NIST Special Publication 1200-18

GC/MS Measurement of Nanomaterial-Induced 

DNA Modifications in Isolated DNA

Version 1.0 

 This publication is available free of charge from: 

http://dx.doi.org/10.6028/NIST.SP.1200-18 

Bryant C. Nelson 

Elijah J. Petersen 

Pawel Jaruga 

Miral Dizdaroglu
NIST Special Publication 1200-18

GC/MS Measurement of Nanomaterial-Induced 

DNA Modifications in Isolated DNA

Version 1.0 

Bryant C. Nelson 

Elijah J. Petersen 

Material Measurement Laboratory 

Biosystems and Biomaterials Division 

Pawel Jaruga 

Miral Dizdaroglu 

Material Measurement Laboratory 

Biomolecular Measurement Division 

This publication is available free of charge from: 

http://dx.doi.org/10.6028/NIST.SP.1200-18 

November 2015 

U.S. Department of Commerce 

Penny Pritzker, Secretary 

National Institute of Standards and Technology 

Willie May, Under Secretary of Commerce for Standards and Technology and Director
Certain commercial entities, equipment or materials may be identified in this 

document in order to describe an experimental procedure or concept adequately. 

Such identification is not intended to imply recommendation or endorsement by the 

National Institute of Standards and Technology, nor is it intended to imply that the 

entities, materials or equipment are necessarily the best available for the purpose.

National Institute of Standards and Technology Special Publication 1200-18

Natl. Inst. Stand. Technol. Spec. Publ. 1200-18, 17 pages (November 2015) 

CODEN: NSPUE2 

This publication is available free of charge 

from: http://dx.doi.org/10.6028/NIST.SP.1200-18
ii

FOREWORD

This special publication is one in a series stemming from the National Nanotechnology Initiative 

(NNI) Nano-EHS Research Strategy which identified Nanomaterial Measurement Infrastructure 

as one of the essential areas of research needed in order to develop an effective risk assessment 

and management plan regarding various aspects of nanotechnology in consumer products as it 

pertains to human health, exposure and the environment. The National Institute of Standards and 

Technology (NIST) was identified as a lead agency in the development of measurement 

strategies for the robust development to assess the potential effects of engineered nanomaterials 

and their fate in the environment. One important endpoint for measuring the potential human 

health and ecological effects of nanomaterials is the extent of modification that may occur on 

DNA bases, nucleosides or nucleotides. These modifications are considered to be DNA damage 

and may be relevant for the risk assessment of nanomaterials in biological systems. 

The current protocol presents a method to measure nanomaterial induced DNA damage levels 

using gas chromatography/mass spectrometry with isotope-dilution. Updates to this protocol may 

be released in the future. Visit http://nist.gov/mml/np-measurement-protocols.cfm to check for 

revisions of this protocol, or new protocols in the series. We also encourage users to report 

citations to published work in which this protocol has been applied. 
1 

1. Introduction

Making accurate measurements of the environmental fate and environmental and biological 

effects of engineered nanomaterials (ENMs) is critical for reliable risk assessment of these 

materials. It has been shown that ENMs can induce DNA damage through oxidative stress in 

living systems and/or through direct binding of ENMs to DNA. However, the unique behaviors 

of ENMs may cause measurement artifacts during the determination of their DNA damaging 

potential [1]. For example, results from several DNA damage studies using the traditional 

Comet assay have reported artifacts from the presence of ENMs [2-6]. Potential explanations for 

this phenomenon are that ENMs may induce DNA damage during processing after the exposure 

period has concluded or that the ENMs may be in the Comet tail and mistaken for DNA. These 

types of measurement artifacts preclude the accurate measurement of DNA damage and 

prevents researchers from clarifying the fundamental mechanisms of ENM induced DNA 

modifications [2]. 

In this document, we describe a protocol to quantitatively measure a range of DNA lesions using 

gas chromatography/mass spectrometry (GC/MS) with isotope-dilution and NIST standard 

reference material (SRM) 2396 (Oxidative DNA Damage Mass Spectrometry Standards). This 

method circumvents many of the artifacts observed in the Comet DNA strand break assay by 

directly quantifying molecular level DNA damage (i.e., DNA lesion) for a range of oxidatively 

induced damage products. Isotope-dilution mass spectrometry methods have been recently 

utilized by our laboratory to successfully measure DNA damage both in vitro and in vivo caused 

by gold nanoparticles [7], copper oxide nanoparticles [8], single-wall carbon nanotubes [9], iron 

oxide nanoparticles [10], titanium dioxide nanoparticles [11] and silver nanoparticles [12]. 

