The determination of 1,4-dioxane in cosmetic products by gas chromatography with tandem mass spectrometry

Accepted Manuscript Title: The Determination of 1,4-Dioxane in Cosmetic Products by Gas Chromatography with Tandem Mass Spectrometry Author: Wanlong Zhou PII: DOI: Article Number:

S0021-9673(19)30784-8 https://doi.org/10.1016/j.chroma.2019.460400 460400

Reference:

CHROMA 460400

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

16 April 2019 22 July 2019 23 July 2019

Please cite this article as: Zhou W, The Determination of 1,4-Dioxane in Cosmetic Products by Gas Chromatography with Tandem Mass Spectrometry, Journal of Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.460400 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The Determination of 1,4-Dioxane in Cosmetic Products by Gas Chromatography

with Tandem Mass Spectrometry Wanlong Zhou∗

∗E-mail address: [email protected] (W. Zhou).

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Highlights

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U. S. Food and Drug Administration, Office of Regulatory Science, CFSAN, 5001 Campus Drive, College Park, MD 20740, USA

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 GC-MS/MS using pulsed split injection was used to determine 1,4-dioxane in cosmetics.  The UAE was used for preparing liquid samples without sample cleanup.  The method was fully validated using a variety of cosmetic matrices.  1,4-dioxane was detected in 47 out of the 82 products ranged from 0.23 to 15.3 μg/g.

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Abstract

1,4-dioxane is a potential human carcinogen and contaminant produced during the

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manufacturing process from specific cosmetic ingredients, such as certain detergents and emulsifiers. As such, 1,4-dioxane is not identified on product ingredient labels. To assess

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the concentration of 1,4-dioxane in cosmetic products, a gas chromatography-tandem mass spectrometry (GC–MS/MS) method using pulsed split injection and electron ionization was developed and validated. For liquid cosmetic products such as shampoo

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and lotion, test portions were extracted using fast ultrasound-assisted extraction (UAE) procedure without sample cleanup. For solid products (e.g. beauty bars), a C18 solid phase extraction (SPE) procedure was optimized to reduce potential interferences. The corresponding stable isotopically labeled analogue (1,4-dioxane-d8) was selected as an internal standard to compensate for matrix effects and sample recovery. Method recovery experiments were performed in lotion, oil gel, hair detangler, bubble bath and beauty bar

2 (solid) sample matrices with recoveries of 84–108% and relative standard deviations less than 5% at three spike concentrations. Method limits of detection (LOD) and quantification (LOQ) for 1,4-dioxane were determined at 0.2 μg/g and 0.5 μg/g, respectively. The method was successfully used to determine 1,4-dioxane in 82 leave-on and rinse-off cosmetic products marketed toward children including bath products, hair

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treatment, lotions, beauty bars, washes, shampoos, and other products. 1,4-dioxane was detected in 47 of the 82 products with an average concentration of 1.54 μg/g (Range:

0.23-15.3 μg/g). The method can increase sample throughput and reduce matrix-induced interferences.

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Keywords. 1,4-dioxane, pulsed injection, GC-MS/MS, cosmetics, sample preparation

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1. Introduction

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1,4-dioxane is a heterocyclic organic compound commonly used as a solvent in the manufacturing of products such as paints, varnishes, lacquers, rubber, etc. It is also

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used as a laboratory reagent and a stabilizer for chlorinated solvents [1]. In cosmetics products, 1,4-dioxane can be formed as a byproduct during the manufacturing process of

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certain ingredients, including detergents, foaming agents, emulsifiers, and solvents identified by the prefix, word, or syllables “PEG,” “Polyethylene,” “Polyethylene glycol,”

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“Polyoxyethylene,” “-eth-,” or “-oxynol-”. Polyethoxylated raw materials are widely used in cosmetic products as emulsifiers, foaming agents, and dispersants [2]. They are made

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by the polymerization of ethylene oxide, usually with fatty alcohols, to form polyethoxylated alcohols, which are then used to synthesize other sulfated surface active

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agents. During the ethoxylation process, 1,4-dioxane can be formed as an undesired byproduct by the dimerization of ethylene oxide. Because1,4-dioxane is a by-product and not a cosmetic ingredient, it is not identified on product ingredient labels. Based on animal carcinogenicity test data by oral administration in mice, rats, and guinea-pigs, 1,4-dioxane was classified as a potential human carcinogen (Group 2B) by the International Agency for Research on Cancer (IARC) in 1999 [3]. The U.S.

3 Environmental Protection Agency (EPA) also concluded in 2013 that 1,4-dioxane is “likely to be carcinogenic to humans” in view of the evidence of multiple tissue carcinogenicity in several 2-year bioassays performed in rats, mice, and guinea pigs [1]. 1,4-dioxane is listed in Annex II of the EU Cosmetic Regulation: List of substances prohibited in cosmetic products [4]. The European Commission Scientific Committee on

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Consumer Safety (SCCS) determined that a trace level of 1,4-dioxane in cosmetic

products of ≤10 ppm (µg/g) is safe [5]. The U.S. Food and Drug Administration (FDA) has not established or recommended a specific limit on the levels of 1,4-dioxane in cosmetics.

Since the 1980s, FDA has recommended that manufacturers use the “vacuum

stripping” technique as a way of reducing 1,4-dioxane [6] in their products. Additionally,

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since the late 1970s, the FDA has periodically monitored the levels of 1,4-dioxane in

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cosmetics products [2], with the last survey being completed in 2010 [7]. To update our monitoring data, a new survey has been undertaken.

