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. 2025 May 22;17(10):651–660. doi: 10.1080/17576180.2025.2506352

Validated GC-MS methods to detect ethylene, diethylene, and triethylene glycol in serum, plasma, and urine samples

Nicolas Abrigo a,b,*, Susan Daniela Selaya a,c,*, Adil Mohammad a, Diaa Shakleya a, Dustin G Brown a, Jinhui Zhang a, Valerie Pratt d, Jody E Green d, Amanda Degunia e, Michael Bennett e,f,g, Kaitlyn Bloom e, Lynnette Rogers f,g, Aiman Q Khan f,g, Robert O Heuckeroth e,h, Patrick J Faustino a,✉
PMCID: PMC12160607  PMID: 40401362

ABSTRACT

Background

While Polyethylene glycol 3350 (PEG 3350) is approved by the USFDA for short term use by adults, it is commonly recommended for use in constipated children. Multiple reports of adverse events in children taking PEG 3350 raised safety concerns suggesting that low molecular weight species of PEG 3350 might be absorbed from the gut and cause side effects such as ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG).

Research design and methods

This article documents the development, validation, and application of analytical methods using GC-MS and GC-MS/MS for the quantitation of EG, DEG, and TEG in human plasma, serum, and urine.

Results

The analytical range for EG, DEG, and TEG was 2–20 µg/mL. The sample preparation process involves derivatization using N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane in each biological matrices. Deuterated internal standards for each of the analytes were included to provide accurate quantitation of the glycol analytes.

Conclusions

The validated methods were applied to analyze samples a pilot study of children taking PEG 3350. DEG and TEG were detected at levels below the limit of quantitation. In summary, a platform of analytical methods was developed to evaluate glycol analogs in urine, serum, and plasma clinical samples.

KEYWORDS: Polyethylene glycol 3350, GC-MS, GC-MS/MS, ethylene glycol, diethylene glycol, triethylene glycol, clinical samples

Plain Language Summary

This study looks at the safety of Polyethylene Glycol 3350 (PEG 3350), a commonly used medication to treat constipation in adults and children. Though PEG 3350 is approved by the USFDA for short-term use in adults, concerns have arisen regarding its safety in children, particularly because PEG 3350 may break down into smaller substances that could be absorbed into the body and cause harm. These smaller substances, like ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG), were the focus of this research work. The researchers developed and tested new methods to measure these substances in blood and urine using advanced laboratory techniques called GC-MS and GC-MS/MS. They found that these substances could be detected in very small amounts, with a specific range of measurement between 2 µg/mL and 20 µg/mL. They used a special process to prepare the samples and included internal standards to ensure the results were accurate. In a pilot study involving children taking PEG 3350, the levels of DEG and TEG were found to be very low, below the detectable threshold. Overall, this study developed effective ways to test for these potentially harmful substances in clinical samples, which could help better understand the safety of PEG 3350 in children.

1. Introduction

Constipation is a common problem for adults and children worldwide [1–3]. While acute constipation can be treated with dietary changes and laxatives, many children experience long-term constipation [4]. Among the most common treatments for chronic constipation are drug products that contain polyethylene glycol 3350 (PEG 3350)-containing laxatives. PEG 3350 is an active ingredient commonly used in laxatives that draws water into the colon and prevents water absorption to soften stool and increase the frequency of bowel movements. Although PEG 3350 is approved by the United States Food and Drug Administration (FDA) for short term use by adults and children 17 years of age or older, PEG 3350 is recommended off label to young children (e.g., toddlers) for prolonged periods to treat constipation [5–7].

The use of PEG 3350 by children raised potential safety concerns after the FDA received reports of adverse events in children taking PEG 3350-containing laxatives. PEG 3350 is a non-absorbable drug product with high molecular weight. However, one concern was that the PEG 3350 polymer might contain low molecular weight species that could be absorbed. Low molecular weight compounds of interest include ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG). These three glycols are considered safe at low levels but can be toxic at higher concentrations, including documented mass poisonings worldwide [8–11]. Since the manufacturing process for ethoxylated products, such as PEG 3350, may lead to residual amounts of EG, DEG, and TEG in the final product [12], the United States Pharmacopeia National Formulary (USP-NF) includes a limit test for EG and DEG of not more than (NMT) 0.10% for impacted monographs [12,13]. Thus, additional research was necessary to determine the levels of these low molecular weight glycols from PEG 3350 based laxatives in urine and serum.

One key problem is that EG, DEG, and TEG are difficult to accurately measure when high sensitivity and specificity are required in complex matrices. In particular, there were no established quantitative methods for these molecules that could be reliably applied to human plasma, serum, and urine. Additionally, there were no reliable methods that could analyze low volume samples obtained from children taking PEG 3350 [14]. Analytical methods are available to detect EG, DEG, and TEG in human biomatrices and many of the methods include a derivatization step for the glycols to improve detection in mass spectrometry procedures [15–34]. However, prior methods were not validated according to the FDA Bioanalytical Method Validation Guidance for Industry, or these methods were not adequately sensitive, specific, or reliable [35].

