Quantification of 19 Aldehydes in Human Serum by Headspace SPME

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Environmental Measurements Methods

Quantification of 19 aldehydes in human serum by headspace SPME/GC/high-resolution mass spectrometry Lalith K Silva, Grace A. Hile, Kimberly M. Capella, Michael F. Espenship, Mitchell M Smith, Víctor R. De Jesús, and Benjamin C. Blount Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02745 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Quantification of 19 aldehydes in human serum by headspace SPME/GC/high-resolution

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mass spectrometry

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Lalith K. Silva*, 1, Grace A. Hile1, Kimberly M. Capella1, Michael F. Espenship1, Mitchell M.

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Smith1, Víctor R. De Jesús1, and Benjamin C. Blount1 1

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Division of Laboratory Sciences, National Center for Environmental Health,

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Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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* Corresponding Author: Lalith K. Silva, PhD

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Division of Laboratory Sciences, National Center for Environmental Health, CDC,

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4770 Buford Highway, NE, Mail Stop F47, Atlanta, GA 30341

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Phone: 770.488.3559; Fax: 770.488.0181; email: [email protected]

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The findings and conclusions in this report are those of the authors and do not necessarily

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represent the official position of the Centers for Disease Control and Prevention. Use of trade

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names is for identification only and does not imply endorsement by the Centers for Disease

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Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human

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Services.

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Running Title: Quantification of 19 aldehydes in human serum

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Key Words: Aldehydes, human serum, Schiff base protein adducts, acid hydrolysis, gas

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chromatography, high-resolution mass spectrometry, solid-phase microextraction

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Abstract Sources of human aldehyde exposure include food additives, combustion of organic

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matter (tobacco smoke), water disinfection byproducts via ozonation, and endogenous processes.

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Aldehydes are potentially carcinogenic and mutagenic, and chronic human aldehyde exposure

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has raised concerns about potential deleterious health effects. To aid investigations of human

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aldehyde exposure, we developed a novel method to measure 19 aldehydes released from Schiff

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base protein adducts in serum using controlled acid hydrolysis, solid-phase microextraction

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(SPME), gas chromatography (GC), and high-resolution mass spectrometry (HRMS).

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Aldehydes are released from Schiff base protein adducts through acid hydrolysis, and are

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quantified in trace amounts (µg/L) using stable isotope dilution. Detection limits range from 0.1

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to 50 µg/L, with calibration curves spanning three orders of magnitude. The analysis of fortified

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quality control material over a three-month period showed excellent precision and long-term

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stability (3-22% CV) for samples stored at -70°C. The intra-day precision is also excellent (CV,

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1-10%). The method accuracy ranges from 89-108% for all measured aldehydes, except acrolein

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and crotonaldehyde, two aldehydes present in tobacco smoke; their analysis by this method is not

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considered robust due in part to their reactivity in vivo. However, results strongly suggest that

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propanal, butanal, isobutanal, and isopentanal levels in smokers are higher than levels in non-

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smokers, and thus may be useful as biomarkers of tobacco smoke exposure. This method will

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facilitate large epidemiological studies involving aldehyde biomonitoring to examine non-

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occupational environmental exposures.

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Introduction Environmental aldehydes arise from a number of sources, as they (1) are formed in water

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as disinfection byproducts of ozonation1 (2) are used as food additives,2 and (3) result from

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pyrolysis of organic matter such as fuel,3 wood,4 and tobacco.4-6 In addition, the body generates

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aldehydes endogenously7 from the reaction of free radicals with cell membrane lipids.

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Upon exposure, aldehydes readily diffuse through cell membranes and covalently bind to

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cellular macromolecules, disrupting function and potentially causing mutations.8, 9 Studies

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involving laboratory animals indicate aldehydes such as formaldehyde, crotonaldehyde, and

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hexanal are toxic and carcinogenic, with associations with cancers of the liver, lungs, and

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reproductive organs.6, 10 Initial studies of aldehyde carcinogenicity in humans link aldehyde

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exposure to respiratory distress, pulmonary diseases, and some cancers.8, 10-13 To address these

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adverse health outcomes, an assessment of human aldehyde exposure is needed. Measurement of

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an internal dose or biomarker of exposure is a key aspect of assessing exposure to environmental

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toxicants that may cause adverse health outcomes.14, 15 Therefore, we developed an analytical

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method to quantitate 19 aldehydes in human serum. We selected these 19 aldehydes because they

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are known to be present in tobacco smoke and other environmental sources. The high throughput

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and ruggedness of this method enables quantification of aldehydes present in the U.S. population

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at trace levels.

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Aldehydes present in serum as free non-covalently bound molecules can be analyzed

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directly. However, because a significant amount of aldehydes are typically bound to proteins and

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other biomolecules,16 a chemical step, such as hydrolysis, is necessary to release them for

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detection. Methods using acidic or basic sample pre-treatments are capable of hydrolyzing the

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bound fraction of aldehydes from proteins that exist as Schiff base adducts, thus allowing an 3 ACS Paragon Plus Environment

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estimation of total aldehydes.17 There are published methods for measuring aldehydes in human

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plasma, serum, and blood.16, 18, 19 Those methods that target bound aldehydes are generally

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performed with the addition of hydrochloric acid (HCl) to hydrolyze the Schiff base adduct

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bonds. However, care must be taken to minimize loss of the more volatile aldehydes once

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hydrolysis is complete. One approach to extract and pre-concentrate volatile aldehydes is to form

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a nonvolatile derivative, which is further derivatized for analysis by gas chromatography/mass

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spectrometry (GC/MS). For example, a method for formation of o-pentafluorobenzyl-oxime

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(PFB-oxime) derivatives was adapted by Luo et al. in 1995 and used to quantify 22 bound

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aldehydes in plasma, urine, and tissue samples.20 This approach permitted isolation and pre-

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concentration of aldehydes and prevented the loss of the more volatile aldehydes before

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trimethylsilylation and analysis by GC/MS. Similarly, in 2004, Deng et al. used a PFB-oxime

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derivatization approach, but formed oximes of volatile aldehydes in the headspace of human

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blood samples on a solid-phase microextraction (SPME) fiber followed by trimethylsilylation.19

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These methods produced detection limits between 1 -5 µmol/L. In 1994, Yeo et al. quantified

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femtomole levels of malondialdehyde (MDA) in plasma, tissue, and sperm using hydrolysis of

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MDA protein adduct (Schiff base) and GC/MS instrumentation.16 Each of these published

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methods, however, consists of complex, multi-step procedures and involves derivatization for

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quantification, throughput, or ruggedness. Therefore, there is a need for a more robust method to

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assess aldehyde levels in human populations.