2. Principles and Scope

This protocol is proposed for the measurement of DNA lesion levels using GC/MS with isotopedilution [13] after exposure of isolated DNA to ENMs. Methods for determination of DNA 

lesion levels after cellular or whole organism exposure to ENMs are similar but are not described 

in the present protocol. Not all possible forms of DNA damage can be measured by this 

approach. This protocol focuses specifically on oxidatively-induced damage to DNA bases. The 

methodology, and specifically the instrumentation described in the present protocol, is based on 

the accurate measurement of DNA base lesions using a single quadrupole mass analyzer in 

selected ion monitoring (SIM) mode (see Table A). The monitored SIM ions for each base 

lesion are specifically given in section 6.2 below. A similar protocol for the quantification of 

DNA base lesions that describes the use of a triple quadrupole mass analyzer and multiple 

reaction monitoring transitions for the measurement of ENM induced DNA damage also exists,

but this protocol exists as a separate document. Single quadrupole mass analyzers cost 

approximately one-third the price of triple quadrupole mass analyzers, therefore, the current 

methodology is inherently more cost accessible for more users. In addition, the instrumental 

expertise needed to properly operate and optimize a single quadrupole instrument is not as 

extensive as the expertise required to properly operate a triple quadrupole instrument. Usage of 

stable isotope-labeled internal standards (ISTDs) from SRM 2396 enables absolute identification 

and quantification of the following DNA lesions: 4,6-diamino-5-formamidopyrimidine 

(FapyAde), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-

OH-Ade), 5-hydroxycytosine (5-OH-Cyt), 5-hydroxyuracil (5-OH-Ura), 5-

(hydroxymethyl)uracil (5-(OHMe)Ura), thymine glycol (ThyGly) and 5-hydroxy-5-
2 

methylhydantoin (5-OH-5-MeHyd). Other important DNA lesions such as 8-hydroxyguanine (8-

OH-Gua) can also be measured with this approach by hydrolyzing 8-hydroxy-2-deoxyguanosine15N5 ISTD in SRM 2396 to 8-hydroxyguanine15N5 (8-OH-guanine-15N5) or stable isotopelabeled versions of 8-OH-Gua may be synthesized directly [14]. In this procedure, 8-OHguanine-15N5 is obtained by hydrolysis of 8-hydroxy-2-deoxyguanosine-15N5 with 60% formic

acid at 140 °C for 30 min followed by lyophilization. Subsequently, 8-OH-guanine-15N5 is

dissolved in 10 mM NaOH and its concentration in solution is determined using UV 

spectrophotometry prior to use. 

Table A DNA lesions detected and quantified by different MS procedures. 

Lesion # GC/MS1

(bases by SIM)

GC-MS/MS 

(bases by MRM)

LC-MS/MS 

(nucleosides by MRM)

1 FapyAde FapyAde 8-OH-dGuo

2 FapyGua FapyGua R-cdAdo

3 8-OH-Ade 8-OH-Ade S-cdAdo

4 5-OH-Cyt 5-OH-Cyt R-cdGuo

5 5-OH-Ura 5-OH-Ura S-cdGuo

6 5-(OHMe)Ura 5-(OHMe)Ura

7 ThyGly ThyGly

8 5-OH-5MeHyd 5-OH-5MeHyd

9 8-OH-Gua 8-OH-Gua

1. Base lesions detected and quantified in the present protocol.

3. Terminology

This protocol complies with definitions relevant to nanotechnology as set forth in the ASTM 

International E2456 [15] and is consistent with the draft standard ISO TS 80004-1:2010 [16]. 

nanoparticle—sub-classification of ultrafine particle that is characterized by dimensions in the 

nanoscale (i.e., between approximately 1 nm and 100 nm) in at least two dimensions; also 

referred to as “nano-object” in ISO TS 80004-1:2010 [16]. 

primary particle — the smallest discrete identifiable entity associated with a particle system; in 

this context, larger particle structures (e.g., aggregates and agglomerates) may be composed of 

primary particles. 

aggregate — a discrete assemblage of primary particles strongly bonded together (i.e., fused, 

sintered, or metallically bonded). 

