Ethylene Glycol Residue Determination by Gas Chromatography: Methods, Standards, and Applications

Ethylene glycol (EG) residue determination by gas chromatography (GC) provides sensitive and accurate quantification of this toxic residual in medical devices, pharmaceuticals, food contact materials, and consumer products. This technical article explains the analytical principle, sample preparation procedures, GC-FID and GC-MS method parameters, and the regulatory limits defined by ISO 10993-7, ICH Q3C, and major pharmacopoeias. It serves as a practical reference for manufacturers, quality engineers, and regulatory professionals who require reliable EG residual testing and compliance assessment.

Technical Overview

What Is Ethylene Glycol and Why Is It Controlled?

Ethylene glycol (1,2-ethanediol, CAS No. 107-21-1) is a colorless, odorless, hygroscopic, and viscous liquid that is fully miscible with water and many polar organic solvents. It is widely used as a raw material for polyester (PET) production, as a coolant and antifreeze agent, and as a solvent or intermediate in chemical and pharmaceutical synthesis. In the medical device industry, ethylene glycol is generated as a hydrolysis by-product when products are sterilized with ethylene oxide (EtO), and it can remain in or on the device after sterilization and aeration.

Because ethylene glycol is metabolized in the human body to glycolic and oxalic acids, excessive exposure can cause central nervous system depression, metabolic acidosis, cardiopulmonary dysfunction, and acute renal failure driven by calcium oxalate crystallization. Residual EG is therefore strictly controlled by international standards and pharmacopoeias, and accurate quantification is an essential element of product safety evaluation and market release.

Principle of Gas Chromatographic Determination

Gas chromatography separates ethylene glycol from co-extracted matrix components on a capillary column, while a flame ionization detector (FID) or mass spectrometer (MS) provides sensitive and selective detection. Samples are extracted with water or aqueous methanol, the extract is injected directly or after derivatization, and the EG content is quantified by external-standard or internal-standard calibration. Results are typically reported in mg/kg (ppm), µg/g, or mg per device, and are compared against the applicable regulatory limit to determine conformity.

Testing Samples

Medical Devices Sterilized with Ethylene Oxide

EtO-sterilized products are the most common source of ethylene glycol residue testing requests. Typical samples include breathing circuits and anesthesia consumables, hemodialysis cartridges and blood circuits, wound dressings and gauze products, syringes, infusion and transfusion sets, catheters, surgical drapes, and single-use sterile packaging components. Both production batches and validation lots from sterilization cycle qualification studies can be tested.

Pharmaceutical Raw Materials and Finished Products

Testing is performed on active pharmaceutical ingredients, synthetic intermediates, polyethylene glycol (PEG)-based excipients, formulated drug products, and process residues where ethylene glycol may be present as a solvent impurity or degradation-related compound. Diethylene glycol (DEG) is frequently monitored in parallel as a closely related impurity.

Food Contact Materials and Packaging

PET bottles, polyester films, coatings, adhesives, and multilayer laminated packaging materials are tested for ethylene glycol migration and residual monomer content, supporting compliance with food contact regulations such as GB 9685 and equivalent international frameworks.

Cosmetics, Textiles, and Industrial Materials

Wet wipes, nonwoven fabrics, hygiene products, cosmetic formulations, polyester resins, coolants, brake fluids, and antifreeze products may also require EG residual or content analysis for quality control, safety assessment, and supplier verification purposes.

Testing Items

Testing programs are tailored to the product type, regulatory context, and risk profile. Commonly requested items include:

• Ethylene glycol (EG) residual content, reported as mg/kg, µg/g, mg/device, or ppm
• Ethylene oxide (EtO) residual content, typically determined by headspace GC
• 2-Chloroethanol (ethylene chlorohydrin, ECH) residual content
• Diethylene glycol (DEG) and related glycol impurities
• Comprehensive residual solvent panels in accordance with ICH Q3C and pharmacopoeial requirements
• Method suitability, extraction efficiency, and stability studies for sterilization validation projects

Testing Methods

Reference Standards and Regulatory Limits

Testing is performed with reference to ISO 10993-7 (and its Chinese equivalent GB/T 16886.7) for ethylene oxide sterilization residuals, ICH Q3C and USP General Chapter <467> for residual solvents, and GB/T 14233.1 for chemical analysis of infusion, transfusion, and injection equipment. Under ICH Q3C, ethylene glycol is a Class 2 solvent with a permitted daily exposure (PDE) of 6.2 mg/day and a concentration limit of 620 ppm for pharmaceutical products. ISO 10993-7:2008 specifies tolerable exposure limits for ethylene glycol of 5 mg per device for limited exposure (up to 24 hours), 3 mg for prolonged exposure (24 hours to 30 days), and 0.4 mg for long-term exposure (more than 30 days); users should always confirm values against the current edition of the standard.

Sample Preparation and Extraction

Solid samples are cut into small pieces and extracted following the principles of ISO 10993-12. A representative portion (typically 1 to 5 g) or a complete device is immersed in a measured volume of purified water or aqueous methanol and extracted by sonication or mechanical shaking for 30 to 60 minutes. For regulatory submission purposes, simulated-use extraction or exhaustive extraction at elevated temperature may be applied to establish worst-case residual levels. The extract is filtered through a 0.45 µm membrane before injection. When the internal-standard method is used, an internal standard such as 1,4-butanediol or 1,3-propanediol is added prior to extraction.