This article details the development of analytical methods using GC-MS and GC-MS/MS to quantitate levels of EG, DEG, and TEG in human plasma, serum, and urine. The methods were validated according to the FDA Bioanalytical Method Validation Guidance for Industry [35] and then applied to evaluate a small cohort of pediatric biospecimens (serum and urine) collected by investigators at the Children’s Hospital of Philadelphia (CHOP). These validated methods will be essential for use in a future larger study designed to evaluate plasma and urine of children prescribed PEG 3350 to treat constipation.

2. Materials and methods

2.1. Chemicals and reagents

The EG (R093Q0), DEG (R05070), and TEG (F0M074) reference standards were purchased from the United States Pharmacopeia (USP). Ethylene-d4 glycol, acetonitrile anhydrous, dimethylformamide anhydrous, ethylene glycol bis(trimethylsilyl ether), and N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA-TMCS) were purchased from Sigma Aldrich (St. Louis, MO). Diethylene glycol bis(trimethylsilyl ether) was purchased from Tokyo Chemical Industry (Portland, OR). Diethylene-d8 glycol was purchased from Cambridge Isotopes Laboratories (Tewksbury, MA). Triethylene-d4 glycol was purchased from Medical Isotopes, Inc. (Pelham, NH). Acetonitrile and high purity water were purchased from EMD Millipore (Burlington MA). Table S1 lists the standards and solvent information including the manufacturer, catalog #, lot #, and purity, where applicable. Triethylene glycol bis(trimethylsilyl ether) was synthesized in-house and the procedure is included in the supplemental information document. The characterization of triethylene glycol bis(trimethylsilyl ether) is presented in Figures S1-S5.

2.2. Human plasma, serum, and urine

Human plasma, serum, and urine (Na2EDTA) from at least 6 donors of mixed gender were purchased from BioIVT (Westbury, NY) to serve as control human biomatrices during method development and validation. Human biospecimens were provided in aliquots of 1 mL. Plasma and urine were passed through a 0.2 µm filter by the vendor, but serum was not filtered. Each analyte type was pooled prior to use in our studies. Samples were stored at −80 °C prior to analysis.

A cohort of human serum and urine samples were collected by Children’s Hospital of Philadelphia (CHOP) investigators under a study approved by CHOP’s Institutional review board (IRB 14–011454). After informed consent was signed, blood and urine were obtained at the CHOP Center for Human Phenomic Science (CHPS) from volunteers who had been taking PEG 3350 daily for 1 to 8 years as part of their personal medical therapy. For this study, a blood sample was drawn before PEG 3350 ingestion in the morning followed by 6 additional samples at 0.25, 0.75, 1.5, 3.5, 5.5, and 7.5 hours after the morning PEG 3350 dose. Urine samples were collected at 2 and 8 h after the same morning PEG 3350 dose. Blood was collected in 10 mL glass red cap tubes (VWR, catalog # 366430) and sat at room temperature for 30 min to clot before samples were centrifuged (3000 × g, 10 min, room temperature) to pellet the clot. Supernatant serum was aliquoted into 1.8 mL NUNC CRYO Tubes (Thermo Fisher, Waltham, MA catalog # 374500). Urine samples were collected in 120 mL sterile specimen cups (VWR, Radnor, PA Catalog # 89508–714) and 5 mL aliquots were prepared in Cryobank 5 mL 2D Rack CS240 tubes (VWR, Catalog # 366430). Blood and serum were stored at −80 °C until time of analysis except when samples were shipped overnight on dry ice between institutions. These biospecimens were then analyzed by FDA investigators using the newly developed methods.

2.3. Instrumentation and GC conditions

Two analytical methods were developed for the detection of EG, DEG, and TEG in human biomatrices. Method A used an Agilent GC 7890A coupled with a Gerstel MPS Autosampler and an Agilent MSD 5977B. Method B used an Agilent 8890 GC coupled with an Agilent ALS 7693A and an Agilent MSD 7000D. Both methods used a J&W DB-Select 624 UI 30 m x 0.32 mm x 1.8 µm GC column (Agilent 123-0334UI) for separation of analytes and an ultra-inert split liner (Agilent 5190–2295). Amber vials with a fixed insert volume of 300 µL were purchased from Agilent (Santa Clara, CA, 5188–6594). Amber glass vials and amber glass bottles were used for preparation and storage of standards and samples. Reacti-Therm heating and stirring modules were purchased from Thermo Fisher. Stir bars (3×10 mm) made of polytetrafluoroethylene were used.