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Developing an improved analytical method is challenging since aldehydes are difficult to

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quantify due to their volatility and high reactivity. The method reported here relies on carbonyl

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groups’ reactivity with amino groups in proteins to make stable aldehyde protein adducts.17, 21

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There are two types of aldehyde-bound protein adducts: 1,4-Michael addition aldehyde protein 4 ACS Paragon Plus Environment

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adducts and Schiff base aldehyde protein adducts. Typically, unsaturated aldehydes form both

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1,4-Michael addition and Schiff base aldehyde protein adducts, while saturated aldehydes form

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Schiff base aldehyde protein adducts.21 We developed a method to quantify 19 aldehydes (C2-

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C10 saturated, five unsaturated including acrolein and crotonaldehyde, and three aromatic) in

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human serum by forming Schiff base aldehyde protein adducts. The method hydrolyzes the

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protein adducts to release the native aldehydes, extracts them from the sample headspace by

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SPME, and utilizes stable isotope dilution to quantify them by gas chromatography/high-

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resolution mass spectrometry using an adapted method.22 However, formaldehyde could not be

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quantified by this method due to interference from methanol, which was used for all standards

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and internal standards stock solution as solvent.

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Experimental

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Materials

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Purge-and-trap grade methanol was purchased from Burdick and Jackson (Muskegon,

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MI). High-performance liquid chromatography (HPLC) grade water was purchased from J.T.

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Baker (Phillipsburg, NJ). HPLC-grade water, from a single lot, was used to prepare all solutions,

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blanks, and standards. Unlabeled analytes (Table 1) were purchased from Sigma-Aldrich (St.

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Louis, MO). Stable isotope-labeled analogs (Table 1) were specially synthesized by CanSyn

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Chemical Inc. (Toronto, Canada), flame-sealed in glass ampoules, and stored at -70°C. The

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isotopic purity was verified to be >99% for all compounds, and the chemical purity was ≥99%.

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Clear glass 10-mL headspace vials were manufactured by La-Pha-Pack (Werner

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Reifferscheidt GmbH, Germany). Stainless steel washers (size M10) were purchased from

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Hillman Fastener Corp. (Cincinnati, OH). Headspace vial septa (20 mm-diameter, 1.0-1.3 mm 5 ACS Paragon Plus Environment

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thickness), made of polydimethylsiloxane and coated with polytetrafluoroethylene (0.1-0.15 mm

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thickness) and open-center aluminum seals, were obtained from Supelco, Inc. (Bellefonte , PA).

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The headspace vial septa were processed to reduce volatile organic compound (VOC)

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contamination as described previously.22 Glass ampoules were purchased from Wheaton Science

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Products (Millville, NJ). Carboxen/polydimethyl siloxane (80µm) SPME fibers were purchased

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from Supelco Inc. and heated at 250°C for two hours before use for analysis.

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Standard solutions

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The primary standard stock solution and the internal standard (ISTD) stock solution were

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prepared by diluting neat chemicals in purge-and-trap grade methanol. Subsequent dilutions were

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made in purge-and-trap methanol to produce intermediate stock solutions. Intermediate stock

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solutions were sealed in glass ampoules and stored at -70°C. On the day of analysis, ampoules

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containing intermediate stock solutions of the target aldehydes were opened and further diluted

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in helium-sparged distilled water23 to produce the nine solutions used for calibration curves. The

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lower and upper calibration levels for the 19 target aldehydes is shown in Table 2. In addition, a

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spiking solution containing the stable isotope-labeled analytes was prepared from the

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intermediate ISTD stock solution by further dilution with helium-sparged distilled water.23 To

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each sample vial, a 40-µL aliquot of the labeled spiking solution was added.

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Quality control materials

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Two quality control (QC) pools (QL and QH) were prepared by spiking desired levels of

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aldehydes in a non-smoker human serum lot purchased from Tennessee Blood Service

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(Memphis, TN). The resulting QL and QH samples were then tested to confirm the target

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concentrations of all analytes. The QC pools were dispensed in 1-mL aliquots into 2-mL cryo6 ACS Paragon Plus Environment

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vials. These QC cryo-vials were numbered according to the dispensing order and stored at -70°C

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for future use. Each QC pool was characterized (N = 20 independent determinations) for target

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aldehydes over a three-month period.

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Blank analysis

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Aldehyde contamination was evaluated by analysis of blanks prepared from VOC-free

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water. VOC-free blank water was produced in our laboratory by sparging overnight with filtered

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ultra-high purity grade helium, distillation, and flame sealing.23 On the day of analysis, water

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blanks were spiked with ISTD and analyzed with each batch of unknowns and QC samples. If

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the blank contained aldehyde levels exceeding the low reporting level, the run was flagged as

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contaminated for that aldehyde and those results were rejected.

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Proficiency testing

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Absolute assay accuracy was verified by blinded analysis of these aldehydes prepared in

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water. Four proficiency testing (PT) samples containing all aldehydes were prepared as dilutions

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of the stock solution to achieve the final concentrations to cover the calibration range for each

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aldehyde. PT samples were blind-coded by an independent QC officer. PT samples were run bi-

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annually and following major instrument maintenance. An analyte passed PT if blind-analyzed

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concentrations fell within 25% of target values according to gravimetric calculations.

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Serum sample collection

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Blood samples were drawn from anonymous healthy adult volunteers by venipuncture

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(red-top vacutainers for serum, Becton, Dickinson and Company; Franklin Lakes, NJ,) in

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accordance with an approved human subjects protocol. The specimens were obtained from

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Tennessee Blood Services. Blood in the vacutainers was mixed by manually inverting 10 times,

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and then allowed to sit for 30 minutes at room temperature to clot before spinning and

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separation. Serum separation results in the removal of blood cells, including red blood cells,

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white blood cells and platelets, and coagulation factors by centrifuge at 3000 rpm for 10 minutes.

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Separated serum samples were stored at -70°C until analysis, and they were typically analyzed

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within one week of sample collection.

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Preparation of samples, blanks, and water standards for daily analysis

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Prior to daily analysis, serum samples were thawed and mixed on a hematology mixer

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(Fisher Scientific, Inc., Pittsburgh, PA). A 0.25-mL aliquot was removed from the cryo-vial and

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the sample was dispensed into a 10-mL SPME vial. Subsequently, the serum sample was spiked

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with a 40-µL aliquot of the labeled ISTD solution, crimp-sealed, and mixed with a vortexer (S/P

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Multi-Tube Vortexer, Baxter Diagnostics, Inc., Minster, OH) for five minutes. A water-based

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blank and the serum-based QCs were prepared in a similar manner. Following sample

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preparation, vials were placed in a Peltier cooler sample tray (15°C) on the CombiPAL

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autosampler. Standards prepared as described above were analyzed with each analytical run.