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There are numerous methods developed to determine 1,4-dioxane in water [8, 9],

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food [10] and environmental samples [11] using gas chromatography-flame ionization detector (GC-FID) [2, 10], gas chromatography-mass spectrometry (GC-MS) [8], and gas

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chromatography-tandem mass spectrometry (GC-MS/MS) [11]. A few studies have been

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previously reported to determine 1,4-dioxane in cosmetic raw materials [2, 12] and finished cosmetic products [2, 13, 14] by high performance liquid chromatography (HPLC) [13] , GC-MS [14] and GC-FID [2]. Since HPLC methods suffer from a higher

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LOQ of 6.5 ppm [13], GC-MS and GC-FID emerged as the most commonly used techniques. To minimize matrix interferences and achieve trace level quantification of

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1,4-dioxane, researchers have investigated different sample preparation procedures, including liquid-liquid extraction (LLE) followed by solid-phase extraction (SPE) [2, 14-

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18], headspace extraction [19-21], headspace solid-phase microextraction (HS-SPME) [22-24], single-drop microextraction [12], and azeotropic atmospheric distillation [2]. Direct injection of the diluted samples was also tested [25, 26]. All published sample preparation procedures have their limitations. For example, SPE and azeotropic atmospheric distillation are time-consuming, specialized autosamplers are needed for performing headspace and HS-SPME application, a long heating time is required prior to

4 GC analysis for headspace extraction [19], expensive fibers with limited lifetime are essential for SPME procedures [12], and manual injection of the extractant is needed for single-drop microextraction methods [12]. In direct injection methods, the injection port and columns were severely contaminated by non-volatile residues [25, 26]. The use of pulsed splitless injection for the determination of pesticide residues in

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vegetables and water samples has been reported because it can lower detection limits and reduce matrix-induced effects [27, 28]. However, pulsed injection has not been reported for the determination of 1,4-dioxane in any matrix. To increase sample throughput and minimize matrix interferences, a sensitive and selective GC–MS/MS method using

pulsed split injection was developed to determine 1,4-dioxane in cosmetics products. A fast ultrasound-assisted extraction (UAE) procedure without sample cleanup was

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developed to determine 1,4-dioxane for liquid cosmetic products (e.g., body wash,

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shampoo, lotion, cream). Compared to SPE procedures, the sample throughput using a common ultrasonic bath can be increased by three times because more than a dozen

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samples can be prepared simultaneously. For solid products (e.g., beauty bar), further

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cleanup of the extract was necessary and a previously reported SPE procedure [14] was modified to reduce sample processing. A stable isotopically labeled analog of 1,4-

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dioxane (1,4-dioxane-d8) was implemented as a surrogate internal standard (internal

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standard , IS) [29] to compensate for analyte recovery and matrix effects. The method was validated in a variety of cosmetic matrices and has been successfully applied to analyze 82 commercial cosmetic products (79 liquid products + 3 solid products)

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marketed for children and containing the ingredients associated with 1,4 dioxane contamination (see above). The products cover most common children cosmetic

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categories, e.g., bath products, hair treatment, lotions, and washes. This survey will allow

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the FDA to assess the prevalence of 1,4-dioxane in cosmetic products.

2. Experimental

2.1. Chemicals 1,4-dioxane and 1,4-dioxane-d8 were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade hexane (HEX) and dichloromethane (DCM) were obtained from EMD Millipore Corporation (Billerica, MA, USA) and Acros (New Jersey, USA),

5 respectively. Pesticide grade acetone, LC-MS grade acetonitrile (ACN) and methanol were acquired from Fisher Scientific (Fair Lawn, New Jersey, USA). All of the chemicals were used without further purification. 2.2. Preparation of standard solutions The primary stock solutions of 1,4-dioxane and 1,4-dioxane-d8 were separately

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prepared at approximately 1,000 µg/mL in methanol, respectively. The stock solutions

were stored at -30 °C into amber narrow mouth bottles. Working stock solutions for the native and labeled 1,4-dioxane were prepared by dilution of the primary stock solutions in ACN. Eight standard calibration solutions ranged from 50 to 50,000 ng/mL with

constant IS concentration of 1,000 ng/mL were prepared by diluting the working stock

solutions using ACN. The standards were transferred into amber GC-MS vials with crimp

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top caps and stored at -30 °C until analysis.

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2.3. Sample preparation

For liquid cosmetic products, approximately 250 mg of product was accurately

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weighed into a 15-mL centrifuge tube. After adding 250 µL of IS (10.0 µg/mL 1,4-

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dioxane-d8) to each of the tubes, the tubes were capped tightly and gently vortexed for 3 min. Two mL of ACN was added to the tubes. The final volume for the extracted solution

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is approximately: 250 μL (product) + 250 uL (IS) + 2mL (ACN), i.e., ~ 2.5 mL. The

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actual volume will vary with amount weighed and the density as well as the solubility of individual products. The internal standard added to all samples would correct for small sample to sample variation. After vortexing for 3 min to disperse the sample and

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sonicating for 30 min, the tubes were vortexed again for 3 min and centrifuged at 15,557 rcf (11,000 rpm) for 10 min using an Eppendorf 5804 centrifuge (Hamburg, Germany).

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The supernatant was filtered through a 0.2 µm PTFE filter (Pall Life Sciences, Port Washington, NY) directly into a GC-MS sample vial.