Method A used a 3 µL injection volume. The inlet was set to a temperature of 220 °C with a split ratio of 10:1. The GC oven temperature started at 60 °C for 1 min, then increased at 15 °C/min to 200 °C, then increased at 50 °C/min to 240 °C, and held at 240 °C for 1.3 min. The GC run time was 12.43 min with a post run time of 6.6 min. The column flow was 2 mL/min of Helium. The MS Source temperature was 230 °C and the MS Quad temperature was 150 °C. Mass detection by selected ion monitoring (SIM) was used with retention time (RT) to identify product peaks. The retention times, quantifier ions, and qualifier ions for the compounds of interest are listed in Table 1.

Table 1.

Compound information for method A and Method B.

  Method A: GC-MS
 Method B: GC-MS/MS
Compound RT (min) Quantifier
ion (m/z)		 Qualifier
ion (m/z)		 RT (min) Precursor
ion (m/z)		 Product
ion (m/z)
Ethylene glycol 6.8 191.1 147.1 8.6 191 147
Ethylene-d4 glycol 6.8 195.1 147.1 8.6 195 147
Diethylene glycol 9.8 117.1 103.1 10.8 191 146.8
Diethylene-d8 glycol 9.8 121.1 195.1 10.8 195 147
Triethylene glycol 11.8 117.1 161.1 12.7 161 72.9
Triethylene-d4 glycol N/A N/A N/A 12.7 163 73
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Method B used a 1 µL injection volume. The inlet was set to a temperature of 250 °C with a split ratio of 20:1. The GC oven temperature started at 40 °C for 2 min, then increased at 20 °C/min to 160 °C, then increased at 20.5 °C/min to 250 °C, and held at 250 °C for 3 min. The GC run time was 15.3 min. The column flow was 1.2 mL/min of Helium. The MS Source temperature was 230 °C and the MS Quad temperature was 150 °C. Mass detection by multiple reaction monitoring (MRM) was used with retention time to identify the product peaks. The retention times, quantifier ions, and qualifier ions for the compounds of interest are listed in Table 1.

2.4. Preparation of mixtures

The compounds listed in Table 2 were prepared as individual 1 mg/mL solutions in water (EG, DEG, TEG, ethylene-d4 glycol, diethylene-d8 glycol, triethylene-d4 glycol). These 1 mg/mL solutions were used to make mixtures A1, A2, B1, B2, and C. Mixture A1 (8 mL) and mixture B1 (8 mL) were prepared using water and had a final concentration of 100 µg/mL of EG, DEG, and TEG. Mixture A2 (8 mL) and mixture B2 (8 mL) were prepared using water and had a final concentration of 10 µg/mL of EG, DEG, and TEG. Mixture C (8 mL) was prepared using deionized water (DI) water and had a final concentration of 100 µg/mL of ethylene-d4 glycol, diethylene-d8 glycol, and triethylene-d4 glycol.

Table 2.

System suitability data for method A in serum.

    Ethylene glycol
 Diethylene glycol
 Triethylene glycol
SST
Parameter		 Specification Day
1		 Day
2		 Day
3		 Day
1		 Day
2		 Day
3		 Day
1		 Day
2		 Day
3
RT (min) N/A 6.83 6.83 6.83 9.76 9.76 9.76 11.83 11.83 11.83
RSD <2.0% 0.00 0.02 0.03 0.00 0.02 0.02 0.01 0.01 0.01
Area RSD <5.0% 0.38 3.39 1.77 0.33 3.56 1.91 1.28 3.18 2.43
Theoretical Plates >2000 68735 80536 88853 101511 104160 107720 306727 337349 302414
USP
Tailing
Factor		 <2.0 0.97 0.98 1.02 0.97 0.96 0.95 1.02 1.01 1.01
RSD <10.0% 3.15 4.84 9.58 4.43 7.88 7.90 4.45 5.95 3.56
Resolution >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0
Capacity
Factor		 k’ > 3 6.18 6.18 6.17 9.25 9.25 9.25 11.42 11.42 11.42
RSD <2.0% 0.00 0.03 0.03 0.00 0.02 0.02 0.01 0.01 0.01
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N/A = Not applicable. RT = Retention time. SST = System Suitability Test. AUC = Area Under the Curve. RSD = Relative Standard Deviation. k’ = capacity factor.

2.5. Preparation of system suitability standards

The system suitability test solution was prepared using purified versions of the ethylene glycol bis(trimethylsilyl ether), diethylene glycol bis(trimethylsilyl ether), and triethylene glycol bis(trimethylsilyl ether) standards. The compounds were prepared as individual 1 mg/mL solutions in anhydrous acetonitrile. Then 26.6 μL of the 1 mg/mL solution for ethylene glycol bis(trimethylsilyl ether), 18.9 μL of the 1 mg/mL solution for diethylene glycol bis(trimethylsilyl ether), and 15.7 μL of the 1 mg/mL solution for triethylene glycol bis(trimethylsilyl ether) were combined with 1.939 mL of anhydrous acetonitrile. The solution containing all three bis(trimethylsilyl ether) glycols was diluted 10-fold for the final system suitability test standard solution.