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Protein adducts hydrolysis in serum

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Our method reproducibly hydrolyzes aldehyde adducts covalently bound to proteins by

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individually incubating samples prior to analysis with 0.1 M HCl.16 The incubation of samples is

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controlled by two PAL autosampler arms (CombiPAL and PrepPAL) using the software program

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Chronos (Axel Semrau, Sprockhövel, Germany). The PrepPAL arm with a 1000 µL syringe

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withdraws 330 µL of 0.1 M HCl from a reservoir and expels the acid into a SPME vial

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containing serum (250 µL) and ISTD (40 µL). The PrepPAL then takes the SPME vial with acid 8 ACS Paragon Plus Environment

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and places it into agitatorMx for 20 minutes at 30°C and 350 rpm. The PrepPAL moves the

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SPME vial from agitatorMx into agitator1. The CombiPAL arm equipped with the SPME fiber

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then extracts aldehydes from the headspace at 50°C for 10 minutes. We systematically optimized

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the hydrolysis conditions (hydrolysis time, temperature, mixing method, rpm of agitator during

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hydrolysis) (Table 3) to reproducibly measure aldehydes and to prevent artifactual formation.

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Instrumental analysis

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A Thermo Scientific TRACE 1310 gas chromatograph (Thermo Fisher Scientific,

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Rodano-Milan, Italy) coupled to a high-resolution magnetic sector mass spectrometer (DFS,

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Thermo Finnigan, Bremen, Germany) was used to analyze all samples. A CombiPAL

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autosampler and extra PAL autosampler arm (CTC Analytics AG, Zwingen, Switzerland) were

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mounted on the GC system; this system performed automated sample heating, hydrolysis, SPME

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extraction, and GC injection. Aldehydes adsorbed on the SPME fiber during sample extraction

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were subsequently desorbed into a splitless GC inlet (200°C) during sample injection. Target

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analytes were separated on a DB-624 capillary column (25-m; 0.25-mm; 1.12-µm film; 6%

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cyanopropyl-phenyl, 94% dimethylsiloxane stationary phase; J & W Scientific, Folsom, CA).

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Ultra-high purity research-grade helium (99.9999%) was used as the column carrier gas for all

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analyses and held at a constant flow rate of 0.70 mL/min. The GC injector was operated in

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splitless mode for the first 2.0 min to permit cold-trapping of aldehydes at the head of the GC

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column, which was cooled to -100°C by use of liquid nitrogen. The injector was then switched to

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split mode at a purge flow of 70 mL/min. After the two-minute wait, the cryo-trap was

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ballistically heated to 220°C to focus the analytes into the column to be chromatographically

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separated by use of the following GC ramping program: hold at -1°C for 3 min; ramp at

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50°C/min to 30°C; hold at 30°C for 1 min; ramp at 10°C/min to 200°C; ramp at 50°C/min to

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220°C; hold at 220°C for 3 min. The total GC run time was 25 minutes.

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We achieved about 2,000 injections with each individual SPME fiber. The potential

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contamination of SPME fibers from analytes present in the laboratory environment and analyte

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carryover from sample to sample was addressed by leaving the fiber in the heated GC inlet

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during the entire analysis while the heated zone was purged with helium at 70 mL/min (split

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mode). This procedure effectively removed all volatile chemicals from the fiber assembly,

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eliminating carryover from the previous injection and preventing the contamination of the fiber

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from laboratory air.

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Target analytes were analyzed by DFS in positive ion electron-impact (EI) mode, with

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multiple-ion-detection at 10000 mass resolution (5% valley definition). For each analyte, the

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intensity of a quantification ion, a confirmation ion, and an ISTD analog ion (Table 1) was

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monitored by the mass spectrometer. The 19 target analytes and their stable isotope-labeled

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analogs were divided into 11 groups for multiple ion detection analysis. We controlled the cycle

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time for each time window, allowing for the acquisition of 10-12 data points across each peak.

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Quality assurance

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For each analyte, three ion signals were collected: quantification, confirmation, and the

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corresponding labeled ISTD analog (Table 1). The response of the labeled analogs was evaluated

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on the basis of absolute peak area signal. The signal-to-noise ratio was also used as a check of

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instrument response. The analyte peak identity was further evaluated by comparing GC retention

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time (RT) and the confirmation-to-quantification ion ratio in unknown samples to reference

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standards. The standard deviation of the confirmation-to-quantification ion ratio for aldehydes 10 ACS Paragon Plus Environment

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was approximately 5% across the entire linear range. For unknown serum samples, a

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confirmation-to-quantification ion ratio that differed by more than ± 25% from the value

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determined from the calibration standards resulted in rejection of the data for the affected

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analytes in that analytical run. A blank water sample was used in each analytical run to test for

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contamination. If the blank contained analyte levels higher than the limit of detection (LOD), the

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run was flagged as contaminated for that analyte. Additionally, at the start of each analyses, a

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SPME fiber sampling of laboratory air was performed to assess airborne contaminants

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qualitatively.

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Limit of detection (LOD)

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A total of 60 runs containing water blanks and four varying analyte concentration serum

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samples were used to calculate the LOD24 for this assay. These four varying concentration

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samples consisted of a serum blank and three low concentration, analyte-fortified samples were

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prepared from a human serum sample and used in the LOD evaluation.

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Method accuracy and precision

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Method accuracy and precision were assessed by analysis of QC materials and analyte-

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fortified human serum. Human serum was anonymously collected from three healthy volunteers

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with no known recent exposure to the target compounds. The serum was fortified with known

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amounts of target analytes. These serum samples were subsequently analyzed, and measured

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amounts were compared with the spiked amounts.

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Smoker vs. non-smoker classification

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We compared aldehyde levels of individuals classified as a smoker versus those who are classified as non-smokers. We used an established smoking biomarker, 2, 5-dimethylfuran 11 ACS Paragon Plus Environment

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(2DF), measured in whole blood, to determine a person’s smoking status. A blood 2DF value ≥

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0.014 µg/L indicates an individual is an active smoker.25 JMP statistical analysis software (SAS;

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Cary, NC) was used to generate a receiver operating curve (ROC) to determine a smoking cutoff

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point, based on categorical blood 2DF smoking status and continuous serum 2DF values.

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Results and Discussion

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We developed a sensitive analytical method for quantifying 19 aldehydes at low parts-

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per-billion (µg/L) levels in human serum to assess human exposure. We used controlled acid

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hydrolysis to release aldehydes existing as Schiff base aldehyde protein adducts. We optimized

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key parameters including sample volume, serum pH, hydrolysis temperature and time, mixing

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method, and SPME extraction temperature in order to minimize the artifactual formation of

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aldehydes (Table 3). The resulting method allows us to reliably measure aldehyde levels in

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serum with high reproducibility, precision, and accuracy.