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For solid products (e.g., beauty bar), approximately 200 mg of product was

accurately weighed into a 15-mL centrifuge tube. After adding 200 µL of IS (10.0 µg/mL 1,4-dioxane-d8) to each of the tubes, the tubes were capped tightly and gently vortexed for 3 min. Two mL of 80:20 HEX:DCM was added to the tubes, followed by vortexing for 3 min to disperse the sample in solution, sonicating for 30 min, vortexing for 3 min, and centrifuging at 15,557 rcf for 10 min. To clean up sample extracts, the extraction

6 supernatant was then loaded with a plastic pipette onto a HyperSep™ C18 SPE Cartridge (3mL, 500 mg, Thermo Scientific HyperSep) that had been conditioned under mild vacuum with 2 mL of ACN and 2 mL of 80:20 HEX:DCM. The cartridge was washed with 2 mL of 80:20 HEX:DCM. To help remove excessive HEX:DCM, a pipette bulb was used to “dry” the remaining sample which could not be removed by vacuum. 1,4-dioxane

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was eluted from the SPE cartridge into a GC-MS vial using 2 mL of ACN. 2.4. Instrumentation

The data were acquired using an Agilent 7890A GC System coupled to an Agilent 7000 Triple Quadrupole MS (Santa Clara, CA, USA). MassHunter GC-MS Acquisition (Version B.07.00, Agilent) and Quantitative Analysis (Version B.06.00, Agilent) were

used to control the GC-MS/MS system and process data. An Agilent HP-5 MS UI column

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(30 m × 0.25 mm × 0.25 µm) was implemented for chromatographic separation. The

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injector temperature was fixed at 185 °C and a pulsed split injection mode was used with helium as a carrier gas at a split ratio of 10:1. The injection pulse pressure was set to 10

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psi for 1 min followed by a constant pressure of 3.5 psi. The injection volume for each

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sample was 1.0 µL. The oven temperature was held at 40 °C for 4 min, then ramped at 5 °C/min to 60 °C, and then at 50 °C/min to 220 °C, and then held at this temperature for 1

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min. The total run time was 12 min. The MS was operated in positive electron ionization

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(EI) mode. The MS source temperature was set at 230 °C. To increase analyte selectivity and minimize background interference, a tandem mass spectrometry method in selected reaction monitor (SRM) mode was used in this

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study. Ion transitions and collision energy values for each transition were determined by injecting individual test solutions of 1,4-dioxane and 1,4-dioxane-d8, respectively. The

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product scan spectra for 1,4-dioxane and 1,4-dioxane-d8 are shown in supplemental Figure 1. Three transitions were selected and acquired for 1,4-dioxane and 1,4-dioxane-d8.

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The m/z 88 → 57 transition for 1,4-dioanxe and m/z 96 → 62 for IS were used for quantitation. A longer dwell time (150 ms) was applied to the quantitation transition (m/z 88 → 57) to achieve a stronger signal as compared to a 50 ms dwell time for all other transitions. All transitions, optimized delta EMV (electron multiplier voltage), dwell time, and collision energy for each analyte are listed in Table 1. After 1,4-dioxane was eluted

7 off the column, the delta EMV was set to 0 (8 min) to increase the lifetime of the multiplier. 2.5. Method validation 2.5.1. Linearity, intraday, and interday instrument precision studies The peak area ratio of 1,4-dioxane (m/z 88 → 57) to IS (m/z 96 → 62) was used

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for quantitation. To reduce errors in the lower end of a calibration curve, a weighting

factor of 1/x2 was applied for the calibration curve, where x represents the concentration ratio of 1,4-dioxane to 1,4-dioxane-d8. The detailed calculation for selecting the best

weighting factor for the calibration curve can be found in Kiser et al.’s paper and FDA Guidance for Industry Bioanalytical Method Validation [30, 31].

Intraday instrument precision studies were performed by sequentially injecting the

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calibration standard solutions in triplicate within a single day. For interday instrument

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precision studies, a set of standard solutions was placed in the autosampler (at room temperature, ~ 25 °C) and injected on consecutive days. The caps were replaced after

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each injection to avoid evaporation. The calibration curve was performed daily.

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2.5.2. Recovery studies

For the recovery study, known amounts of 1,4-dioxane were fortified into

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weighed samples at three concentrations (1.5, 7.5, 50 μg/g). The same sample preparation

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procedures for liquid and solid samples were followed as discussed above (2.3.). Sample recovery and percent standard deviations were calculated for three replicate sample

(solid).

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preparations for each matrix: lotion, oil gel, hair detangler, bubble bath, and beauty bar

2.6. Product selection

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A list of cosmetic 327 products marketed toward children and containing

ingredients associated with 1,4 dioxane contamination was assembled through searching

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market share databases Mintel Global New Products, Gladson, and Label Insight. After removing replicates and errors a list of over 200 different products was created. From the smaller list, 82 unique cosmetic products available on the US market were selected and purchased for analysis. The 82 products were classified as cosmetics based on the Federal food, drug, and cosmetic act (FD&C Act) [32]. The products were divided into five categories: 1) bath products, 2) hair treatment, 3) lotions 4) washes, and 5) other products

8 (Table 4). These categories do not represent legal definitions, but were created based on similar physical characteristics and performance during method development and validation. More detailed product information including product number, category, physical form (liquid/solid), and description are listed in Supplemental Table 1 for all 82 products surveyed. The website links for the databases (Mintel Global New Products,

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Gladson/Syndigo, and Label Insight) were also included as footnotes in Supplemental Table 1. 2.7 QA/QC for survey samples

Instrument check solutions and independent check solutions were prepared using a stock solution different from that used to prepare the calibration solutions. The

instrument check solutions were analyzed directly following the calibration standards, as

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the last in the run, and every 10th sample or a minimum of once per batch to monitor

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retention time and instrument accuracy. All samples analyzed were bracketed by the instrument check solutions to evaluate instrument deviations.