2.6. Preparation of calibration standards

The calibration curve for EG, DEG, and TEG covered the range of 2–20 µg/mL. Ten calibration curve levels were prepared with a final volume of 1 mL at each level. Mixture A1 was used to prepare the levels at 20, 17.5, 15, 12.5, and 10 µg/mL. Mixture A2 was used to prepare the levels at 7.5, 5, 4, 3, and 2 µg/mL. The internal standards (ISTDs) ethylene-d4 glycol, diethylene-d8 glycol and triethylene-d4 glycol were included to achieve a final ISTD concentration of 7.5 µg/mL for all calibration curve levels (Table S2).

2.7. Preparation of quality control standards

Quality control (QC) standards were prepared at four concentrations over the analytical range in water with a final volume of 4 mL. The high QC (HQC) was prepared at the concentration of 15 µg/mL by diluting mixture B1. The mid QC (MQC) was prepared at the concentration of 7.5 µg/mL by diluting mixture B1. The low QC (LQC) was prepared at the concentration of 4 µg/mL by diluting mixture B2. The lower limit of quantitation QC (LLQC) was prepared at the concentration of 2 µg/mL by diluting mixture B2. The internal standards (ISTD), ethylene-d4 glycol, diethylene-d8 glycol, and triethylene-d4 glycol were included to achieve a concentration of 7.5 µg/mL for all quality control levels (Table S3).

2.8. Preparation of internal standard control

The ethylene-d4 glycol, diethylene-d8 glycol, and triethylene-d4 glycol internal standards were added to water to achieve a concentration of 7.5 µg/mL (Table S4). Note that during validation of method A, the triethylene-d4 glycol was not available so the internal standard diethylene-d8 glycol was used for the TEG analyte.

2.9. Plasma or serum sample preparation

Plasma or serum (50 µL) and standard (50 µL of calibration or QC) were combined in a 1.5 mL microcentrifuge tube and mixed by vortex for 20 s. Acetonitrile (600 µL) was added to the solution and mixed again by vortex for 30 s. The solution was centrifuged at 18,750 relative centrifugal force (CFG) for 20 min at 4 °C. The supernatant was transferred into 4 mL glass vials containing a mini-stir bar. The samples were dried at 35 °C under a stream of nitrogen for 30 min while stirring. If the mini stir bar could not easily be dislodged from the bottom of the vials by tapping on the benchtop lightly, then 5 min of drying time was added. The vials were removed from the heating module and capped until the derivatization step.

2.10. Urine sample preparation

Urine (50 µL) and standard (50 µL of calibration or QC) were combined in a 4 mL glass vial with a mini-stir bar and vortexed for 10 s to mix. Acetonitrile (600 µL) was added to the solution. The solution was vortexed for 30 s. The samples were dried at 35 °C under a stream of nitrogen for 30 minutes while stirring. If the mini stir bar could not easily be dislodged from the bottom of the vials by tapping on the benchtop lightly, then 5 min of drying time was added. The vials were removed from the heating module and capped until the derivatization step.

2.11. Derivatization of mixture using BSTFA-TMCS

Anhydrous acetonitrile (100 µL) and BSTFA-TMCS (100 µL) were added to each vial. Vials (4 mL amber, CG-4908-A-01) were capped tightly and mixed by vortex for 20 s. The derivatization reaction occurred at 80 °C for 30 min while stirring. The vials were removed from heat and placed in vial racks that were pre-chilled in a −80 °C freezer. The vials were placed in the −20 °C freezer for 3.5 min. The vials were then centrifuged at 400 × g for 2 min at 4 °C to pool the sample. Anhydrous N,N-dimethylformamide (60 µL) was added to each vial. The vials were capped and mixed by vortex for 10 s. The vials were then centrifuged at 400 × g for 2 min at 4 °C to pool the sample. The vial caps were removed, and the solution was stirred for 3.5 min at 25 °C under a stream of nitrogen. The vials were capped and centrifuged at 400 × g for 2 min at 4 °C to pool the sample. The entire sample (approximately 100 µL) was transferred to the autosampler vials (300 µL insert volume, 5188–6594) for direct injection into the GC.

2.12. Method validation

Method validation followed the requirements of the FDA Bioanalytical Method Validation Guidance for Industry [35]. Method validation characteristics included selectivity, specificity, limit of quantitation, linearity, range, accuracy, precision, stability, and recovery. Method A (GC-MS) validation was completed in the human serum biomatrix with three separate validation days. Method B (GC-MS/MS) validation was completed in the human urine biomatrix with three separate validation days. A partial validation in human plasma and human urine using Method B was also completed according to the FDA bioanalytical guidance with a one validation day for each biomatrix.

2.13. Method application

Method A (GC-MS) was applied to test human serum samples obtained at seven timepoints after ingestions of PEG 3350, as provided by CHOP investigators. Method B (GC-MS/MS) was applied to test human urine samples also provided by CHOP investigators. A system suitability test, calibration curve, and quality control standards were evaluated for each day of testing. The spike recovery of QC standards in the patient samples was evaluated.