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We evaluated two different DB-624 capillary GC columns—a 60-m, 0.25 mm I.D., 1.40

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µm film thickness column and a 25-m, 0.20 mm I.D., 1.12 µm film thickness column—to

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resolve the aldehydes adequately. Peak separation was most adequate with the 25-m column.

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The use of the 25-m column with a carefully optimized GC thermal gradient successfully

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resolved all 19 aldehydes (Figure 1) from potentially interfering contaminants [e.g.,

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cyclohexanol (m/z = 100.0883, RT=14.80 min.) from heptanal (m/z = 114.1039, RT=14.90

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min.)]. A power of 10,000 resolution for the high resolution mass spectrometry provided

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additional selectivity. Multiple ion monitoring mode provided additional sensitivity. We

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optimized the GC injector temperature (200°C) and the SPME extraction time (10 min) for

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aldehydes measured in a 25-minute GC run time; the total cycle time per sample was 36 minutes.

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The method allows for the analysis of 30 samples per day. The combination of chromatographic

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resolution and selective headspace extraction of the aldehydes above the liquid sample

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substantially reduced the chemical noise and helped to achieve detection limits below 1 µg/L for

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most aldehydes.

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The lowest sample volume that could still produce a robust signal was 0.25 mL serum.

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While higher sample volumes produced more mass spectrometric signal, the use of low specimen

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volumes is preferred to minimize specimen handling in the laboratory. This is particularly

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important in large-scale population-based studies, where multiple assays may be performed using

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the same specimen aliquot.

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The calibration curves for both water and serum samples were linear, ranging over three

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orders of magnitude with coefficients of determination exceeding 0.9900 for all analytes in Table

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2. By weighting the data by the reciprocal of the concentration, we were able to maintain an

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excellent linear fit, even at low concentrations. The slopes for the calibration curves varied by

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less than 10% between the matrices (Figure 2, e.g. butanal is 6%). These results allowed us to

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make the calibration standards in water to eliminate potential errors associated with the varying

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background aldehyde levels commonly found in human serum.

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The quantification ranges and LODs were determined with known standard levels for all

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19 aldehydes (Table 2) in serum. These LODs are the lowest of all published methods18 for

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analysis of aldehydes in human serum. This method is simple and does not involve a lengthy

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derivatization step, thus allowing for low limits of detection.

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The intra-day stability (short term) of the aldehydes in serum was studied for 24 hours on a sample Peltier cooled tray maintained at 15°C. Short-term stability is essential because daily 13 ACS Paragon Plus Environment

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sample runs lasting up to 24 hours must have good reproducibility. All 19 analytes have

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coefficients of variation less than 10% over a 24-hour period (Figure 3). To evaluate inter-day

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stability, fortified QC materials stored at -70°C were analyzed over a period of three months. We

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did not observe a decrease in aldehyde levels over time at sample storage conditions. The

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analyses yielded inter-day coefficients of variation ranging from 5% to 12% in serum QC pools

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for all aldehydes, except acetaldehyde (22%), acrolein (15.5%), and crotonaldehyde (13.5%)

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(Table 4).

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In addition, data from QC samples demonstrate reproducibility over time. Table 4 shows

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the mean and the coefficients of variation (CV%) measured using low concentration (QC Low)

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and high concentration (QC High) QC pools from more than 20 independent runs over a three-

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month period. Aldehyde coefficients of variation are within 15% for most analytes for both QC

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pools. The method was the least precise for acetaldehyde (22.3% CV for QC High; 16.7% CV

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for QC Low). While acetaldehyde, acrolein, and crotonaldehyde are known to be present in

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tobacco smoke, their analysis by this method is not considered robust due in part to their

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reactivity in vivo.26-28 These three aldehydes are rapidly metabolized in the body, reducing their

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availability as native compounds in serum. Thus, we do not consider this method adequately

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sensitive for analysis of acetaldehyde, acrolein, and crotonaldehyde in serum.

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Accuracy and precision were evaluated using PT samples and spiked human serum QC

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samples. Percent error of aldehydes for a typical PT run for low concentration (PT Low) and

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high concentration (PT High) PT samples is shown in Table 5. The percent error of PT samples

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and spiked serum samples was calculated by taking the difference between measured and target

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values and dividing by the target value. The percent error was within ±11% for all aldehydes,

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except PT Low for octanal and nonanal, where the concentration was below the LOD. The

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percent error was within ±11% for all aldehydes for PT High.

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The accuracy of this method in human serum was analyzed by examining the percent

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recovery of human serum spiked with known amounts of aldehydes. Spiked human serum at

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medium concentrations in triplicate and un-spiked human serum in triplicate were prepared for

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analysis. Results were calculated separately for the three medium level concentration-spiked

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serum samples after subtracting un-spiked background levels. Blank correction was necessary

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due to relatively high endogenous concentrations of some aldehydes in the serum. Recoveries for

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most aldehydes were within ±11% (Table 5). However, the recovery was lower for unsaturated

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aldehydes such as acrolein (percent error 80%) and crotonaldehyde (percent error 30%). The

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lower recovery may be partially due to the formation of non-Schiff base protein adducts, such as

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1,4- Michael addition products.21

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Hydrolysis of Schiff base acetaldehyde protein adducts has been reported to form

320

artifactual acetaldehydes.29 Therefore, we optimized parameters that influence the artifactual

321

formation of aldehydes during hydrolysis of proteins adducts (Table 3) in order to minimize the

322

artifactual formation. The extent of aldehyde release from acid hydrolysis partly depends on the

323

pH of serum samples (online supporting information Figure S1). We determined that acidifying

324

human serum samples to pH 3 maximizes the release of aldehydes from Schiff base protein

325

adducts. The desired sample pH can be obtained using different types and concentrations of acid.

326

Concentrated acids change sample viscosity, possibly due to protein denaturation and

327

precipitation, whereas dilute acids contribute to sample homogeneity. Also, we tested two

328

common strong acids, hydrochloric acid and sulfuric acid; a uniform solution was formed during

329

the hydrolysis step using hydrochloric acid, while sulfuric acid made the solution heterogeneous. 15 ACS Paragon Plus Environment

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330

Consequently, we chose to use larger volumes of dilute HCl (330 µL, 0.1 M HCl). Three other

331

variables—time on the tray after acid addition, hydrolysis temperature, and mixing method—are

332

also crucial to control, since they affect the concentration of aldehydes released into the

333

headspace over time. Some aldehyde concentrations increase 2- to 3-fold over 24 hours on the

334

sample tray at 15°C after acid addition (Figure S2). Hexanal increases 3-fold, pentanal increases

335

4-fold, and propanal increases 2-fold. Control samples that ran with 20 minutes acid hydrolysis

336

showed that aldehyde levels remained constant (Figure S2, open symbols). These data suggest

337

that aldehydes continue to release into the headspace over time while samples sit on the tray at

338

15°C.