Note—The adjective "primary", when used in conjunction with the term aggregate, is employed in 

the present context to indicate the smallest achievable dispersed particle entity. 

agglomerate—assemblage of particles (including primary particles and/or smaller aggregates) 

held together by relatively weak forces (e.g., van der Waals, capillary, or electrostatic), that may 

break apart into smaller particles upon further processing. 

Note—Although we define them as distinct entities, the terms aggregate and agglomerate have 

often been used interchangeably to denote particle assemblies. 

dispersion—used in the present context to denote a liquid (aqueous) in which particles are 

homogeneously suspended, or the process of creating a suspension in which discrete particles are 

homogeneously distributed throughout a continuous fluid phase; implies the intention to break 

down agglomerates into their principal components (i.e., primary particles and/or aggregates). 
3 

4. Materials and equipment

4.1 Reagents 

4.1.1 NIST SRM 2396 

4.1.2 Distilled and deionized 18 MΩ water (ddH2O), e.g., Millipore Milli-Q; sterile 

DNase/RNase-free water is recommended (e.g., Ambion, non-DEPC treated) 

4.1.3 Dried genomic DNA (e.g., calf thymus DNA, Sigma-Aldrich) or oligomeric DNA (e.g., 

Integrated DNA Technologies) 

4.1.4 Absolute anhydrous ethanol, (e.g., Sigma-Aldrich, <0.005 % water) 

4.1.5 Acetonitrile (e.g., Sigma-Aldrich with a minimum purity of 99.9 %, anhydrous) 

4.1.6 Fpg/Nth buffer (50 mmol/L sodium phosphate, 100 mmol/L potassium chloride, 1 

mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1 mmol/L dithiothreitol (DTT), and 

adjusted to pH 7.4 using ddH2O)

4.1.7 DNA repair enzymes (e.g., Fpg or Nth, Trevigen) 

4.1.8 Liquid nitrogen 

4.1.9 Pyridine (e.g., Sigma-Aldrich, stored in a amber glass vial with a few pellets of KOH to 

help remove water) 

4.1.10 Bis(trimethylsilyl)trifluoroacetamide + 1 % trimethylchlorosilane (BSTFA + TMCS, e.g., 

ThermoScientific Corp) 

4.1.11 High purity (99.999 %) nitrogen gas (e.g., Airgas Inc.) 

4.1.12 ENMs (e.g., NIST SRM 8011, 8012, 8013, etc.) 

4.2 Materials 

4.2.1 15 mL centrifuge tubes 

4.2.2 Glassware for making ICP-MS measurements 

4.2.3 Calibrated pipettes and disposable tips 

4.2.4 Quartz cuvettes for UV/Vis measurements 

4.2.5 Drierite desiccant 

4.2.6 2 mL glass autosampler vials with caps and low volume (300 μL) inserts 

4.2.7 Chelex 100 resin (e.g., Sigma-Aldrich) 

4.3 Equipment 

4.3.1 Fixed angle refrigerated centrifuge that is capable of spinning up to 20 000 x g and which 

is suitable for 2 mL Eppendorf sample tubes 

4.3.2 Vortex mixer 

4.3.3 Double beam UV/Vis absorbance spectrophotometer or another instrument capable of 

quantifying DNA concentrations 

4.3.4 Speed Vac system capable of holding 2 mL Eppendorf sample tubes or 2 mL glass 

autosampler vials or a vacuum desiccator 

4.3.5 Horizontal shaker with speed control 

4.3.6 Orbital rotator with speed control 

4.3.7 3500 MWCO dialysis membrane 

4.3.8 Lyophilizer 
4 

4.3.9 2 mL Eppendorf sample tubes 

4.3.10 4 °C refrigerator 

4.3.11 -20 oC freezer 

4.3.12 -80 oC freezer 

4.3.13 Oven that can reach 120 oC 

4.3.14 Single quadrupole gas chromatography/mass spectrometry (GC/MS) system with an 

electron ionization source. The system should have a mass range of 10 u to 1050 Da, a 

scan speed of 10 000 Da/s and mass axis stability ≥ 0.1 Da/48 h 

4.3.15 Single quadrupole inductively coupled plasma/mass spectrometry (ICP/MS) system with 

nickel cones and a Conikal nebulizer or a suitable single quadrupole inductively coupled 

plasma/optical emission spectrometry (ICP/OES) system 

5. Treatment of and removal of ENMs from laboratory prepared DNA stock

solutions. 