3. Results and discussion

3.1. Method optimization

A derivatization agent was selected to increase the molecular weight and increase the volatility of targeted analytes. Silylation using BSTFA results in trimethylsilyl substitution of alcohols and is commonly used in gas chromatography [36,37]. While BSTFA is an efficient derivatization agent, water will decompose the trimethylsilyl reagent and derivatives [38]. Therefore, dry needles, syringes, and other lab supplies (i.e., vials and pipette tips) were used for the reactions. The aqueous human biomatrices were dried completely before the derivatization reaction. BSTFA with 1% TMCS showed improved derivatization efficiency over BSTFA alone. Additionally, a 50:50 ratio between BSTFA-TMCS and acetonitrile improved the post-reaction stability of the glycols. The time and temperature for the derivatization reaction was optimized to 30 min at 80 °C. Decreasing the reaction time and lower reaction temperatures led to incomplete derivatization of the glycols. Increasing the reaction time and higher reaction temperatures led to degraded samples. The volume was optimized to be 200 µL total solvent. Volumes at 100 µL and lower led to poor reproducibility between samples. Constant stirring during derivatization and drying steps proved to be critical. A lack of stirring led to poor reproducibility between samples. The microcentrifuge tubes were evaluated for the presence of EG, DEG, and TEG. No detection of analyte levels above the matrix baseline was detected for the tested tubes.

Headspace extraction for gas chromatography can provide greater efficiency and cleaner injections of the volatile components. Headspace injections worked well for the silylated glycol reference standards, but in the presence of BSTFA a significant decrease in instrument response was observed. BSTFA is highly volatile (boiling point of 45–55 °C) which may have led to the BSTFA outcompeting the glycols for the headspace of the vial. Direct injection into the gas chromatograph instrument showed no decrease in signal in the presence of BSTFA, but the stability of the glycols in the presence of BSTFA still remained a concern.

A post-reaction drying step to remove the BSTFA reagent was attempted. Drying the samples fully so that no solvent remained led to a significant loss of the derivatized glycols and poor repeatability precision. Short drying times led to a high amount of BSTFA remaining in the sample and poor autosampler stability. N,N-dimethylformamide (DMF) was added to the vials due to low volatility of DMF with expectation that the BSTFA would selectively evaporate, while the glycol analytes would remain. The drying step was optimized to 3.5 min under a stream of nitrogen. The derivatization reaction was also attempted using N,N-dimethylformamide instead of acetonitrile, but this produced a side reaction artifact. The artifact was caused by the reaction of N,N-dimethylformamide with BSTFA at elevated temperatures and interfered with the retention of EG [39]. For this reason, we included a step post-reaction for cooling the samples before adding the N,N-dimethylformamide.

The derivatization and drying steps introduced variability between samples, but the inclusion of deuterated internal standards resolved this problem. The best results for accuracy and precision were achieved when the relative response for each glycol was calculated using the respective deuterated version of the glycol (e.g., EG with EG-d4, DEG with DEG-d8, and TEG with TEG-d4). Lastly, the system suitability standards were the silylated derivatives for EG, DEG, and TEG. The silylated derivative for TEG was not available commercially and was synthesized in-house. The procedure provided 97.9% purity of the silylated TEG without the use of column chromatography for purification. The procedure for producing silylated TEG can be found in the supplemental information document.

3.2. System suitability

A system suitability test to evaluate instrument performance was conducted for each day of analysis. The system suitability test followed USP < 621> to evaluate the instrument parameters for retention time (RT), peak area (detector response), theoretical plates, tailing factor, resolution, and capacity factor based on six replicate injections [40]. The system suitability results for validation of Method A in serum are presented in Table 2 and Method B in urine are presented in Table S5.

3.3. Selectivity

The human biomatrices were evaluated for interferences at the retention time of the analyte and internal standards. Interference with EG was observed to be no more than 35% in the serum blank and no more than 25% in the urine blank (Table S6). Interference with DEG and TEG was no more than 10% in the serum blank and no more than 20% in the urine blank.

3.4. Specificity

The separation of EG, DEG, and TEG analytes by retention using gas chromatography is displayed in Figure 1. The figure depicts the identification of EG, DEG, and TEG in urine (blue), serum (green), and plasma (red). The three glycols were identified by mass spectrometry through their unique quantifier and qualifier ions.

Figure 1.

Figure 1.

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Specificity of EG, DEG, and TEG in urine, serum, and plasma (retention times of EG, DEG, and TEG are reflective of method B; refer to RT provided in table 1 and SST data provided in table S5).

3.5. Limit of quantitation

The peaks for EG, DEG, and TEG displayed a signal-to-noise ration >10 at the LOQ in serum, urine, and plasma (Table S7).

3.6. Linearity and range

The linearity for the three glycols was established using a 10-concentration level calibration curve over the range of 2–20 µg/mL. The three glycols displayed linearity with r2 ≥ 0.99 for each day of validation (Table 3).