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A spike recovery sample was prepared for every 20th sample analyzed. However,

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at least one recovery sample per matrix and a minimum of 1 sample per batch was analyzed. Replicate (triplicate) sample preparations were performed for every 20th

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sample analyzed, however at least one replicate sample per matrix and a minimum of one

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sample per batch was analyzed.

2.8 Identification and confirmation of the analytes

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The FDA CVM guidance 118 (Guidance for industry mass spectrometry for confirmation of the identity of animal drug residues) was applied to confirm the identity of 1,4-dioxane in a sample [33]. In this situation, the acceptability range of retention

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times (RT) should not exceed 2% when compared to the average retention times found in the calibration standards for 1,4-dioxane. The transition ion ratios (Intensity of

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confirmatory transition / Intensity of quantitation transition × 100%) for 1,4-dioxane and 1,4-dioxane-d8 between samples and standard solutions should agree within ±20%. Sample concentrations (g/g) were calculated based on the determined concentration (ng/mL), sample weight, and extraction volume.

3. Results and discussion

9 3.1. Mass spectrometry optimization The most reported technique for analyzing 1,4-dioxane is GC-MS with selected ion monitoring (SIM) because the method can be easily performed on widely available single quadrupole GC-MS systems. Although GC-MS methods have better selectivity than GC-FID, these methods are limited by the interferences caused by solvents and

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blank SPE cartridges [8, 9]. The interferences were previously reported when hexane or

dichloromethane was used as a solvent to determine 1,4-dioxane in cosmetics using GCMS with an HP-5 column [14]. A contaminant was also reported in some blank SPE cartridges and dichloromethane solvent in Carrera’s study for 1,4-dioxane in

environmental waters by GC-MS/MS with an Rxi-624Sil MS column [11]. The inference caused from blank solvents and SPE cartridge was also observed in this study (data not

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shown). The possible reason is that the molecular weight (88 Dalton) of 1,4-dioxane is

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similar to the molecular weight of common solvents and matrix impurities which result in interferences. To improve analyte selectivity, an SRM method was implemented and

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3.2 Gas Chromatography optimization

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collision energies were optimized for each 1,4-dioxane and IS transition (Table 1)..

The Agilent HP-5 MS UI column was selected for this study providing desired

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peak symmetry for 1,4-dioxane and IS. Because the pulsed splitless injection can lower

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detection limits, reduce the matrix-induced effect, and significantly reduce contamination of the inlet liner [27], the GC parameters were initially optimized using a pulsed splitless injection mode to increase sensitivity and permit a lower analyte signal detection. Pulsed

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splitless injection mode worked for neat standard solutions (Figure 1A). However, an extracted cosmetic product displayed peak tailing and reduced peak height (Figure 1B).

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In split injection mode, pulsed split injection was compared to a non-pulsed injection. Using pulsed split injection improved peak shape and signal-to-noise ratio by more than 6

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times (not shown). In pulsed split injection mode, high pressure (10 psi) was applied for 1 min after injection permitting a higher load on column and thereby resulting in a stronger signal. After that period, normal pressure (3.5 psi) was applied to the column. Injection mode conditions were optimized including pulsed pressure (10 psi), pulsed time (1 min), inlet temperature (185 oC), and split ratio (10:1) and tested for both standard neat solution (data not shown) and the cosmetic samples (Figure 1C).

10 3.3. Optimization for sample preparation procedures Different sample preparation procedures have been reported for analyzing cosmetic products for 1,4-dioxane. The procedures include SPE [2, 14-18], headspace extraction [19-21], and HS-SPME [22-24], and azeotropic atmospheric distillation [2]. SPE is the most commonly used procedure for sample preparation. However, the reported

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SPE procedures are very time-consuming, such as recommending a second SPE

cartridge/procedure [2] or a second centrifugation step [14]. The implementation of

pulsed split injection and GC-MS/MS precluded the need for complex sample cleanup, therefore, a simple UAE extraction procedure using labeled IS without sample cleanup was developed for liquid cosmetic products including bath, hair treatment, lotion, oil, wash, cream, and shampoo products. Both acetone and ACN extraction solvents

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demonstrated success for selected products. ACN was selected for validation because it

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has a higher boiling point. This is the first UAE procedure to determine 1,4-dioxane in liquid cosmetics products.

