Detecting Ethylene Glycol and Diethylene Glycol Impurities

Polyethylene glycol (PEG), a polyether compound derived from petroleum, has diverse industrial/ biological/medical applications and is one of the most popular polymeric materials.

By polymerizing ethylene oxide, PEG is routinely synthesized. In this process, polymers of ethylene oxide are formed when ethylene oxide reacts with ethylene glycol (EG) in the presence of a catalyst.1  Commonly, the structure of PEG is expressed as H-(O-CH2-CH2)n-OH (where n represents the number of monomer units).

PEG is soluble in both aqueous and organic solvents and possesses various physical properties based on its molecular weight (MW). This characteristic means that it is widely used in many industrial applications. More notably, PEG is predominantly used in cosmetics and pharmaceutical products as a Food and Drug Administration (FDA) approved excipient.

It is possible to use PEG as a permeation enhancer, solubilizer, thickener, and coating agent for tablets capsules and as a surface modifier in a range of drug delivery formulations.2  A clear, colorless, viscous liquid that can easily dissolve many hydrophobic drugs, PEG, with a molecular weight of 400 (PEG 400), is widely used as a solvent for many oral and topical pharmaceutical products, specifically in ophthalmic solutions to treat dry eyes.3 

The FDA traditionally considers PEG safe. However, toxic impurities such as ethylene oxide, 1,4-dioxane, ethylene glycol (EG) and diethylene glycol (DG) are commonly encountered4,5 depending on the synthetic route employed during the polymerization process. Both EG and DG are toxic to human health and were found to be nephrotoxic in some cases. 

United States Pharmacopoeia (USP) monograph for PEG has specified Gas Chromatography with Flame Ionization Detection (GC-FID) as the most appropriate analytical method to detect and quantify these impurities.6 Specifically, the determination of EG and DG impurities based on the molecular weight (MW) of the PEG sample under test is specified by the monograph. 

This article focuses on PEG samples with an MW of less than 450, where USP specifies a GC-FID using a packed column as the analytical method of choice. A stainless-steel (SS) packed column with a 12% G13 phase (D-Sorbitol, packed on a S1NS solid support) is recommended by USP. Therefore, a custom-made Restek packed column equivalent was used for this application. 

The results from the analysis of Ethylene Glycol and Diethylene Glycol in Polyethylene Glycol 400 sample are outlined in this article, according to USP requirements executed with the PerkinElmer GC 2400™ System equipped with a packed column injector and FID as a detector. The results show improved productivity and lab time optimization. The analytical workflow is managed by PerkinElmer SimplicityChrom Chromatography Data System (CDS) Software, and it supports compliance with Title 21 of the Code of Federal Regulations (CFR), Part 11. 

Instrumentation 

Coupled with a packed column injector and a 12% G13 SS packed column on S1NS support, the PerkinElmer GC 2400 System offers a robust sample introduction system to perform the analysis.

A PerkinElmer AS 2400™ Liquid Sampler and an FID Detector were used to configure the GC 2400 System, creating a reliable platform for quantifying EG and DG impurities in PEG. Real-time monitoring is enabled by the PerkinElmer Simplicity Vision, which runs on the detachable touchscreen or any PCs, from any location on the same network. This cohesion optimizes the time and ultimately increases the lab’s productivity.

PerkinElmer GC 2400 System.

Figure 1. PerkinElmer GC 2400 System. Image Credit: PerkinElmer

Experimental 

In the packed column injector, a  1.5 m x 3 mm SS packed column with 12% G13 phase on S1NS support was installed and subsequently conditioned according to standard practices.

Table 1 lists the GC conditions required for the analysis which are as per USP monograph. For standard and sample preparation, water was used as a solvent. For standard preparation, USP-grade EG and DG were purchased from Millipore Sigma. To be used as a sample, commercial grade PEG 400 was purchased from Millipore Sigma.

Table 1. Chromatography conditions. Source: PerkinElmer

GC Parameters  
Instrument GC 2400 System
Column 1.5 m x 3 mm SS packed column with 12% G13 on support S1NS
GC Oven Parameters Initial Final
140 °C 140 °C (40 minutes)
AS 2400 Liquid Sampler Parameters  
Syringe Size 5 μL (N6402556)
Injection Volume 2.0 μL
Injection Speed Normal
Number of Plunges 6
Sample Wash 2
Sample Wash Volume 50%
Pre-wash 0
Post-Wash 0
Viscosity Delay 2 seconds
Injector Parameters  
Type Packed Column Injector, Septum Flow: 3 mL/minute
Temperature 250 °C
Carrier/mode Helium/Constant Flow mode
Flow Rate (mL/min) 50 mL/minute
FID Detector Parameters  
Type FID with Packed Column Adapter (N6406057)
Temperature 280 °C
Hydrogen 30 mL/minute
Air 400 mL/minute
Makeup Gas Nitrogen 25 mL/minute
Data rate 10 pt/second