Table 3.

Linearity and range data for method A (serum) and method B (urine).

    Serum
 Urine
Analyte (Range)   Equation r2 Equation r2
Ethylene Glycol
(2–20 µg/mL)		 Day 1 y = 0.929230*x + 0.115932 0.9997 y = 0.112116*x + 0.087507 0.9988
Day 2 y = 1.049876*x + 0.026000 0.9999 y = 0.115988*x + 0.135071 0.9977
Day 3 y = 1.014570*x + 0.134806 0.9996 y = 0.125492*x + 0.069141 0.9953
Diethylene Glycol
(2–20 µg/mL)		 Day 1 y = 1.024441*x + 0.019229 0.9998 y = 0.103324*x + 0.045221 0.9993
Day 2 y = 1.049876*x + 0.026000 0.9999 y = 0.106003*x + 0.041149 0.9991
Day 3 y = 1.042413*x + 0.027105 0.9999 y = 0.101723*x + 0.026351 0.9989
Triethylene Glycol
(2–20 µg/mL)		 Day 1 y = 1.133419*x − 0.009132 0.9956 y = 0.097809*x + 0.054428 0.9998
Day 2 y = 1.075734*x + 0.000140 0.9965 y = 0.107903*x + 0.052464 0.9998
Day 3 y = 1.137555*x − 0.016646 0.9916 y = 0.158699*x + 0.068845 0.9986
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3.7. Intraday accuracy and precision

The intraday accuracy and intraday precision (repeatability) of the method were determined using QC standards at four concentrations. The QC standard concentrations were prepared using n = 6 samples for each day of validation. The intraday accuracy in serum ranged from 92.4–101.0% for EG, 97.0–100.3% for DEG, and 94.4–109.3% for TEG. The intraday precision in serum ranged from 0.6–5.7% for EG, 0.3–1.1% for DEG, and 3.2–8.1% for TEG. The intraday accuracy in urine ranged from 89.9–106.8% for EG, 90.9–100.7% for DEG, and 90.4–101.2% for TEG (Table 4).

Table 4.

Intraday accuracy and intraday precision for method A (serum) and method B (urine).

Biomatrix and QC level   Ethylene glycol
 Diethylene glycol
 Triethylene glycol
  Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Serum
LLQC (2 µg/mL)		 Accuracy 100.9 92.4 95.6 99.1 97.0 97.4 101.1 102.7 102.0
%RSD 3.2 4.6 5.7 0.7 1.0 1.1 4.7 5.8 3.7
Serum
LQC (4 µg/mL)		 Accuracy 101.0 97.9 98.4 100.3 97.2 98.0 101.5 102.4 94.4
%RSD 1.5 1.9 1.5 0.4 0.5 0.3 4.6 7.2 4.8
Serum
MQC (7.5 µg/mL)		 Accuracy 100.6 98.6 100.0 100.1 97.8 98.8 100.7 101.0 99.6
%RSD 1.3 0.6 1.3 0.4 0.4 0.4 8.0 6.5 6.5
Serum
HQC (15 µg/mL)		 Accuracy 100.8 100.6 100.5 100.2 99.0 99.5 102.4 109.3 99.1
%RSD 0.7 0.6 0.7 0.3 0.3 0.4 6.5 3.2 8.1
Urine
LLQC (2 µg/mL)		 Accuracy 106.8 89.9 102.6 90.9 92.7 93.1 90.4 94.5 95.9
%RSD 8.8 8.5 11.8 2.5 6.6 8.0 3.2 5.3 4.8
Urine
LQC (4 µg/mL)		 Accuracy 102.0 92.3 98.0 98.6 98.8 96.2 99.3 98.7 98.3
%RSD 6.0 2.7 4.1 5.4 3.0 10.3 1.9 0.9 2.5
Urine
MQC (7.5 µg/mL)		 Accuracy 103.0 99.6 96.7 100.7 98.9 96.4 101.2 98.9 99.5
%RSD 3.7 4.5 2.0 3.4 2.4 2.1 0.9 0.9 1.1
Urine
HQC (15 µg/mL)		 Accuracy 101.0 99.3 98.0 98.8 97.3 97.0 100.3 98.4 99.0
%RSD 1.2 1.0 0.7 0.8 1.6 1.9 0.8 0.8 1.0
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LLQC = Lower Limit Quality Control. LQC = Low Quality Control. MQC = Middle Quality Control. HQC = high Quality Control. RSD = Relative Standard Deviation.

3.8. Intermediate precision

The intermediate precision of the method was determined using QC standards at four concentrations. The values represent the %relative standard deviation (RSD) of n = 18 samples completed over three days of validation. The intermediate precision in serum ranged from 0.6–5.7% for EG, 0.6–1.4% for DEG, and 4.6–7.1% for TEG (Table S8). The intermediate precision in urine ranged from 1.6–11.9% for EG, 1.6–6.6% for DEG, and 1.2–5.0 for TEG.