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While simple extraction worked well for liquid samples, solid samples such as

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beauty bars required additional cleanup steps to produce acceptable chromatographic results (Figure 2). An SPE procedure including extraction solvents developed by Song et

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al. [14] was adapted and optimized to prepare solid matrices, such as beauty bar (solid

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soap). Different SPE cartridges including HyperSep C18, Supelco ENVI-18, UCT EEC18, PRiME HLB6cc, Envi-Carb-Plus were evaluated. The HyperSep™ C18 SPE cartridge was selected offering reduced matrix interference (Figure 2) and flexibility for various

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flow rates without affecting sample recoveries. The SPE procedure developed by Song et al. (Song 1996) was modified by including a wash step using 2 ml of 80:20 HEX:DCM

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after loading the extraction solution. The second centrifuge step in Song’s procedures was eliminated to improve productivity. To minimize the influence of HEX and DCM on

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analyte elution time, a pipet bulb was used to “dry” the cartridge and remove excess HEX and DCM from the cartridge before eluting the analytes from the cartridge. Since 1,4dioxane was eluted directly to a 2-mL vial using 2 mL of ACN for GC-MS injection, the step to transfer 50 µL of the elution solution in Song’s procedure was also omitted. The optimized SPE procedure performed well for solid cosmetic products and had a higher sample throughput. The comparison of the peak shape for a solid matrix (beauty bar)

11 before and after SPE treatment is shown in Figure 2. To determine if there were differences between the UAE and SPE methods, other liquid matrices including lotion, body wash were also tested by SPE. Similar recoveries for liquid testing matrices were achieved for the solid matrices. UAE procedure resulted in overall better recoveries than those obtained by SPE procedure for the same liquid matrices (Supplemental Table 2).

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Because pulsed split injection with a split ratio of 10 was applied, the complex

sample matrices even without cleanup had limited contamination to the inlet and columns. The same inlet and the same column performed well without obvious deterioration for the whole survey period including the method validation and survey of samples. In

combination with a pulsed split injection mode, a simple UAE procedure was suitable for all liquid matrices tested.

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3.4. Method validation

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The method was validated in accordance with the “Guidelines for the validation of chemical methods for the FDA FVM (Foods and Veterinary Medicine) program” [34].

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An eight-point calibration curve was created for the quantitation of 1,4-dioxane based on

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the peak area ratio of 1,4-dioxane to the IS. The calibration curve had a good linearrange from 50 to 50000 ng/mL. The coefficient of determination (r2) was greater than 0.99. The

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calibration equation is shown as “y = 0.694x -0.114”. The intraday and interday

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instrument precision studies show that the standards were stable in the autosampler for at least three weeks at room temperature. All stock and working solutions were stable at -30 °C for over 6 months.

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The method limits of quantitation (LOQ, 0.5 μg/g, equivalent/corresponding to the lowest point on the calibration curve, 50 ng/mL) for both liquid and solid matrices

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was confirmed by spiking the analyte into two different sample matrices (lotion and beauty bar) in triplicate, where the determined concentration was within 20% of the

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expected concentration and within a precision of ≤20%, and the S/N ratio of the quantitative transition was calculated to be at least 10:1. The method limits of detection (LOD, 0.2 μg/g) was confirmed by spiking the different concentrations of analyte into two different sample matrices (lotion and beauty bar), where the peaks were reliably distinguished from the noise of the blank matrices. The S/N ratios of the confirmatory transitions were at least 3:1.

12 Recovery studies were carried out using different matrices including lotion, oil gel, hair detangler, bubble bath, and beauty bar at three spiking levels: low (1.50 μg/g, equal to 3X LOQ), medium (7.5 μg/g), and high (50 μg/g). A known amount of the analyte was spiked into a sample matrix and carried through the sample preparation process in triplicate. Since some matrices contain detectible 1,4-dioxane, the amount incurred in a

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sample before fortification was subtracted. The recovery results for lotion, oil gel, hair detangler, bubble bath, and beauty bar (solid soap) are summarized in Table 2. As

mentioned previously, all beauty bar samples went through SPE procedure as described

in section 2.3. Sample recoveries ranged from 87.4 to 108% for liquid products and 83.6 to 87.3% for solid products requiring SPE. The relative standard deviations were less than 5% for all tested matrices.

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3.5. Cosmetic product survey

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The validated method was used to determine 1,4-dioxane in 82 leave-on and rinse-off cosmetics products marketed toward children including bath products, hair

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treatment, lotions, beauty bars, washes and shampoos, and other products. Seventy-nine

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out of the 82 products (96%) were liquid products which were extracted using the UAE procedure, while 3 out of the 82 products (4%) were solid products which went through

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the SPE clean-up procedure. Representative chromatograms and SRM (MRM) mass

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spectra for a bath product sample containing 1,4-dioxane are shown in Figure 3 and supplemental Figure 2, respectively. The survey results including 1,4-dioxane concentration, product category, and the labeled surfactants of interest are listed in Table

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3. 1,4-dioxane was detected in 47 out of the 82 products (57%) with an average concentration of 1.54 µg/g (Range: 0.23-15.3 μg/g). Only two products (2%) exceeded 10

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μg/g of 1,4-dioxane, one at 10.3 μg/g and a second at 15.3 μg/g. Of the products containing detectable 1,4-dioxane, PEG (polyethylene glycol) and laureth surfactants

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were the most listed ingredient (surfactant). Of the 58 products that listed at least one PEG ingredient, 42 products measured detectable concentrations of 1,4-dioxane. Six out of the 36 products that listed laureth surfactant as an ingredient measured detectable concentrations of 1,4-dioxane. The survey results summarized by product category are shown in Table 4. For all samples with detectable concentrations of 1,4-dioxane, analyte retention times and

13 transitions ion ratios met identification criteria. Most rinse-off cosmetic bath products (19 out of the 21 products) and wash products (23 out of the 24 products) tested had detectable 1,4-dioxane concentrations that ranged from 0.31 to 15.3 μg/g. For leave-on cosmetic products tested, only 2 out of the 14 hair treatment products and 2 out of the 20

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lotion products reported detectable concentrations of 1,4-dioxane < 2 μg/g.