339

In an attempt to complete the reaction using acid hydrolysis, the temperature of samples

340

after acid addition was increased from 15°C to 37°C. In order to isolate the effects of

341

temperature increase on samples, the time on tray was controlled and samples were run

342

immediately after incubation. Increasing hydrolysis temperature to 37°C did not complete or

343

stabilize the hydrolysis. Rather, increasing the temperature resulted in a 2- to 3-fold increase in

344

aldehyde concentration (Figure S3). Thus, higher temperature hydrolysis should be avoided to

345

minimize artifactual aldehyde formation. In other attempts to complete the acid hydrolysis, we

346

investigated sonicating serum samples at 30°C after adding acid to decrease reaction time.

347

Samples that underwent 1, 2, 3, and 6 hours of sonication showed a 2- to 4-fold increase in

348

measured aldehyde concentrations (Figure S4). The resulting large increase may be due to

349

artifactual formation of aldehydes caused by harsh mixing conditions. This observation, paired

350

with the previous observations regarding higher temperatures, led us to use milder mixing

351

conditions (30°C shaking at 350 rpm).

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Rather than choosing to complete the hydrolysis reaction, we successfully stabilized

353

aldehyde release using a dual rail PAL autosampler to control time on the 15°C sample tray after

354

acid addition, hydrolysis temperature, and mixing method (Table 3). In order to reproducibly

355

measure aldehyde levels in this method, it is crucial to either complete the acid hydrolysis

356

reaction or control key variables that stabilize the reaction. We chose to control and stabilize the

357

reaction since the use of high temperatures and harsh mixing conditions did not complete the

358

reaction.

359

Concentrations of all aldehydes were measured after varying periods of acid hydrolysis

360

(1, 3, 5, 10, 15, 20, 30 min) to find the optimum hydrolysis time. All measured aldehyde

361

concentrations were averaged together for each hydrolysis time period. Each average value was

362

normalized at the maximum hydrolysis time of 30 min. The concentrations begin to level off

363

around 20 min of hydrolysis (Figure S5); this optimum hydrolysis time was selected for this

364

method.

365

We found that increasing the temperature of the agitator during headspace extraction

366

yielded an increase in adsorption of analytes onto the fiber. However, when the temperature was

367

above 60°C, the serum samples began to denature. Consequently, we determined that headspace

368

extraction at 50°C yielded excellent analytical sensitivity without compromising the integrity of

369

the samples.

370

We optimized the fiber extraction time for this method by analyzing the SPME extraction

371

time at 5, 8, 10, and 12 minutes. Optimum extraction time is defined as the time required for

372

partitioning equilibrium between the sample and headspace, as well as adsorption equilibrium

373

between the headspace and fiber coating. The impact of extraction time was assessed using

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374

spiked serum samples (N=3) at the same concentration as QC Low samples (Figure S6). The

375

response signal improved with increased extraction time (2-12 min); we chose an extraction time

376

of 10 minutes to maximize adsorption of all analytes and to minimize analyte adsorption

377

competition on the SPME fiber.

378

Aldehydes listed in this method are in tobacco and/or tobacco smoke, or used in flavor

379

formulations. The US Food and Drug Administration classifies acetaldehyde, acrolein, propanal,

380

and crotonaldehyde as Harmful and Potentially Harmful Constituents (HPHC) of tobacco

381

smoke.30 Additionally, 2DF is a useful biomarker for identifying active/daily smokers.25 Thus we

382

measured serum 2DF using the same method to aid interpretation of the measured aldehyde data.

383

The resulting serum 2DF values correlated strongly (r=0.94) with previously measured blood

384

2DF values. We measured aldehydes levels in serum samples collected from non-smokers and

385

smokers. The mean concentrations of 19 aldehydes for non-smokers (N = 5) and smokers (N = 5)

386

are shown in Figure 4. The levels of known tobacco-related aldehydes31 (propanal, butanal,

387

isobutanal, and isopentanal) were categorized according to blood 2DF levels, a biomarker of

388

tobacco smoke exposure.32 We also categorized the smokers and non-smokers according to

389

measured 2DF levels in human serum. Mean concentrations of 2DF in serum for non-smokers

390

and smokers were 0.047 ± 0.023 and 0.220 ± 0.076 µg/L, respectively. Mean concentrations of

391

2DF in blood for non-smokers and smokers were 0.007 ± 0.000 and 0.119 ± 0.053 µg/L,

392

respectively. Our results indicate that smokers have higher levels of propanal, isobutanal, and

393

isopentanal than non-smokers. Preliminary data suggest that our method has adequate sensitivity

394

to measure levels of tobacco smoke-related aldehydes found in the serum of smokers. Aldehyde

395

levels correlated directly with 2DF, suggesting tobacco exposure as a primary source for

396

environmental aldehyde exposure (Figure 4). Moreover, we successfully implemented this assay 18 ACS Paragon Plus Environment

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397

for the analysis of 2400 human samples as part of a large population-based study (unpublished

398

data). Measurement of 19 aldehydes in human serum will better characterize aldehyde exposure

399

among smokers in the U.S. population. Furthermore, this work will establish baseline exposure

400

levels for aldehydes in the U.S. population, allowing for future comparative analyses.

401

We developed and validated a novel analytical method to quantify 19 aldehydes released

402

from Schiff base protein adducts in human serum. This is the first report of a method that

403

successfully performs automated hydrolysis of aldehyde-protein adducts, resulting in free

404

aldehydes that can be detected by high-resolution mass spectrometry to achieve low limits of

405

detection. The main advantage of this method is the use of stable isotope-labeled analog internal

406

standards for each analyte. The use of appropriate ISTDs is important for accurate quantification

407

of very low aldehyde levels since the measured analytes have individual differences in reactivity,

408

competition effects associated with different matrices and fiber coatings, and MS ionization

409

efficiencies. This method is well suited for high-throughput analysis to examine environmental

410

aldehyde exposures in population-based studies such as the National Health and Nutritional

411

Examination Survey33.

412

Acknowledgements:

413

The authors would like to acknowledge and thank Dr. David M. Chambers of the CDC for his

414

valuable input and discussions of data for this study. This study was partially funded by the US

415

Food and Drug Administration Center for Tobacco Products.

416

Conflict of Interest:

417

The authors declare no conflict of interest.

19 ACS Paragon Plus Environment

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Tables and figures: Table 1: Target aldehyde exact masses with Multiple Ion Detection (MID) windows and their respective labeled internal standard for serum aldehyde method.