3.9. Stability

The autosampler stability of the derivatized samples at room temperature was evaluated in serum for 24 hours, urine for 72 hours, and plasma for 72 hours (Table S9). The four quality controls standards were prepared as n = 3 and the relative response of EG, DEG, TEG was compared to the initial timepoint. Stability in serum ranged from 92.4–100.6% for EG, 97.0–99.0% for DEG, and 101.0–109.4% for TEG. Stability in urine ranged from 97.3–107.2% for EG, 100.5–104.1% for DEG, and 98.8–99.6% for TEG. Stability in plasma ranged from 100.4–104.5% for EG, 98.8–101.0% for DEG, and 97.3–98.2% for TEG.

The freeze-thaw stability (−80 °C) was evaluated in serum, urine, and plasma (Table S10). The stability of EG, DEG, and TEG was evaluated using two concentrations for each biomatrix. Three freeze-thaw cycles were completed for each biomatrix. The accuracy of EG, DEG, and TEG ranged from 93–115% in serum, 92–104% in urine, and 87–98% in plasma.

3.10. Extraction efficiency/recovery

A precipitation step with acetonitrile was included in the procedure for serum and plasma samples. The extraction recovery was evaluated by preparing samples where the QC standards were added before or after the precipitation step. The accuracy and precision for the LQC, MQC, and HQC standards are presented in Table S11. There was less than 3% difference between the accuracy of QC standards added before or after the precipitation step. The precision was less than 2.5% for all samples.

3.11. Robustness

The robustness of the methods was evaluated through small adjustments to the method parameters and derivatization reaction. Adjustments for Method A (serum) were made to the injection volume, reaction time, and derivatization reagent (Table S12). Adjustments for Method B (urine) were made to the reaction temperature, reaction time, GC split ratio, and inlet temperature (Table S13).

3.12. Plasma and serum partial validation for method B

The GC-MS/MS method was applied to plasma and serum through a partial validation as part of a method transfer. The partial validation consisted of a 1-day validation for each biomatrix instead of a 3-day validation. The results for LOQ, linearity, accuracy, and precision are included in the Tables S7, S14, and S15.

3.13. Pilot study

Blood and urine samples collected by CHOP investigators from five volunteers (Table S16) were evaluated using the validated analytical methods. Volunteers were 13–17 years old (mean 14.6 years) and their body mass index (BMI) ranged from 16.9 to 36.5 (mean 21.98). All volunteers were normotensive, had regular pulse and respirations, and were afebrile. One volunteer had a history of bowel obstruction and acute pancreatitis. Another volunteer had a history of gastroesophageal reflux and delayed gastric emptying (Table S16). All volunteers had been taking PEG 3350 to treat constipation for 1–8 years prior to this study. Biospecimens included 7 serum samples (one prior to PEG 3350 ingestion) and 2 urine samples from each subject obtained at times indicated in Table 5. A system suitability test, calibration curve, and quality control standards were included for each day of analysis. EG, DEG, and TEG were below the quantifiable analytical range of 2–20 µg/mL for all of the serum and urine samples tested (6). However, 5 out of 450 data points were provided number values as Grubb’s Test (ESD Method) outliers with a two-sided significant level of p < 0.05. Spike recovery in these pilot study samples was conducted for two timepoints in serum and two timepoints in urine. The known concentrations for the LQC, MQC, and HQC were spiked into the pilot study samples and then compared to the pilot study samples unspiked to determine the percentage of recovery. The spike recovery results are included in Tables S17 and S18.

Table 5.

Pilot study results in serum and urine (data presented as µg/mL).

  Ethylene glycol
 Diethylene glycol
 Triethylene glycol
Patient A B C D E A B C D E A B C D E
Serum
0 h		 BQ BQ BQ BQ BQ BQ BQ BQ 0.11** (BQ) BQ BQ BQ BQ 0.70** (BQ) BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
0.25 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
0.75 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
1.5 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ 0.04** (BQ) BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
3.5 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
5.5 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ 0.44** (BQ)
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
Serum
7.5 h		 BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ 0.74** (BQ) BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ
BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ BQ B
Urine
2 h		 ND BQ ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
Urine
8 h		 ND ND ND ND ND ND ND ND ND ND ND ND BQ ND ND
ND ND ND BQ ND ND ND ND ND ND ND ND ND ND ND
ND ND ND BQ ND ND ND ND ND ND ND ND BQ ND ND
ND ND ND BQ ND ND ND ND ND ND ND ND ND ND ND
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BQ = Detected but the concentration below the quantitation limit at LLQC. ND = Not detected. For BQ data points without numerical data the analytical response was near the limit of detection and specificity could not be established.

**= Number values provided for Grubb’s Test or the extreme studentized deviant (ESD) method outliers with a two-sided significant level of p < 0.05.