4. Conclusion

This is the first method to analyze 1,4-dioxane in cosmetics using a GC-MS/MS method with pulsed split injection without extensive sample prep procedures. The

approach could increase sample throughput and reduce sample matrices contamination to

the inlet and column. The method has successfully been used to determine 1,4-dioxane in

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82 leave-on and rinse-off cosmetics products marketed toward children including bath

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products, hair treatment, lotions, beauty bar, washes and shampoos, and other products.

Acknowledgements

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The authors wish to thank Drs. Christine H. Parker, Gregory O. Noonan, John W. Gasper, Jean C. Hubinger, Nakissa Sadrieh, Linda M. Katz, Perry G. Wang, Travis A. Canida,

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John D. Ihrie, Sarvin Moghaddam for helpful discussions and support.

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cosmetic products, 2015; 1–18. [6] (accessed 28.6.18)

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https://www.fda.gov/Cosmetics/ProductsIngredients/PotentialContaminants/ucm1

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01566.htm Byproduct”

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U.S. Food & Drug Administration, “1,4-Dioxane in Cosmetics: A Manufacturing

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[7] H.J. Chou, P.G. Wang, W. Zhou, and A.J. Krynitsky, “Determination of 1,4dioxane in cosmetic products.” Poster session presented at 124th AOAC Annual Meeting; 2010 Sept. 26-29; Orlando, FL

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[8] D. Song, S. Zhang, Rapid determination of 1,4-dioxane in water by solid-phase extraction and gas chromatography-mass spectrometry, J. Chromatogr. A 787

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(1997) 283-287.

[9] (accessed 28.6.18)

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https://cfpub.epa.gov/si/si_public_record_report.cfm?Lab=NERL&dirEntryId=19 9229 United States Environmental Protection Agency, Method 522 - Determination of 1,4-dioxane in drinking water by solid phase extraction (SPE) and gas chromatography mass spectrometry (GC/MS) with selected ion monitoring (SIM)), 2008.

15 [10] W. Guo, H. Brodowsky. Determination of the trace 1,4-dioxane. Microchem. J, 64 (2) (2000), 173–179. [11] G. Carrera, L. Vegué, M.R. Boleda, F. Ventura, Simultaneous determination of the potential carcinogen 1,4-dioxane and malodorous alkyl-1,3-dioxanes and alkyl-1,3-dioxolanes in environmental waters by solid-phase extraction and gas

SC RI PT

chromatography tandem mass spectrometry. J. Chromatogr. A, 1487 (2017), 1–13. [12] M. Saraji, N. Shirvani, Determination of residual 1,4-dioxane in surfactants and cleaning agents using headspace single-drop microextraction followed by gas

chromatography–flame ionization detection, Int. J. Cosmet. Sci. 39 (2017) 36–41 [13] S. Scalia, M. Guarneri, E. Menegatti, Determination of 1, 4-dioxane in cosmetic

products by high-performance liquid chromatography. Analyst. , 115(1990), 929-

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[14] D. Song, S. Zhang, W. Zhang, K. kohlhof, Rapid quantitative determination of 1,4-dioxane in cosmetics by gas chromatography/mass spectrometry, J. Soc.

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Cosmet Chem.,47, (1996) 177-184.

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[15] D.B. Black, R.C. Lawrence,; E.G. Lovering, J.R. Watson, Gas-liquid chromatographic method for determining 1,4-dioxane in cosmetics, J. Assoc. Off.

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Anal. Chem., 66 (1983), 180-183.

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[16] S. Scalla, F.Testoni, G.Frisina, M. Guarneri, Assay of 1,4-dioxane in cosmetic products by solid-phase extraction and GC-MS. J. Soc.Cosmet. Chem. 43 (1992), 207–213.

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[17] S. Scalia, G. Frisina, Solid-phase extraction procedure for the assay of 1,4dioxane in cosmetic products, Sample Prep. Biomed. Environ. Anal., [Proc.

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Chromatogr. Soc. Int. Symp.] (1994), 219-26.

[18] F. Tanabe, A., Kawata, K., Determination of 1,4-dioxane in household detergents

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and cleaners. J. AOAC Int. 91 (2008), 439–444.

[19] S. Rastogi, Headspace analysis of 1, 4-dioxane in products containing polyethoxylated surfactants by GC-MS. Chromatographia, 29 (1990), 441–445. [20] A. Wala-Jerzykiewicz, J. Szymanowski, Headspace gas chromatography analysis of toxic contaminants in ethoxylated alcohols and alkylamines, Chromatographia 48 (1998) 299-304.

16 [21] M. Tahara, T. Obama, Y. Ikarashi, Development of analytical method for determination of 1,4-dioxane in cleansing products, Int. J. Cosmet. Sci. 35 (2013), 575–580. [22] C.B. Fuh, M, Lai, H. Tsai, C.M. Chang, Impurity analysis of 1,4-dioxane in nonionic surfactants and cosmetics using headspace solid-phase microextraction

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coupled with gas chromatography and gas chromatography-mass spectrometry, J. Chromatogr. A, 1071 (2005), 141-145.

[23] S.S.H. Davarani, New aluminium hydroxide coating on fused silica fiber 1,4-

dioxane in surfactants and detergents using HS-SPME-GC, Chromatographia 75 (2012), 371–377.

