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after acid extraction. With the large laser spot diameter and multiple scans (n≥15) of each sample,

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the variation also decreased. A large number of replicates ensured the reliability of the method,

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although the process was time consuming. The Al2O3 matrix is commonly found in environment

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and is very stable in air. Al2O3 does not interfere with Raman scattering for TiO2.

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Various organic compounds added in commercial products can create strong fluorescence effects

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on Raman scattering and the intense background can hide Raman peaks scattered from target

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materials. In addition, the fluorescence background is also found when particles contain

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amorphous TiO2 or have defects in the crystal structure (oxygen vacancies in the nanocrystal

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structure).40, 41 In this study, some samples such as RS07, RS08, RS10, and RS11 showed a 15



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strong fluorescence background. RS07 samples had more than 4000 background intensity in the

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first scan. With strong florescence effects, the peak intensity at 143 cm-1 was not correlated with

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the calibration curve. To decrease background intensity, the photobleaching method, which is

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based on constant sample irradiation for a long period of time using a same light source, was

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attempted.42 After multiple scans at the same position, background counts were reduced to

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around 1000 counts. However, the peak height (h) at 143 cm-1 for each scan was not influenced

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by the fluorescence background, but no significant changes were found in each scan. For those

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samples showing strong fluorescence background, samples were irradiated at least three times

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before acquiring a spectrum. With calculation of the peak height at 143 cm-1, acceptable values

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for RS07, RS08, RS10, and RS11 were obtained and summarized in Table 1.

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SiO2 is a food additive widely used as an anti-caking agent in food. The U.S. FDA approved the

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use of up to 2.0% SiO2 by weight of the food (U.S. FDA 21CFR172.480).43 As shown in Table 1,

331

RS02 label claimed to contain both TiO2 and SiO2. While TiO2 particles were extracted in nitric

332

acid, SiO2 were also isolated and mixed with Al2O3 matrix before Raman analysis. To evaluate

333

any effect of SiO2 on TiO2 quantitation, SiO2 nanoparticles (1.98%) were mixed with TiO2

334

(1.50%) and measured by Raman (Figure 4). A negligible fluorescence background was

335

observed at this concentration, and an average recovery value of TiO2 was 98.6%. In addition,

336

peak positions of the Al2O3 matrix and SiO2 were not overlapped with anatase TiO2. However,

337

peaks for rutile TiO2 were very close to anatase phase and the main peak of anatase at 143 cm-1

338

was overlapped with rutile phase.

339

Anatase and rutile phase of TiO2 are commonly used in food and cosmetic products,

340

respectively.22, 44 In cases of mixtures of anatase and rutile TiO2 in samples, the rutile peak 16


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341

interfered with the peak intensity of anatase TiO2 at 143 cm-1 resulting in poor recovery. When

342

TiO2 containing anatase and rutile (80:20, nanoComposix) was measured by Raman, the average

343

recovery of anatase with the current calibration curve was 20.1% (n=3). If the TiO2 is extracted

344

from the sample and crystallographic data indicate the presence of both anatase and rutile TiO2,

345

Raman quantitative methods would not be appropriate. Although this study did not include any

346

products containing both anatase and rutile forms of TiO2, consideration of the crystalline

347

structures is important when Raman quantitation method is applied.

348

Concentrations of TiO2 particles were accurately quantitated using the Raman method developed

349

in the current study. By comparing the Raman results with ICP-MS, the accuracy of the method

350

was evaluated. Our results demonstrated that the Raman technique is feasible for both qualitative

351

and quantitative analysis of TiO2 in food products. Several factors such as extraction method,

352

fluorescence effect, purity of material, crystallinity, mixing with new matrix can affect the final

353

results. A suitable extraction/precipitation method which does not alter the properties of material,

354

but reduces fluorescence effects is required to improve the accuracy of the Raman method for

355

quantitative analysis.