5.1 Preparation of DNA stock solutions 

5.1.1 To a known mass of dried DNA in a 15 mL plastic centrifuge tube, add a known volume 

fraction of ddH2O so that the final concentration of DNA in the tube is ≤ 500 μg/mL.

5.1.2 Place the tube on an orbital rotator in a 4 °C refrigerator and gently mix the DNA 

solution for 72 h to ensure complete solubilization.

5.1.3 Treat the solubilized DNA with Chelex 100 resin to remove heavy metals and divalent 

transition metals. 

5.1.4 Dialyze (3500 MWCO membrane, 25 nm to 30 mm diameter) the DNA against ddH2O at 

4 °C for 24 h and change the water at least twice during this time period. 

5.1.5 Determine the concentration of the solubilized DNA using UV/Vis spectrophotometry (1 

absorbance unit equals 50 μg/mL double-stranded DNA at 260 nm). For the stock DNA 

solution, it is recommended that the user also check the purity of the DNA solution by 

quantitatively evaluating the total RNA and protein levels in the sample. An estimate of 

RNA contamination can be quickly obtained by evaluating the 260 nm/280 nm ratio for 

the sample. A ratio of 1.8 to 2.0 indicates pure DNA while values higher than 2.0 

suggests RNA contamination. Total RNA contamination can be quantitatively evaluated 

using the Qubit fluorescence assay [17]. Proteins absorb at 280 nm but the absorbance at 

280 nm has a minimal effect on the 260 nm/280 nm ratio due to the much higher 

extinction coefficient of nucleic acids at both 260 nm and 280 nm. However, protein 

levels in the DNA solution can be quantitatively assessed using either the Lowry [18] or 

Bradford [19] protein assays. 

5.1.6 Store the DNA stock solution at 4 °C until needed or proceed directly to treating the 

DNA with ENMs based on the experimental design. 

5.2 Treatment of DNA solutions with ENMs 

5.2.1 To 2 mL Eppendorf sample tubes, add an appropriate volume of DNA stock solution so 

that the total mass of DNA in each tube is 100 μg. 

5.2.2 Add a specified volume of dispersed ENM and additional dispersant into each sample 

tube so that an appropriate range of ENM sample concentrations are obtained. 
5 

5.2.3 Prepare both positive and negative control samples using an identical procedure, as well 

as a dispersant-only control sample. All test and control samples should be prepared in 

triplicate. Prepare extra sets of triplicate control samples that are both unspiked and 

spiked (50 mg/L) with the highest ENM concentration under investigation. These 

samples will be used for verification/quantification of ENM removal via ICP-MS or ICPOES (see below). 

5.2.4 Treat all of the samples as necessary (i.e., expose to light/dark, heat/cold, adjust pH, 

sonicate, etc.,) for the specified period of time based on the experimental design. 

5.3 Removal of ENMs from DNA solutions 

5.3.1 Removal of the ENMs from the DNA samples is performed based upon centrifugation 

followed by ICP-MS quantitative verification of ENM removal. In some instances, it 

may be appropriate to utilize ICP-OES analyses if elemental detection sensitivity is not a 

limiting factor. 

5.3.2 Centrifuge the samples at 16 000 g (14 000 rpm) for 1 h @ 4 °C to pellet the ENMs. This 

procedure has been shown to be suitable for metal (i.e., AuNPs [7]) and metal-oxide (i.e., 

TiO2 NPs [11]) ENMs with a minimum diameter of 10 nm. Using this centrifugation 

procedure,  98 % of the ENMs can be removed from solubilized DNA samples. If less 

than 98 % of the ENMs are removed from the DNA, then one simply increase the 

centrifugation time. (Appendix shows validation data for removal of AuNPs (Figure 1) 

and removal of TiO2 NPs (Table 1). Please see references above for additional details. 

5.3.3 Transfer the DNA supernatants into 30 kDa molecular-weight-cutoff (MWCO) 

centrifugal filter units and centrifuge the samples at 7000 g for 15 min @ 4 °C. 

5.3.4 Wash the filter membrane with ddH2O then reverse-elute the DNA into a clean 1.5 mL 

Eppendorf tube. 

5.3.5 Determine the concentration of the eluted DNA solution using UV spectrophotometry (1 

absorbance unit = 50 μg DNA / mL at 260 nm) and store the collected samples at 4 °C or 

proceed directly to the addition of the isotopically labeled internal standards to each of 

the samples (see below). The accuracy of the DNA quantification method can be tested 

using NIST SRM 2372 (DNA Quantitation Standard). 