Aldehyde

Labeled Internal Standard

Acetaldehyde Acrolein Propanal Isobutanal Butanal Isopentanal Crotonaldehyde 2,5-Dimethylfuran Pentanal Hexanal Furaldehyde trans-2-Hexenal Heptanal Benzaldehyde Octanal trans-2-Octenal o-Tolualdehyde Nonanal trans -2-Nonenal Decanal

Acetaldehyde-13C2 Acrolein-13C3 Propanal-13C Isobutanal-13C Butanal-13C Isopentanal-13C Crotonaldehyde-d6 2,5-Dimethylfuran-13C2 Pentanal-13C Hexanal-13C Furaldehyde-13C trans-2-Hexenal-13C Heptanal-13C Benzaldehyde-13C7 13 Octanal-13 C trans-2-Octenal-13C o-Tolualdehyde-13C Nonanal-13C trans-2-Nonenal-13C Decanal-13C

MID Window (m/z) 1 2 2 3 3 4 4 5 5 6 7 7 8 9 9 9 10 10 11 11

Lock Mass (m/z) 39.9618 39.9618 39.9618 68.9947 68.9947 68.9947 68.9947 68.9947 68.9947 68.9947 68.9947 68.9947 68.9947 99.9931 99.9931 99.9931 118.9915 118.9915 118.9915 118.9915

Calibration Mass (m/z) 68.9947 68.9947 68.9947 99.9931 99.9931 99.9931 99.9931 99.9931 99.9931 99.9931 99.9931 99.9931 99.9931 118.9915 118.9915 118.9915 130.9915 130.9915 130.9915 130.9915

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Retention Time (min) 4.55 6.14 6.23 7.47 8.22 9.58 9.75 10.20 10.49 12.66 13.85 14.02 14.67 16.23 16.55 17.77 18.10 18.27 19.43 19.87

Quantitation Ion (m/z) 42.0100 56.0257 58.0413 72.0570 72.0570 71.0491 70.0413 96.0570 57.0335 82.0777 96.0206 83.0491 81.0699 106.0413 110.1090 97.0648 119.0491 114.1039 111.0804 109.1012

Confirmation Ion (m/z) 41.0022 55.0178 57.0335 57.0335 57.0335 58.0413 68.0257 95.0491 58.0413 72.0570 67.0178 97.0726 86.0726 105.0335 99.0804 108.0934 120.0570 124.1247 122.1090 128.1196

Internal Standard Ion (m/z) 45.0290 58.0279 59.0447 73.0603 73.0603 72.0525 75.0727 (D 98.0637 59.0447 83.0811 97.0239 84.0525 82.0732 83.0587 101.0916 98.0681 121.0603 115.1073 112.0838 129.1229

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Table 2: Limits of detection (LODs) for 19 aldehydes and 2,5-dimethylfuran measured in µg/L.

Analyte

Acetaldehyde

Method LOD Lowest calibration Lower reportable Upper reportable (µg/L) standard (µg/L) level (µg/L) level (µg/L) 50.6

14.2

50.6

3540

Acrolein

2.16

0.627

2.16

418.

Propanal

1.16

0.186

1.16

124.

Isobutanal

0.109

0.071

0.109

47.4

Butanal

0.313

0.062

0.313

41.1

Isopentanal

0.119

0.075

0.119

50.2

Crotonaldehyde

0.147

0.032

0.147

21.6

2,5-Dimethylfuran

0.038

0.006

0.038

3.89

Pentanal

0.316

0.255

0.316

170.

Hexanal

0.693

0.507

0.693

338.

Furaldehyde

1.24

0.181

1.24

121.

trans-2-Hexenal

0.290

0.032

0.290

43.3

Heptanal

0.312

0.040

0.312

26.4

Benzaldehyde

0.461

0.061

0.461

40.9

Octanal

0.660

0.094

0.660

62.8

trans-2-Octenal

1.12

0 0.200

1.12

134.

o-Tolualdehyde

0.142

0.013

0.142

Nonanal

2.63

0.255

2.63

170.

trans-2-Nonenal

2.68

0.204

2.68

136.

Decanal

3.90

0.128

3.90

85.2

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Table 3: Optimized parameters of key variables in aldehyde method using controlled acid hydrolysis.

Variable

Parameters Tested

Optimal

Sample Volume (mL)

3, 0.5, 0.25

0.25

Acid Type

HCl, H2SO4

HCl

Acid Concentration (N)

0.1, 1.0, 2.0, 10

0.1

Fiber Extraction Temperature (°C)

30, 40, 50, 60

50

Incubation Temperature (°C)

15, 30, 37, 60

30

Hydrolysis time (min)

1, 5, 7, 10, 15, 20, 30

20

Mixing Method

Sonication, Shaking

Shaking

Sample Extraction Time (min)

5, 8, 10, 12

10

Incubation Speed (rpm)

300, 350, 375, 400

350

rpm: revolutions per minute. HCl: hydrochloric acid. H2SO4: sulfuric acid.

23 ACS Paragon Plus Environment

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Table 4: Reproducibility of analysis of aldehydes and 2,5-dimethylfuran in quality control (QC) samples made in human serum (N = 20) over three months.

Analyte

Serum QC Low Mean CV% (µg/L)

Serum QC High Mean CV% (µg/L)

Acetaldehyde

149

16.7

266

22.3

Acrolein

31.1

15.5

55.6

8.63

Propanal

8.28

5.81

17.9

6.90

Isobutanal

3.39

3.71

8.14

3.81

Butanal

2.46

5.25

5.85

4.89

Isopentanal

3.55

4.89

9.19

4.46

Crotonaldehyde

0.302

13.3

0.570

13.5

2,5-Dimethylfuran

0.271

9.96

0.660

11.1

Pentanal

11.5

4.68

26.0

3.29

Hexanal

36.6

4.18

79.3

3.85

Furaldehyde

10.5

4.76

28.0

3.42

trans-2-Hexenal

4.20

12.0

9.36

9.88

Heptanal

3.28

4.55

6.96

3.06

Benzaldehyde

4.39

7.77

10.8

5.20

Octanal

5.51

4.76

14.0

4.67

trans-2-Octenal

14.2

8.01

28.1

7.26

o-Tolualdehyde

0.741

4.94

2.03

4.48

Nonanal

22.6

7.54

48.5

5.89

trans-2-Nonenal

15.6

5.57

23.9

5.12

Decanal

5.85

7.29

15.2

6.53

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Table 5: Percent error for proficiency test (PT) solutions and spiked recovery in human serum (N=3) for 19 aldehydes and 2,5-dimethylfuran.