[24] M.A. Farajzadeh, P.Nassiry, M.R.A. Mogaddam1, Development of a new

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dynamic headspace liquid phase microextraction method, Chromatographia 79

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[25] B.A. Waldman, Analysis of 1,4-dioxane in ethoxylated compounds by gas

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Chem. 33 (1982), 19–25.

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chromatography/mass spectrometry using selected ion monitoring. J. Soc. Cosmet.

[26] M.P. Italia, M.A. Nunes, Gas chromatographic determination of 1,4-dioxane at

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the parts-per-million level in consumer shampoo products, J. Soc. Cosmet. Chem.,

[27] L. Tong, X. Ma , C. Li, Application of gas chromatography-tandem mass spectrometry (gc-ms-ms) with pulsed splitless injection for the determination of

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multiclass pesticides in vegetables, Anal. Lett. 39 (2006), 985–996, [28] B. Kovacevik, Z. Zdravkovski, S. Mitrev, Pesticide analysis in water samples

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[29] (accessed 5.6.19) http://kermitmurray.com/msterms/index.php/Surrogate_internal_standard, K.K. Murray, R.K. Boyd, M.N. Eberlin, G.J. Langley, L. Li, Y. Naito, Terms from IUPAC recommendations 2013, Pure Appl. Chem., 85 (2013), 1515-1609. [30] M.M. Kiser, J.W. Dolan, Selecting the best curve fit, LC-GC N. America 22 ( 2) (2004,) 112-117.

17 [31] (accessed 31.5.19) https://www.coursehero.com/file/21419052/FDA/ U.S. Food & Drug Administration, “Guidance for industry bioanalytical method validation”. [32] (accessed 6.6.19)

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https://www.fda.gov/cosmetics/cosmetics-laws-regulations/it-cosmetic-drug-orboth-or-it-soap

U.S. Food & Drug Administration, “Is it a cosmetic, a drug, or both? (or is it soap?) [33] (accessed 28.6.18)

ment/GuidanceforIndustry/ucm052658.pdf

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https://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforce

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U.S. Food & Drug Administration, Center for veterinary medicine, Guidance for industry mass spectrometry for confirmation of the identity of animal drug

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residues, 2003.

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[34] (accessed 28.6.18)

https://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM273418.pdf

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U.S. Food & Drug Administration, Center for veterinary medicine, Guidelines for

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the validation of chemical methods for the FDA FVM (Foods and Veterinary

A

CC

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Medicine) program), 2015.

18 List of Figures: Fig. 1. Extracted ion chromatogram for 1,4-dioxane in a standard neat solution (A) and shampoo product extraction solution using pulsed splitless injection mode (B) and pulsed split injection mode (C).

CC

EP

TE

D

M

A

N

U

SC RI PT

The concentration of 1,4-dioxane is approximately 600 ng/mL in acetone.

Fig. 2. Extracted ion chromatogram for 1,4-doxane in a solid matrix (beauty bar) before (A) and after SPE treatment (B).

A

The concentration of 1,4-dioxane in the bar is 0.39 µg/g.`

D

M

A

N

U

SC RI PT

19

Fig. 3. Extracted ion chromatograms for a bath product containing 1,4-dioxane:

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Quantifier of 1,4-dioxane (A, m/z 88 → 57); Qualifier of 1,4-dioxane (B, m/z 88 → 58); Quantifier of IS (C, m/z 96 → 62); and Qualifier of IS (D, m/z 96 → 64).

A

CC

EP

The concentration of 1,4-dioxane in the bath sample is 2.97 µg/g.

D

TE

EP

CC

A

SC RI PT

U

N

A

M

20

N U SC RI PT 21

M

A

Table 1 SRM scan parameters

CC E A

1,4-Dioxane-d8 (IS)

Precursor Product ion, Dwell ion, m/z m/z Time, ms

Collision Energy, V

Use

4.27

850

88

57

150

10

Quantifier

4.27

850

88

58

50

5

Qualifier-1

4.27

850

88

44

50

5

Qualifier-2

4.22

850

96

62

50

10

Quantifier_IS

4.22

850

96

64

50

5

Qualifier-IS-1

4.22

850

96

32

50

15

Qualifier-IS-2

PT

1,4-Dioxane

RT (min) Delta EMV

ED

Analyte

N U SC RI PT 22

Spiked Conc., µg/g

Lotion (OCAC-050)

ED

Spiked Level

M

A

Table 2 Sample recovery (Rec., %) and relative standard deviations (%) of 1,4-dioxane in cosmetic matrices. Samples prepared in triplicate for each matrix.

1.5

CC E

Low

PT

Rec. %

RSD, %

Oil Gel (OCAC-058)

Rec. %

RSD, %

Hair Detangler (OCAC-029)

Rec. %

RSD, %

Beauty Bar (solid soap OCAC-087)

Bubble Bath (OCAC-010)

Rec. %

RSD, %

Rec. %

RSD, %

95.7

2.1

93.0

0.9

91.1

2.4

87.4

2.5

83.6

3.7

7.5

94.6

2.3

95.6

1.9

91.6

1.9

99.3

3.0

83.8

1.5

High

50

108

0.6

107

0.8

105

3.8

108

1.3

87.3

0.3

A

Middle

Note: The concentrations of 1,-4 dioxane in oil gel (OCAC-058), bubble bath (OCAC-010), and beauty bar (solid soap OCAC-087) were 0.74, 0.64, and 0.39* µg/g before spiking. *: 1,4-Dioxane concentration between LOD (0.2 µg/g) and LOQ (0.5 µg/g).