5.3.6 Verify the level of ENM removal using only the spiked control and unspiked control 

samples via ICP-MS analysis as follows: add a suitable internal standard to each sample 

and digest each sample using for example: a 3:1 volume fraction mixture of concentrated 

HCl/HNO3 (30 min @ 75 °C with orbital shaking at 1 s-1 (60 rpm)). This digestion

solution will work for AuNPs, but the analyst may need to modify this solution for other 

metal or metal-oxide-based ENMs. 

5.3.7 Cool the control samples down to room temperature and dilute them with 3 % HCl + 1 % 

HNO3 as required for analysis. Note: the sample digestion and dilution solutions will 

change depending on the element of interest. 

5.3.8 Perform elemental analysis on the digested control samples using ICP-MS. Based on the 

elemental calibration response factor, determine the elemental response in the spiked and 

unspiked control samples. Determine the mean level of ENM removal from the DNA 

solutions. 
6 

6.1 DNA lesion quantification procedure 

6.1.1 Transfer the required volume of eluted DNA from the centrifugal filter unit (see above) 

into a 1.5 mL Eppendorf tube so that the tube contains at least 50 μg of DNA. If there is 

not enough DNA sample volume to add 50 μg to each sample, add at least 30 μg. It is 

important to use the same DNA mass for all samples in a sample set. 

6.1.2 Add the relevant isotopically labeled internal standards to each vial. We recommend also 

testing a reference DNA such as calf thymus DNA and an internal standard mixture 

sample during each sample set analysis to test assay performance. These control samples 

are used to confirm the retention time and analyte peak stability of the GC column and to 

help assess the performance of the mass analyzer. The concentration of internal standard 

to add for each of the lesions depends on the type of DNA (i.e., isolated DNA, cellular 

DNA, etc.) and on the dynamic range of the GC/MS instrument. This needs to be tested 

during preliminary experiments with reference DNA. If too low or too high of an internal 

standard concentration is used, it may be challenging to integrate the peaks for the 

internal standard or the calibrated measurement range of the instrument may be exceeded. 

6.1.3 Dry the samples using either a Speed Vac or vacuum desiccator. After the samples are 

dry, either begin the next step or store the samples at 4 oC in a jar with desiccant. 

6.1.4 Add 50 μL of Fpg/Nth buffer to each sample and incubate all samples at room 

temperature with gentle rocking for ≥ 4 h (do not vortex mix samples). The Fpg/Nth 

buffer is prepared by adding the following ingredients to ddH2O (50 mmol/L sodium 

phosphate, 100 mmol/L potassium chloride, 1 mmol/L EDTA, and 0.1 mmol/L DTT) and 

adjusting the pH to 7.4. 

6.1.5 If the lesion levels for 8-OH-Gua are going to be tested, add the internal standard for this 

lesion at this point. 8-Hydroxyguanine is more readily oxidized than the other internal 

standards and is thus added at this step instead of in step 6.1.2. The concentration of the 

8-OH-Gua internal standard to be added should be determined by UV spectrophotometry 

before use due to its limited stability. 

6.1.6 Add Nth and/or Fpg to the samples. These enzymes are commercially available and can 

also be prepared using recombinant methodology [20]. The concentration of enzyme(s) 

to be added should be determined during preliminary experiments by adding a range of 

concentrations and assessing the optimal concentration for maximum excision of 

oxidized lesions. 

6.1.7 Briefly centrifuge the samples at 1000 g for 30 s at room temperature to ensure that all of 

the solution is at the bottom of the container. 

6.1.8 Incubate the samples at 37 C for 1 h (water bath). 

6.1.9 Stop the reaction by adding 150 μL of absolute cold ethanol and briefly vortex mix each 

sample. This step will precipitate the unreacted DNA glycosylases and the undigested 

DNA (DNA containing non-modified bases). 

6.1.10 Incubate all samples at -20 °C for 1 h or overnight. 

6.1.11 Centrifuge all samples at 15 800 g for 30 min at 4 °C.

6.1.12 Transfer supernatant (185 μL) into clear crimp-top glass autosampler vials. Leave the 

undigested DNA at the bottom of the Eppendorf tube.

6.1.13 Remove the ethanol from the autosampler vials by placing the vials in the Speed Vac or 

in a vacuum desiccator. Preliminary experiments can be conducted to find the time 

needed to fully remove the ethanol. 