% Error PT low

% Error PT high

% Error Spiked Serum

Acetaldehyde

-10.3

-9.22

-5.87

Acrolein

4.40

-0.500

-80

Propanal

0.800

1.50

-0.71

Isobutanal

-8.50

-10.5

-0.42

Butanal

-8.10

-2.60

-3.29

Isopentanal

3.40

-3.55

-2.27

Crotonaldehyde

11.3

-4.20

30.2

2,5-Dimethylfuran

0.49

-8.49

11.2

Pentanal

0.800

1.23

-2.79

Hexanal

-0.700

2.70

-5.43

Furaldehyde

-0.300

-2.90

1.51

trans-2-Hexenal

6.10

1.40

-5.77

Heptanal

-5.40

-9.20

3.09

Benzaldehyde

-1.10

-7.38

-0.32

Octanal

18.8

-11.4

-4.85

trans-2-Octenal

-3.40

-8.10

3.64

o-Tolualdehyde

-1.90

2.80

8.48

Nonanal

22.5

-7.10

-1.26

trans-2-Nonenal

-0.50

6.20

-11.0

Decanal

6.00

1.88

-7.17

Analyte

25 ACS Paragon Plus Environment

5.0

0

4 6 8 10 12 14

26

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16

o-Tolualdehyde

trans-2-Octenal

Benzaldehyde

Heptanal

trans-2-Hexenal

Octanal

Nonanal

Furaldehyde

Pentanal

Time, min

Isopentanal Crotonaldehyde 2,5 Dimethylfuran

0_ 6.00

_

Decanal

trans-2-Nonenal

Hexanal

Propanal

Acrolein

5

Butanal

Isobutanal

20 Relative Response

5e

Propanal

10 Acetaldehyde

5

25

Acrolein

Relative Response x 10

Environmental Science & Technology Page 26 of 34

__ 10x

6.60

10x

__

18

Time, min

Figure 1: Total ion chromatogram for fortified human serum for 19 aldehydes and 2,5-dimethylfuran (5 µg/L).

20

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30

Butanal Area Ratio

y = 0.4945x + 5.8146 R² = 0.9988

20

y = 0.4657x + 0.3039 R² = 0.9991

10

water serum

0 0

10

20

30

40

50

Specified Amount (ng/mL)

Figure 2: Calibration curves in water and serum for butanal, using nine calibration standards.

27 ACS Paragon Plus Environment

ld eh Ac yd r e Pr ole Is opa in ob n ut a l a B n Cr Iso ut al a 2, oto pen na 5- n t l Di ald an m e al e t hy hy d lf e Pe ura nt n a Fu He na tr r xa l an al n s - de a l 2 - hy He d e Be H xen e nz pt a l al an de a tra h l ns O yde o- - 2 c t a To -O n lu ct al al en de a tra h l ns No yd - 2 na e -N n on al De ena ca l na l

Ac et a

Percent deviation of quality control samples over 26 hours on sample tray at 15°C

Environmental Science & Technology

10 High quality control sample Low quality control sample

5

0

-5

-10

-15

Figure 3: Stability of 2,5-dimethylfuran and aldehyde release from Schiff base protein aldehyde adducts

using a controlled acid hydrolysis over 26 hours for quality control samples on sample tray maintained

at 15°C.

28

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Figure 4: Significantly higher concentrations of some aldehydes were detected in smoker serum samples (N=5, concentration of 2, 5-dimethylfuran (2DF) in serum = 0.220 ± 0.076 and in blood = 0.119 ± 0.053 µg/L) than in non-smoker serum samples (N=5, concentration of 2DF in serum = 0.047 ± 0.023 and in blood = 0.007 ± 0.000 µg/L).

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Table and Figure Legends Table 1: Target aldehyde exact masses with Multiple Ion Detection (MID) windows and their respective labeled internal standard for serum aldehyde method. Table 2: Limits of detection (LODs) for 19 aldehydes measured in µg/L. Table 3: Optimized parameters of key variables in aldehyde method using controlled acid hydrolysis. Table 4:

Reproducibility of analysis of aldehydes in quality control (QC) samples made in human serum (N = 20) over three months.

Table 5: Percent error for proficiency test (PT) solutions and spiked recovery in human serum (N=3) for 19 aldehydes and 2,5-dimethylfuran. Figure 1: Total ion chromatogram for fortified human serum for 19 aldehydes (5 µg/L). Figure 2: Calibration curves in water and serum for butanal, using nine calibration standards. Figure 3: Stability of aldehyde release from Schiff base protein aldehyde adducts using a controlled acid hydrolysis over 26 hours for quality control samples on sample tray maintained at 15°C. Figure 4: Significantly higher concentrations of some aldehydes were detected in smoker serum samples (N=5, concentration of 2, 5-dimethylfuran (2DF) in serum = 0.220 ± 0.076 and in blood = 0.119 ± 0.053 µg/L) than in non-smoker serum samples (N=5, concentration of 2DF in serum = 0.047 ± 0.023 and in blood = 0.007 ± 0.000 µg/L).

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Supporting Information Figures S1-S6. Figures detail method parameters such as: serum pH, hydrolysis time, incubation time, sonication time, and extraction time to reveal optimum aldehyde concentrations for this method.

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TOC/Abstract Art

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Reference List

1.

2. 3. 4. 5.

6.