 

Consumables

Product Description Part Number
Restek 1.5 m x 3 mm Stainless Steel (SS) Capillary Packed Column with a 12% Sorbitol on Diato- WNAW 60/80 Support (equivalent to 12% G13 on S1NS support) PN# PKC49347
FID Packed Column Adapter, Pkg. 1 N6406057
Advanced Green Inlet Septum, Pkg. 10 N9306218
5 μL Autosampler Syringe, Pkg. 1 N6402556
Graphite Vespel Column Ferrules 1/8 in I.D., Pkg. 10 09920133
2 mL Clear Glass 9 mm Screw Top Vial with Write-On Patch, Liquid Autosampler Vials, Pkg. 100 N9307801
9 mm Blue Screw Caps with PTFE/SIL Liner (Liquid Autosampler Caps), Pkg. 100 N9306202
Triple Filter (Hydrogen and Nitrogen), Pkg. 1 N9306110
Moisture/Hydrocarbon Trap (Air), Pkg. 1 N9306117
Triple Filter (Helium), Pkg. 1 N9306106
GC 2400 Packed Injector Liner, 3 mm I.D., Pkg. 1 N6406035

 

Standard Preparation 

The following solution was used to prepare the standard:

  • 0.50 mg/mL each of USP EG and USP DG in water and was prepared as stated in USP monograph. 

Sample Preparation 

Using commercial grade PEG 400 at 400 mg/mL in water as stated in the USP monograph, a sample was prepared. 

Spiked Sample: EG and DG standards were used to intentionally spike the commercial grade PEG 400 sample to mimic a non-USP grade sample under test. At 400 mg/mL commercial grade PEG 400 in water, a spiked sample was prepared and spiked with approximately 0.70 mg/mL each of EG and DG standard solutions in water. This will be used as the spiked sample. 

Data Acquisition 

SimplicityChrom CDS Software (version 2.0) was used to perform instrument control and data analysis, enabling g streamlined instrument setup, data acquisition, and smooth processing. 

Results and Discussion 

Retention Time and Peak Identification 

A standard chromatogram obtained under the parameters set in Table 1 is shown in Figure 1. When using FD, retention time (RT) identification is critical because it is a universal detector for hydrocarbon analysis. Specifically for packed column injectors, the GC 2400 System’s advanced Pneumatic Pressure Controller (PPC) can perform highly repeatable separations, facilitating the reliable identification of compounds by RT. The RT and % relative standard deviations (%RSD) of 0.08% for EG and 0.09% for DG were obtained, respectively, and are shown in Table 2.

Standard Chromatogram containing 0.50 mg/mL of Ethylene Glycol and Diethylene Glycol in water. Peak 1: ethylene glycol (RT 8.279 min), Peak 2: diethylene glycol (RT 23.984 min).

Figure 1. Standard Chromatogram containing 0.50 mg/mL of Ethylene Glycol and Diethylene Glycol in water. Peak 1: ethylene glycol (RT 8.279 min), Peak 2: diethylene glycol (RT 23.984 min). Image Credit: PerkinElmer

Table 2. Retention time (RT) and Peak Response for Standard Solution. Source: PerkinElmer

  Ethylene Glycol RT (minutes) Diethylene Glycol RT (minutes) Ethylene Glycol Peak Response (pA) Diethylene Glycol Peak Response (pA)
Trial 1 8.283 24.010 16.008 7.977
Trial 2 8.280 24.018 15.955 7.904
Trial 3 8.272 23.961 16.140 8.038
Trial 4 8.291 23.984 16.311 8.101
Trial 5 8.279 23.984 16.191 8.032
Trial 6 8.286 23.978 16.257 7.984
Avg.
RSD
8.282
0.08%
23.989
0.09%
16.144
0.86%
8.006
0.84%

 

Further validating the precise and reproducible temperature and flow control of the GC 2400 System, peak response %RSD for EG and DG were less than 1%. The system suitability (SST) parameters are easy to select using SimplicityChrom CDS Software, and these calculations can be tailored to group or individual peaks alike, which offers more customization and flexibility to the user. 

Sample Analysis 

Commercial grade PEG 400 was employed here as a test sample. First, the sample was prepared at 400 mg/mL in water, and subsequently, the sample was analyzed as per the test conditions specified in Table 1. 

It is specified by USP monograph that if a peak is present in the sample at the RT of EG or DG, it must be quantified on the basis of the standard peak response quantitation method, as presented in the following equations. 