6 DNA lesion measurements using GC/MS
7 

6.1.14 Add 200 μL of ddH2O to each sample and cover the glass vials using tissue and rubber 

bands. The tissue prevents the ice from flying out of the vials. Insert the samples into a 

round glass sample holder and freeze all samples using liquid nitrogen. Pour off the 

excess liquid nitrogen from the round glass vial holder. Alternatively, the samples can be 

frozen in a freezer at -80 ° C for at least half an hour. Dry all samples overnight 

(minimum of 16 h) using a freeze dryer. Freeze drying does not form precipitates, 

instead a film of DNA should cover the bottom quarter of the vial at the end of the drying 

step. 

6.1.15 Store the samples at room temperature in a glass jar containing fresh desiccant or proceed 

directly to derivatization which starts in the next step.

6.1.16 Turn on an oven and let it warm up to 120 ° C. Remove BSTFA from the refrigerator at 

4 ° C and let it warm up for 10 min at room temperature.

6.1.17 Derivatize analytes to trimethylsilyl esters by adding 30 μL of BSTFA and 30 μL of 

pyridine to each sample vial.

6.1.18 Cover each sample with a light stream of high purity nitrogen (99.999%) for 10 s, close 

sample vials and vortex for  5 s. Make sure that the vial caps are on tightly to prevent 

oxygen from getting into the sample vials. Try to twist the caps to make sure they are 

secure. Note: connect an oxygen and moisture removal cartridge to the outlet of the high 

purity nitrogen supply to ensure the purity of the nitrogen entering the samples. 

6.1.19 Incubate samples at 120 C for 30 min. 

6.1.20 Allow samples to cool for approx. 15 min (leave them on a lab bench). 

6.1.21 Transfer samples into GC/MS autosampler vials (containing low-volume inserts) with 

syringe (wash syringe with acetonitrile solution after each sample transfer). 

6.1.22 Cover samples with a light stream of nitrogen for 10 sec and crimp-cap. 

6.1.23 Analyze the samples using GC/MS methodology [21-23].

6.2 GC/MS sample analyses 

6.2.1 The GC/MS sample analyses are performed on a high resolution single quadrupole 

GC/MS system operated in positive ion mode with an electron ionization source. 

6.2.2 The GC column is an HP-Ultra 2 high-resolution fused silica capillary column (12.5 m, 

0.2 mm i.d.) coated with cross-linked 5 % phenylmethylsilicone gum phase (film 

thickness, 0.33 μm) (Agilent Technologies).

6.2.3 Gas chromatography separation of the derivatized lesions is performed with a 

temperature programmed ramp from 130 C to 300 C (130 C, 2 min hold, 8 C per min 

to 207 C, 0 min hold; 10 C per min to 300 C, 4 min hold). The total run time is 24.75 

min. The sample injection amount is 5 L.

6.2.4 Trimethylsilyl derivatives of each DNA lesion and their stable isotope-labeled analogue 

are detected in SIM mode after appropriately tuning and optimizing the MS instrument 

and analyte detection parameters. Each manufacturer’s instrument will have a different 

detection sensitivity for the analytes and thus the analytes will have to be tuned 

independently. Typical instrument parameters that need to be tuned and optimized for 

the derivatized analytes are the following: analyte dwell time, injection mode, split ratio, 

split flow rate, inlet temperature, inlet pressure, MS source temperature, GC/MS interface 

temperature, MS quadrupole temperature, MS quadrupole voltage, repeller voltage, 

extractor voltage, electron energy voltage and MS run time. The relevant SIM ions are: 
8 

m/z 354, 368, 369 and m/z 357, 371, 372 for FapyAde and FapyAde13C,15N2,

respectively; m/z 442, 457 and m/z 445, 460 for FapyGua and FapyGua13C,15N2,

respectively; m/z 440, 455 and m/z 445, 460 for 8-OH-Gua and 8-OH-Gua15N5,

respectively; m/z 352, 367 and m/z 355, 370 for 8-OH-Ade and 8-OH-Ade13C,15N2,

respectively; m/z 342 and m/z 345 for 5-OH-Cyt and 5-OH-Cyt13C,15N2, respectively;

m/z 329 and m/z 335 for 5-OH-Ura and 5-OH-Ura13C4,

15N2, respectively; m/z 343, 358

and m/z 347, 362 for 5-OH-MeUra and 5-OH-MeUra13C2, d2, respectively; m/z 259, 433,

448 and m/z 262, 437, 452 for ThyGly and ThyGly-d4, respectively, and m/z 331 and m/z 

334 for 5-OH-5-MeHyd and 5-OH-5-MeHyd13C,15N2, respectively. [21-23]. The SIM

current profiles are integrated using appropriate peak integration software and the 

measured lesion levels are determined using the SIM area ratios from the DNA lesion of 

interest and its labeled analogue in conjunction with the known amount of the labeled 

analogue initially spiked into each sample.