7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

Richardson, S. D.; Thruston, A. D.; Caughran, T. V.; Chen, P. H.; Collette, T. W.; Floyd, T. L.; Schenck, K. M.; Lykins, B. W.; Sun, G. R.; Majetich, G., Identification of new ozone disinfection byproducts in drinking water. Environmental Science & Technology 1999, 33, (19), 3368-3377. Clark, L.; Aronov, E. V., Human food flavor additives as bird repellents: I. Conjugated aromatic compounds. Pesticide Science 1999, 55, (9), 903-908. Elghawi, U. M.; Mayouf, A. M., Carbonyl emissions generated by a (SI/HCCI) engine from winter grade commercial gasoline. Fuel 2014, 116, 109-115. Lipari, F.; Dasch, J. M.; Scruggs, W. F., Aldehyde Emissions from Wood-Burning Fireplaces. Environmental Science & Technology 1984, 18, (5), 326-330. Kataoka, H.; Sumida, A.; Makita, M., Determination of aliphatic and aromatic aldehydes in cigarette smoke by gas chromatography with flame photometric detection. Chromatographia 1997, 44, (9-10), 491-496. O'Brien, P. J.; Siraki, A. G.; Shangari, N., Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Critical Reviews in Toxicology 2005, 35, (7), 609-662. Bruce, W. R.; Lee, O.; Liu, Z.; Marcon, N.; Minkin, S.; O'Brien, P. J., Biomarkers of exposure to endogenous oxidative and aldehyde stress. Biomarkers 2011, 16, (5), 453-456. Furuhata, A.; Nakamura, M.; Osawa, T.; Uchida, K., Thiolation of protein-bound carcinogenic aldehyde - An electrophilic acrolein-lysine adduct that covalently binds to thiols. Journal of Biological Chemistry 2002, 277, (31), 27919-27926. Lee, C.; Yim, M. B.; Chock, P. B.; Yim, H. S.; Kang, S. O., Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation. Journal of Biological Chemistry 1998, 273, (39), 25272-25278. Xue, R. Y.; Dong, L.; Zhang, S.; Deng, C. H.; Liu, T. T.; Wang, J. Y.; Shen, X. Z., Investigation of volatile biomarkers in liver cancer blood using solid-phase microextraction and gas chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 2008, 22, (8), 1181-1186. Nair, U.; Bartsch, H.; Nair, J., Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: A review of published adduct types and levels in humans. Free Radical Biology and Medicine 2007, 43, (8), 1109-1120. Yazdanpanah, M.; Oudit, G.; Lee, A.; Dawood, F.; Wen, W. H.; Backx, P.; Liu, P., Cytotoxic aldehydes aggravate ventricular dysfunction in an injured heart: Evidence from an iron overload model of heart failure. Journal of the American College of Cardiology 2002, 39, (5), 175A-175A. Yazdanpanah, M.; Luo, X. P.; Lau, R. B.; Greenberg, M.; Fisher, L. J.; Lehotay, D. C., Cytotoxic aldehydes as possible markers for childhood cancer. Free Radical Biology and Medicine 1997, 23, (6), 870-878. Pirkle, J. L.; Sampson, E. J.; Needham, L. L.; Patterson, D. G.; Ashley, D. L., Using biological monitoring to assess human exposure to priority toxicants. Environ Health Perspect 1995, 103 Suppl 3, 45-8. Sampson, E. J.; Needham, L. L.; Pirkle, J. L.; Hannon, W. H.; Miller, D. T.; Patterson, D. G.; Bernert, J. T.; Ashley, D. L.; Hill, R. H.; Gunter, E. W.; et al., Technical and scientific developments in exposure marker methodology. Clin Chem 1994, 40, (7 Pt 2), 1376-84. Yeo, H. C.; Helbock, H. J.; Chyu, D. W.; Ames, B. N., Assay of Malondialdehyde in Biological-Fluids by Gas-Chromatography Mass-Spectrometry. Analytical Biochemistry 1994, 220, (2), 391-396.

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17.

Codreanu, S. G.; Liebler, D. C., Novel approaches to identify protein adducts produced by lipid peroxidation. Free Radic Res 2015, 49, (7), 881-7. 18. Deng, C. H.; Li, N.; Zhang, X. M., Development of headspace solid-phase microextraction with onfiber derivatization for determination of hexanal and heptanal in human blood. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2004, 813, (1-2), 47-52. 19. Deng, C. H.; Zhang, X. M., Simple, rapid and sensitive method for determination of aldehydes in human blood by gas chromatography/mass spectrometry and solid-phase microextraction with on-fiber derivatization. Rapid Communications in Mass Spectrometry 2004, 18, (15), 1715-1720. 20. Luo, X. P.; Yazdanpanah, M.; Bhooi, N.; Lehotay, D. C., Determination of Aldehydes and Other Lipid-Peroxidation Products in Biological Samples by Gas-Chromatography Mass-Spectrometry. Analytical Biochemistry 1995, 228, (2), 294-298. 21. Cai, J.; Bhatnagar, A.; Pierce, W. M., Protein Modification by Acrolein: Formation and Stability of Cysteine Adducts. Chemical Research in Toxicology 2009, 22, (4), 708-716. 22. Silva, L. K.; Wilburn, C. R.; Bonin, M. A.; Smith, M. M.; Reese, K. A.; Ashley, D. L.; Blount, B. C., Quantification of fuel oxygenate ethers in human blood using solid-phase microextraction coupled with gas chromatography-high-resolution mass Spectrometry. Journal of Analytical Toxicology 2008, 32, (4), 273-280. 23. Cardinali, F. L.; Mccraw, J. M.; Ashley, D. L.; Bonin, M. A., Production of Blank Water for the Analysis of Volatile Organic-Compounds in Human Blood at the Low Parts-Per-Trillion Level. Journal of Chromatographic Science 1994, 32, (1), 41-45. 24. Clinical and Laboratory Standards Institute, S. E. C. d. E.-A. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline; 950 West Valley Road, Suite 2500, Wayne, PA 19087, 2012, 2012. 25. Chambers, D. M.; Ocariz, J. M.; McGuirk, M. F.; Blount, B. C., Impact of cigarette smoking on volatile organic compound (VOC) blood levels in the U.S. population: NHANES 2003-2004. Environ Int 2011, 37, (8), 1321-8. 26. Seeman, J. I.; Dixon, M.; Haussmann, H. J., Acetaldehyde in mainstream tobacco smoke: formation and occurrence in smoke and bioavailability in the smoker. Chem Res Toxicol 2002, 15, (11), 133150. 27. Alwis, K. U.; deCastro, B. R.; Morrow, J. C.; Blount, B. C., Acrolein Exposure in U.S. Tobacco Smokers and Non-Tobacco Users: NHANES 2005-2006. Environ Health Perspect 2015, 123, (12), 1302-8. 28. Bagchi, P.; Geldner, N.; deCastro, B. R.; De Jesus, V. R.; Park, S. K.; Blount, B. C., Crotonaldehyde exposure in U.S. tobacco smokers and nonsmokers: NHANES 2005-2006 and 2011-2012. Environ Res 2018, 163, 1-9. 29. Tuma, D. J.; Hoffman, T.; Sorrell, M. F., The Chemistry of Acetaldehyde Protein Adducts. Alcohol and Alcoholism 1991, 271-276. 30. Kux, L., Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke; Established List. In Federal Register 2012; 77:20034–7, http://www.gpo.gov/fdsys/pkg/FR-201204-03/pdf/2012-7727.pdf., Accessed November 2015. 31. Rodgman, A.; Perfetti, T. A., The Chemical Components of Tobacco and Tobacco Smoke. Second ed.; CRC Press, 2012; p 2238. 32. Ashley, D. L.; Bonin, M. A.; Hamar, B.; McGeehin, M., Using the blood concentration of 2,5dimethylfuran as a marker for smoking. International Archives of Occupational and Environmental Health 1996, 68, (3), 183-187. 33. National Health and Nutrition Examination Survey. Available: http:// www.cdc.gov/nchs/nhanes.htm 34 ACS Paragon Plus Environment

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