Detecting Ethylene Glycol and Diethylene Glycol Impurities

Analysis of the Sample

Figure 2 shows the chromatogram of commercial grade PEG 400 sample injection. As can be seen, EG and DG detected in the PEG 400 sample are confirmed on the basis of the RT of EG and DG peak to that of the standard solution. The % content of each of these impurities in the sample was calculated based on the formulas presented before and is shown in Table 3. 

Sample Chromatogram of commercial PEG 400 sample solution at 400 mg/mL in water. Peak 1: ethylene glycol (RT 8.285 min), Peak 2: diethylene glycol (RT 23.767 min).

Figure 2. Sample Chromatogram of commercial PEG 400 sample solution at 400 mg/mL in water. Peak 1: ethylene glycol (RT 8.285 min), Peak 2: diethylene glycol (RT 23.767 min). Image Credit: PerkinElmer

An acceptance criterion of no more than (NMT) 0.25% for the sum of % EG and % DG in the sample is stated in USP monograph. This acceptance criteria is met by the commercial PEG 400 as specified in USP, from the results summarized in Table 3. 

Table 3. Sample Results for PEG 400. Source: PerkinElmer

Analyte Retention Time (min) Sample Peak Response (pA) PEG 400 Sample Concentration (mg/mL) Standard Peak Response (pA) Standard Concentration (mg/mL) % Content
Ethylene Glycol 8.285 2.194 400.331 16.144* 0.495 0.02
Diethylene Glycol 23.767 1.800 8.006* 0.498 0.03
Sum of % Content of Ethylene Glycol and Diethylene Glycol in sample 0.05

 

* Average peak response of 6 consecutive standard injections (refer Table 2).

Spiked Sample Analysis 

The commercial grade PEG 400 sample was intentionally spiked with EG and DG standards to mimic a non-USP grade sample under test and to test the validity of the method. As per the test conditions in Table 1, this spiked sample was analyzed and then the quantification of EG and DG was carried out as explained prior and as specified by USP.

The content results of % EG and % DG found in the spiked PEG 400 sample are presented in Table 4, and it does not meet the USP requirement of NMT 0.25% for the sum of % EG and % DG found. It is shown by the analysis that, for PEG of MW less than 450, the new PerkinElmer GC 2400 System can carry out USP PEG analysis for the limit of ethylene glycol and diethylene glycol. 

Table 4. Sample Results for PEG 400 spiked sample. Source: PerkinElmer

Analyte Retention Time (minutes) Sample Peak Response (pA) PEG 400 Sample Concentration (mg/mL) Standard Peak Response (pA) Standard Concentration (mg/mL) % Content
Ethylene Glycol 8.254 24.280 400.814 16.144* 0.495 0.19
Diethylene Glycol 23.953 11.672 8.006* 0.498 0.18
Sum of % Content of Ethylene Glycol and Diethylene Glycol in sample 0.37

 

* Average peak response of 6 consecutive standard injections (refer Table 2).

Conclusion 

The PerkinElmer GC 2400 System, configured with a packed column injector and an FID Detector, delivers results for the determination of the limit of ethylene glycol and diethylene glycol impurities according to USP PEG monograph.

With %RSD of less than 0.1% and peak response repeatability of 1% or less demonstrating superior performance, the retention time repeatability for this analysis was precise. A practical, customizable user experience with multifunctionality and accessibility options is provided by SimplicityChrom CDS Software. The detachable touchscreen also offers both portability and versatility, which provides busy lab environments with time optimization. 

References 

  1. F.E. Bailey, J.V. Koleske, "Alkylene Oxides and Their Polymers", CRC Press, New York, Dekker; pp 261, 1991. 
  2. A.A D’souza, R. Shegokar, “Polyethylene glycol (PEG): A versatile polymer for pharmaceutical applications”, Expert Opinion on Drug Delivery, Volume 13, Issue 9, pp 1257-1275, 2016. 
  3. Gary N Foulks, “Clinical evaluation of the efficacy of PEG/PG lubricant eye drops with gelling agent (HP-Guar) for the relief of the signs and symptoms of dry eye disease: a review”, Drugs Today (Barc), Volume 43, Issue 12, pp 887-896, Dec 2007. 
  4. FDA. Compounded Curcumin Emulsion Product for Injection by Imprimis Rx: FDA Investigation - Serious Adverse Events Associated with Use. FDA Drug Safety Newsletter: 8 Aug 2017. 
  5. Andersen FA, "Special Report: Reproductive and Developmental Toxicity of Ethylene Glycol and Its Ethers". International Journal of Toxicology, Volume 18, Issue 3, pp 53-67, 1999. 
  6. USP-NF Polyethylene Glycol Monograph: USP43-NF38 P. 5939.v

This information has been sourced, reviewed and adapted from materials provided by PerkinElmer.

For more information on this source, please visit PerkinElmer.

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