356

Developing new analytical methods is scientifically and academically significant. The

357

quantitative methods developed by the He group24, 25, 30 led to the conclusion that Raman

358

spectroscopy is less sensitive and difficult to apply for quantitative analysis. Their methods

359

showed higher sensitivity (2 mg/kg of detection limit for anatase TiO2) compare to our method

360

(60 mg/kg of detection limit). They also demonstrated that the existing methods are accurate and

361

can be applied to detect TiO2 in pure or simple matrix such as table sugar or spiked samples such

362

as pond water. However, the published methods were difficult to directly apply to commercial 17



363

products containing multiple ingredients for survey study or risk assessment. Commercial food

364

products are not as simple as the specimens used in their studies. Foods contain various organic

365

and inorganic compounds and these materials can interfere or reduce the Raman intensity of

366

TiO2. Specially, metal oxide (i.e., ZnO, SiO2, iron oxide, calcium phosphate, etc.) remaining

367

after centrifugation and organic compounds producing strong a fluorescent effect could be main

368

issues with the existing methods to quantitate TiO2 in food products, waste water (environmental

369

samples) or drug products. In contrast, our acid extraction and Raman method could minimize

370

those issues and have better accuracy when the Raman technique applied to quantitate TiO2

371

particles in complex matrices. However, it is very important to note that the accuracy of Raman

372

methods can be reduced dramatically when both anatase and rutile TiO2 exist in one sample. The

373

mixed form of TiO2 was found in commercial products in our previous studies.22, 41 Quantitating

374

mixed TiO2 in complex matrices and improving the sensitivity are still challenging.

375

For a realistic estimation of oral intake of TiO2, further investigations will be explored. It could

376

include more information on the number and types of food products containing TiO2, and the

377

concentration of TiO2 in these products. In addition, tracking the source of raw materials and

378

manufacturing processes producing food grade TiO2 powders could help the identification of

379

presence of nanomaterials in the final products.

380 381

Abbreviations Used

382

ICP-MS, inductively coupled plasma mass spectrometry; XRD, X-ray diffraction; EDS, energy

383

dispersive X-ray spectroscopy; TEM, transmission electron microscope; RSD, relative standard

384

deviation; MDL, Method detection limit; LOQ, limit of quantitation; CCV, continuing 18


Page 18 of 35

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385

calibration verification; ICV, initial calibration verification; RV, recovery value; DV, difference

386

value.

387

Acknowledgement

388

The views expressed in this manuscript are those of the authors and should not be interpreted as

389

the official opinion or policy of the U.S. Food and Drug Administration, Department of Health

390

and Human Services or any other agency or component of the U.S. government. The mention of

391

trade names, commercial products, or organizations is for clarification of the methods used and

392

should not be interpreted as an endorsement of a product or manufacturer. This research was

393

supported in part by an appointment to the Research Participation Program at the Office of

394

Regulatory Affairs/Arkansas Laboratory, U.S. FDA, administered by the Oak Ridge Institute for

395

Science and Education through an interagency agreement between the U.S. Department of

396

Energy and FDA.

397

Disclosure statement

398

No competing financial interests exist.

399

Conflict of interest

400

The authors declare that they have no conflict of interest.

401

Supporting Information

402

This material is available free of charge via the Internet at http://pubs.acs.org.

403

Particle extraction efficiency test and XRD characterization-FESEM images of before and after

404

extraction of TiO2 particles, and XRD peaks of anatase TiO2 before and after extraction; XRD 19



405

patterns of TiO2 particles with various ratio of anatase and rutile; Determination of ICP-MS

406

MDL and LOQ; ICP-MS calibration curve verification; CCV, ICV and spiked sample recovery

407

tests for ICP-MS; Raman calibration curve verification; Determination of Raman MDL and LOQ;

408

Raman spectra measured multiple times (n=10) at the same location of RS07 sample and the

409

corresponding peak height (h) of each scan at 143 cm-1; Raman spectra of anatase/rutile TiO2 and

410

anatase only TiO2 in Al2O3 matrix

20


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26


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Table 1. List of Food Products Investigated in this Study, Summarizing the Particle Size, Size Distribution, and Concentrations (%) of TiO2 Food ID Category

Labela

Particle size (nm) (mean±SD)