4. Conclusions

Collaboratively, many groups have spent years testing alternative strategies, using state-of-the-art mass spectrometry instrumentation, and optimizing every procedure to establish sensitive and selective analysis methods to detect EG, DEG, and TEG in human plasma, serum, and urine. These low molecular weight glycols are notoriously difficult to quantify in human biomatrices and require derivatization for analysis by mass spectrometry. Our methods can provide very reproducible and highly accurate quantitative data for EG, DEG, and TEG concentrations in the 2–20 µg/mL range. The purpose of this work was to determine if these low molecular weight glycols were present and measurable in small sample volumes in blood or urine of children taking PEG 3350. The new GC-MS and GC-MS/MS methods were validated according to the FDA Bioanalytical Method Validation Guidance for Industry [35]. After rigorous analytical method development, we conducted a pilot study with biospecimens from teenagers to determine whether there were detectable and measurable amounts of EG, DEG, TEG in serum or urine after participants took their usual morning PEG 3350 dose (17 grams). Each study participant had been taking this medicine daily for the prior 1–8 years to treat constipation. None of the serum samples tested had EG, DEG, or TEG at levels above the lower limit of quantitation (2 µg/mL). However, DEG and TEG were identified in some serum samples from 3 of the 5 participants at levels below the quantitative limit (BQL). Similarly, none of the urine samples contained EG, DEG, or TEG at levels above the lower limit of quantitation (2 µg/mL). In some urine samples these analytes may have been detected near the limit of detection, but specificity could not be established. The clinical significance (if any) of these low but detectable levels of glycols in serum and/or urine is not yet known. In summary, this pilot study determined that EG, DEG, and TEG were present in pediatric serum and urine samples. The new GC-MS/MS analytical methods will soon be used to test for and measure EG, DEG, and TEG in plasma and urine from a second larger group of children (infants, toddlers, young children and teenagers) enrolled in a current study at Children’s Hospital of Philadelphia (NCT05424757). Details regarding the clinical trial designs can be found at ClinicalTrials.gov.

Supplementary Material

Supplemental Material
IBIO_A_2506352_SM4986.docx (432.7KB, docx)

Acknowledgments

The authors would like to thank FDA project managers Dr. Helen Lee and Ms. Janice Adams-King for their outstanding assistance throughout the study. The authors would also like to thank Dr. Xiaoming Xu and Dr. David Keire for their support of this study.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This study was funded by the U.S. Food and Drug Administration to support the PEG–3350 pilot and clinical studies at Children’s Hospital of Philadelphia (CHOP), as detailed in IRB-14-011454.

Article highlights

  • Derivatization with BSTFA and TMCS: The derivatization agent BSTFA (with 1% TMCS) was chosen to increase the molecular weight and volatility of the glycols (EG, DEG, TEG), enhancing gas chromatography analysis efficiency.

  • Reaction Conditions: The optimal reaction conditions for derivatization were 30 min at 80°C, as shorter times or lower temperatures led to incomplete derivatization, and longer times or higher temperatures caused sample degradation.

  • Sample Preparation and Solvent Optimization: The reaction required dry human biomatrices and precise solvent volume (200 µL), as smaller volumes resulted in poor reproducibility, and constant stirring was crucial for consistency between samples.

  • Headspace vs Direct Injection: Headspace extraction for gas chromatography was less effective in the presence of BSTFA, which led to a decreased response. Direct injection showed better signal stability, though concerns about glycol stability with BSTFA persisted.

  • Drying and Solvent Removal: Drying the samples after derivatization to remove BSTFA was critical; however, over-drying led to significant glycol loss, and insufficient drying resulted in poor sample stability. Nitrogen drying for 3.5 min was optimized to balance this.

  • Internal Standards for Accuracy: The inclusion of deuterated internal standards (e.g., EG-d4, DEG-d8, TEG-d4) ensured accuracy and precision in the analysis, resolving variability introduced by the derivatization and drying steps.

Author contributions

Susan Daniela Selaya: Investigation, Methodology, Validation, Data curation, Formal analysis, Writing – original draft. Nicolas Abrigo: Investigation, Methodology, Validation, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Adil Mohammad: Investigation, Methodology (synthesis and analysis), Data curation, Formal analysis. Diaa Shakleya: Conceptualization, Project administration, Methodology, Formal analysis, Writing – review & editing. Dustin Brown: Investigation, Methodology, Validation, Writing – review & editing. Jinhui Zhang: Investigation, Methodology. Valerie Pratt: Supervision, Project administration. Jody E. Green: Supervision, Investigation, Clinical methodology, Formal analysis, Writing – original draft, Writing – review & editing. Amanda Degunia: Methodology. Michael Bennett: Methodology. Kaitlyn Bloom: Methodology, Lynnette Rogers: Methodology. Aiman Q. Khan: Methodology. Robert O. Heuckeroth: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing – review & editing. Patrick J. Faustino: Conceptualization, Supervision, Project administration, Resources, Writing – review & editing.

Disclosure statement

This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includesemployment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical declaration

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17576180.2025.2506352

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