7 Abbreviations

5-OH-Cyt 5-hydroxycytosine 

5-OH-Ura 5-hydroxyuracil 

5-(OHMe)Ura 5-(hydroxymethyl)uracil 

5-OH-5-MeHyd 5-hydroxy-5-methylhydantoin 

8-OH-Ade 8-hydroxyadenine 

8-OH-Gua 8-hydroxyguanine 

FapyAde 4,6-diamino-5-formamidopyrimidine 

FapyGua 2,6-diamino-4-hydroxy-5-formamidopyrimidine 

BSTFA bis(trimethylsilyl)trifluoroacetamide 

DTT dithiothreitol 

EDTA ethylenediaminetetraacetic acid 

ENM engineered nanomaterial 

NP nanoparticle 

SIM selected-ion-monitoring 

SRM standard reference material 

ThyGly thymine glycol 

UV/Vis ultraviolet-visible 

8 Acknowledgements 

We thank Julian Taurozzi and Vince Hackley of NIST for their helpful advice and 

discussions regarding the dispersion and characterization of the P25 TiO2 NPs (NIST SRM 

1898) utilized in this and other studies and for the use of their LDS system. 
9 References 

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15. ASTM (American Society for Testing Materials) International, E2456-06: Standard terminology

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11

Appendix 

1. Removal of Engineered Nanomaterials from DNA Solutions Using Centrifugation

Techniques 

In the present study, we are strictly concerned with the separation of one discrete size and shape 

of ENM from a solution matrix of completely solubilized ct-DNA in aqueous buffer. Based on 

the constant buoyancy and density of the solution matrix, we can utilize the centrifugal force 

from a fixed-angle rotor centrifuge to separate the ENMs from the DNA solution. We are able to 

effect an almost 100 % separation of ENMs from the DNA solution due to the fact that the DNA 

solution has a lower density (ρ) than the density of the relevant ENMs (ρDNA = 1.7 g/cm3and

ρAuNPs = 19.3 g/cm3

, ρTiO2 NPs = 4.3 g/cm3) [24-27]. Therefore, the ENMs can be forced to

sediment at the bottom of the DNA solution using appropriate centrifugation speeds and times. 

When the centrifugation speed is held constant, and assuming spherical particles for simplicity, 

the appropriate centrifugation time for separating the ENM from the DNA solution can be 

determined empirically or it can be estimated theoretically using the following equations [28]: 

(1) vt =

ω2r(ρρ− ρi

)dρ

2

18η

(2) vt =

dr

dt

(3) tt = 

[lnrmax

rmin

]

vt

,

where vtis the terminal velocity, ω

2

is the angular velocity of the rotor, ρρ and ρi are the

density of the ENM and solution matrix (solubilized ct-DNA), respectively, dρ is the diameter of 

the ENM, η is the viscosity of the solution matrix, rmax and rmin are the maximum and 

minimum rotor radii, respectively, and tt

is the total time for complete separation of the ENM 

from the solution matrix. 
2. Figure 1 

Results from the removal of 10 nm, 30 nm and 60 nm NIST SRM AuNPs from aqueous buffered solutions containing 500 μg/mL ctDNA using centrifugation. 
2

3. Table 1

Removal of NIST P25 TiO2 NPs from 500 μg/mL ct-DNA aqueous buffer solutions using centrifugation.

Sample # Detected Level of TiO2

(μg) after centrifugation Standard Deviation (μg)

Expected Level of TiO2

(μg) before 

centrifugation

%TiO2 Removed

1 7.516 0.019 198 96.2

2 3.165 0.028 198 98.4

3 2.109 0.010 198 98.9

Ti was detected in the aqueous ct-DNA samples using ICP-OES and converted to TiO2 levels in the samples. The data shown are for three independently 

prepared samples that were analyzed 5 times each. The mean percentage of TiO2 removed was 97.8 % ± 1.4 %.