N U SC RI PT M

A

23

CC E

PT

ED

Table 3A 1,4-Dioxane product survey results for cosmetics marketed towards children.

Product Category

1,4-Dioxane (µg/g)

OCAC-133

Lotions

0.23*

PEG-40 hydrogenated Castor oil

OCAC-040

Hair Treatment

0.27*

PEG-40 hydrogenated Castor oil

OCAC-014

Bath Products

0.31*

PEG-150 distearate

OCAC-087

Washes

0.39*

sodium laureth sulfate

OCAC-201

Bath Products

0.42*

PEG-80 sorbitan laurate

OCAC-152

Washes

0.44*

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-084

Washes

0.47*

PEG-150 distearate

A

Product #

Surfactants on Label

N U SC RI PT PEG-80 sorbitan laurate

OCAC-091

Washes

0.50

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-076

Washes

0.51

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-075

Washes

0.55

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-095

Washes

0.61

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-010

Bath Products

0.64

PEG-80 sorbitan laurate

OCAC-101

Bath Products

0.65

PEG-80 sorbitan laurate

OCAC-097

Washes

0.67

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-005

Bath Products

0.71

PEG-150 distearate

OCAC-163

Washes

0.73

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-058

Lotions

0.74

propylene glycol, PEG-40 hydrogenated Castor oil

OCAC-013

Bath Products

0.76

PEG-80 sorbitan laurate

Bath Products

0.79

PEG-80 sorbitan laurate

Bath Products

0.79

PEG-80 sorbitan laurate

Bath Products

0.80

PEG-80 sorbitan laurate

OCAC-079

Washes

0.80

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-125

Bath Products

0.82

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-115 OCAC-104

CC E

OCAC-211

M

Bath Products

ED

OCAC-124

A

0.48*

PT

24

A

*: 1,4-Dioxane concentration between LOD (0.2 µg/g) and LOQ (0.5 µg/g

N U SC RI PT 25

Table 3B 1,4-Dioxane product survey results for cosmetics marketed towards children.

OCAC Product Category

OCAC-082

Washes

OCAC-083

Washes

OCAC-210 OCAC-081

1,4-Dioxane (µg/g) Surfactants on Label

A

Product #

sodium laureth sulfate, PEG-150 distearate

0.94

PEG-80 sorbitan laurate, PEG-150 distearate

Bath Products

1.10

PEG-80 sorbitan laurate, PEG-150 distearate

Washes

1.10

PEG-200 hydrogenated glyceryl palmate

Washes

1.11

PEG-150 distearate

Washes

1.14

PEG-120 methyl glucose dioleate

Bath Products

1.14

PEG-80 sorbitan laurate, PEG-150 distearate

Bath Products

1.17

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-159

Washes

1.17

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-205

Bath Products

1.21

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-022

Bath Products

1.26

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-089

Washes

1.35

PEG-150 distearate

OCAC-073

Other

1.53

sodium laureth sulfate

OCAC-036

Hair Treatment

1.66

PEG-40 hydrogenated Castor oil

OCAC-110

Bath Products

1.69

disodium laureth sulfosuccinate

OCAC-077

Washes

1.88

PEG-80 sorbitan laurate, PEG-150 distearate

OCAC-214

A

ED

CC E

OCAC-017

PT

OCAC-158 OCAC-154

M

0.83

N U SC RI PT 26

Washes

OCAC-092

Washes

OCAC-155

Washes

OCAC-103

Bath Products

OCAC-118

Bath Products

OCAC-090

Washes

2.00

PEG-200 hydrogenated glyceryl palmate, PRG-7 glyceryl cocoate

2.08

PEG-150 distearate, PEG-9

2.72

sodium laureth sulfate

2.79

PEG-80 sorbitan laurate, PEG-150 distearate

2.97

PEG-80 sorbitan laurate

10.3

sodium laureth sulfate

15.3

sodium trideceth sulfate, PEG-80 sorbitan laurate, PEG-150 distearate

A

OCAC-086

M

Washes

PT

ED

OCAC-093

CC E

Table 4 1,4-Dioxane survey results based on product categories

A

Product Category

Bath Products Hair Treatment

2

Description

Number of Product

Products 1 Detected # (%)

Minimum (µg/g)

Maximum (µg/g)

Average (µg/g)

Bubble bath, baby bath, bath dropz (Rinse-off)

21

19 (90)

0.31*

10.3

1.47

Conditioner, detangle, style gel (Leave-on)

14

2 (14)

0.27*

1.66

0.97

N U SC RI PT 27

Baby cream, baby lotion, oil gel (Leave-on)

Lotions

Bath soap, baby wash, shampoo (Rinse-off) Bathtub fingerpaint soap, baby cologne

Washes

A

Other

M

TOTAL 1

20

2 (10)

0.23*

0.74

0.49

24

23 (96)

0.39*

15.3

1.74

3

1 (33)

1.53

1.53

82

47 (57)

0.23*

15.3

1.54

A

CC E

PT

ED

Detected #: The Products Detected # includes the Trace #, whose amount was between LOD (0.2 µg/g) and LOQ (0.5 µg/g). “*: 1,4-Dioxane concentration between LOD (0.2 µg/g) and LOQ (0.5 µg/g).