GC-FID Direct Injection Procedure

Direct liquid injection GC-FID is the routine approach because ethylene glycol is a high-boiling, strongly polar compound. Representative conditions are listed below and can be optimized for specific matrices:

• Column: polyethylene glycol (WAX-type) capillary column, 30 m × 0.32 mm × 0.25 µm, or a 6% cyanopropylphenyl/94% dimethylpolysiloxane phase for combined glycol separations
• Carrier gas: high-purity nitrogen or helium at a constant flow of 1.0 to 2.5 mL/min
• Inlet temperature: 230 to 250°C with a split ratio of 10:1 to 20:1; injection volume 1 µL
• Oven program: 60°C (hold 2 min), ramp at 10°C/min to 220°C (hold 5 min), adjusted to achieve baseline resolution
• FID temperature: 260 to 280°C with standard hydrogen and air gas flows
• Quantification: multi-point external or internal standard calibration covering the expected concentration range

Derivatization and GC-MS Confirmation

For demanding applications requiring lower detection limits, the extract is derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS to form the bis-trimethylsilyl derivative of ethylene glycol. Derivatization significantly improves volatility, peak shape, and detector response. GC-MS in selected ion monitoring mode, using characteristic fragment ions such as m/z 73 and 147, provides confirmatory identification and superior selectivity in complex matrices. Static headspace injection, which is standard for ethylene oxide, is generally not suitable for ethylene glycol due to its low vapor pressure and boiling point of approximately 197°C.

Method Validation and Performance Parameters

Methods are validated in accordance with the relevant pharmacopoeial and ICH analytical validation guidance before routine use. Typical performance characteristics include:

• Specificity: baseline resolution of ethylene glycol from ethylene chlorohydrin, diethylene glycol, solvent peaks, and matrix interferences
• Linearity: correlation coefficient r ≥ 0.999 across the working calibration range
• Limit of detection and limit of quantification: commonly in the range of 0.1 to 1 µg/mL in solution, corresponding to sub-mg/kg levels in samples depending on the extraction ratio
• Precision: repeatability RSD ≤ 5% for replicate preparations
• Accuracy: spike recovery typically within 85% to 115% at low, medium, and high fortification levels
• Robustness: verified against small deliberate variations in carrier gas flow and oven ramp rate

Applications

Ethylene glycol residue testing by gas chromatography supports a broad range of quality, safety, and regulatory activities across multiple industries:

• Validation and routine release testing of ethylene oxide sterilization cycles for medical devices of all risk classes
• Pharmaceutical quality control of residual solvents in APIs, intermediates, and excipients in accordance with ICH Q3C and pharmacopoeial monographs
• Migration and residual monomer testing of PET packaging, films, and food contact materials for food safety compliance
• Safety assessment and stability monitoring of cosmetics, hygiene products, and nonwoven materials
• Quality control of polyester resins, coolants, and functional fluids, along with supplier verification and failure investigation support

FAQ

Why is ethylene glycol not measured by headspace GC together with ethylene oxide?

Ethylene oxide is highly volatile and is well suited to static headspace analysis, whereas ethylene glycol is polar and high-boiling with very low vapor pressure. Static headspace sensitivity for ethylene glycol is poor, so direct liquid injection GC-FID or derivatization followed by GC-MS is the preferred approach. The two analytes are usually determined by complementary methods within the same testing program.

What are the regulatory limits for ethylene glycol residues?

For medical devices sterilized with ethylene oxide, ISO 10993-7 specifies tolerable exposure limits of 5 mg per device (limited exposure), 3 mg (prolonged exposure), and 0.4 mg (long-term exposure). For pharmaceuticals, ICH Q3C classifies ethylene glycol as a Class 2 solvent with a limit of 620 ppm. The applicable limit depends on product contact duration and daily exposure, so the product's intended use must be considered when evaluating conformity.

How much sample is required for testing?

For material-based testing, approximately 2 to 10 g of sample is typically sufficient. For medical devices, a specified number of complete devices or representative units may be required depending on the extraction protocol. Samples should be sealed in airtight containers after sterilization, kept cool, and shipped promptly, particularly when ethylene oxide residuals are to be tested in parallel.

What detection limits can be achieved?

With validated GC-FID methods, detection limits in solution of 0.1 to 1 µg/mL are routinely achievable, which corresponds to low mg/kg or sub-mg/kg levels in samples depending on the sample-to-solvent extraction ratio. Derivatization combined with GC-MS detection extends sensitivity further and is recommended for complex matrices or very low specification limits.

How are the results reported?

Reports present the ethylene glycol concentration in mg/kg, µg/g, or mg per device, together with the method used, calibration information, quality control results, and a statement of conformity against the applicable limit. Where required, measurement uncertainty can be provided, and testing can be performed under ISO/IEC 17025-accredited management systems to support regulatory submissions.

What is the typical turnaround time?

Standard ethylene glycol residue testing is generally completed within 5 to 10 working days after sample receipt, depending on sample complexity, the number of analytes requested, and whether additional validation or confirmatory GC-MS analysis is required. Expedited services may be arranged for urgent release testing.