Particles with less than 100 nm (%)b

Particle size distribution (nm)c

% TiO2 concentration (mean±SD) ICP-MS Raman

%RV2

%DV

RS01

Coffee Cream

Titanium dioxide (color)

141.7±54.3

24.2

40.3-391.3

0.111±0.005

0.126±0.010

113.5

12.7

RS02

Coffee Cream

Titanium dioxide (artificial color)

131.0±51.0

29.4

33.5-318.4

0.140±0.001

0.152±0.043

108.6

8.2

RS03

Cookie

Color added

107.8±39.9

49.8

31.1-286.1

0.185±0.003

0.195±0.110

102.0

2.0

RS04

Yogurt snack

Titanium dioxide

104.8±42.1

53.7

30.7-328.0

0.300±0.002

0.271±0.028

90.3

-10.2

RS05

Yogurt snack

Titanium dioxide

131.5±49.8

28.3

26.9-348.4

0.192±0.005

0.209±0.026

108.9

8.5

Yogurt beverage Hard candy Hard candy

Titanium dioxide (E171)

117.0±55.3

41.0

32.2-463.2

0.015±0.001

0.013±0.002

86.7

-14.3

Titanium dioxide

125.3±47.9

34.0

36.8-310.4

0.051±0.009

0.057±0.003

111.8

11.1

Titanium dioxide (for color)

123.1±47.2

34.8

30.1-307.3

0.275±0.003

0.314±0.013

114.2

13.2

RS09 Chocolate

Titanium dioxide

103.5±34.3

50.4

29.8-244.0

0.462±0.025

0.403±0.081

87.2

-13.6

RS06 RS07 RS08

RS010

Chewy candy

Titanium dioxide

147.8±57.9

21.3

37.2-439.2

0.028±0.004

0.032±0.001

114.3

13.3

RS011

Chewy candy

Titanium dioxide

120.4±48.9

40.3

29.9-326.1

0.025±0.001

0.029±0.001

116.0

14.8

aLabeling

of TiO2 found on products. bTiO nanoparticles with at least one dimension less than 100 nm. The values 2 cSize ranges of the smallest and largest TiO particles found on TEM images. 2

correspond to TEM results in Figure 2.

27



Page 28 of 35

Table 2. Comparison of ICP-MS and Raman Values of TiO2 Working Standards Standard

standard known value (% TiO2)

ICP-MS (mean±SD)

Raman

%RV1

%RV2

%DV

1

1.159

1.130±0.001

1.066

91.9

94.3

-5.55591

2

1.067

1.030±0.003

1.095

102.6

106.3

6.137609

3

1.242

1.260±0.003

1.249

100.5

99.1

-0.51759

28


Page 29 of 35


Table 3. Spiked Sample Recovery Test for Raman Analysis Sample

Spiked sample known value (% TiO2)

Raman results (% TiO2)

%RV1

1

0.932

1.065

114.3

2

1.161

1.275

109.8

3

1.462

1.711

117.0

4

1.063

1.237

116.3

5

1.120

1.201

107.2

Average

112.9

29



Figure Captions Figure 1. XRD patterns of TiO2 isolated from RS01-RS11. Figure 2. TEM images and size distribution histograms of TiO2. The number of TiO2 particles measured for the size distribution analysis was more than 350 for each sample (scale bar=200 nm). Figure 3. (A) Raman spectra and (B) calibration curve with six independent TiO2 working standards (anatase phase). Figure 4. Comparison of Raman spectra: (A) Al2O3 matrix, (B) mixture of anatase TiO2 and SiO2 in Al2O3 (the percent concentration of TiO2 and SiO2 is 1.5% and 1.98%, respectively), (C) anatase TiO2 in Al2O3 (1.8% of TiO2), (D) anatase, (E) rutile and (F) SiO2.

30


Page 30 of 35

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Figure 1

31



Figure 2

32


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Figure 3

33



Figure 4

34


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Table of Contents (TOC)

35


Titanium Dioxide in Food Products: Quantitative Analysis using ICP

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