The Limit of Ethylene Glycol and Diethylene Glycol

Known as methyl ethyl glycol, 1,2-propanediol, or propane-1,2-diol, propylene glycol, is a synthetic, clear, colorless liquid that is widely used as an additive or antifreeze within the food, chemical, pharmaceutical, and cosmetics industries.1,2 

For increasing the solubility and stability of prescribed and over-the-counter medicines, propylene glycol is a vital excipient. Moreover, propylene glycol helps maintain moisture in specific medicines and topical formulations such as creams due to its ability to absorb water.1

Commonly present as impurities in propylene glycol, ethylene glycol and diethylene glycol are toxic to human health.3 United States Pharmacopoeia (USP) revised its monograph for propylene glycol in 2010 in response to these toxic considerations and adhering to the recommendations from US Food and Drug Administration (USFDA).

By specifying the limit for ethylene glycol and diethylene glycol, this monograph4 addresses the toxicity concerns while using propylene glycol as an inactive ingredient to meet quality standards in pharmaceutical products.4,5

Propylene glycol is identified by this updated monograph, which defines a limit test for ethylene glycol and diethylene glycol. This limit test is constructed through Gas Chromatography/Flame Ionization Detection (GC-FID) and an internal standard peak response quantitation method, which allows the user to then measure ethylene glycol and diethylene glycol content in propylene glycol samples. 

The performance of the PerkinElmer GC 2400™ System with FID for the analysis of propylene glycol quality according to the updated USP monograph is outlined within this article while demonstrating the instrument’s superior performance and 40% improvement to the required resolution.  

Instrumentation 

With a capillary split/splitless (CAP) injector and PerkinElmer Elite 624 analytical column, the PerkinElmer GC 2400 System offered a highly-streamlined solution to propylene glycol quality evaluation.

A PerkinElmer AS 2400TM Liquid Sampler and an FID were used to configure the GC 2400 System, facilitating a reliable platform for the quantification of ethylene glycol and diethylene glycol as impurities for USP grade propylene glycol analysis. 

For real-time monitoring and live status checks, PerkinElmer Simplicity Vision runs on the detachable touchscreen when connected to the laboratory network, optimizing time and increasing the lab’s productivity. 

The PerkinElmer GC 2400 System.

The PerkinElmer GC 2400 System. Image Credit: PerkinElmer

Experimental 

According to practices in the PerkinElmer Capillary Column Installation Quick Care guide, a PerkinElmer Elite 624 column 30 m X 0.53 mm X 3.0 μm was installed in the injector and conditioned. Table 1 lists the GC conditions required for this analysis, which are as per USP monograph for propylene glycol.

Millipore Sigma provided the methanol (Purge and Trap grade)  used as a solvent for standard and sample preparation. Millipore Sigma also provided USP-grade propylene glycol, ethylene glycol, diethylene glycol, 2,2,2-trichloroethanol (internal standard), and commercial USP-grade propylene glycol (used as sample). 

Standard Preparation 

The following method was used to prepare a standard: 

2.0 mg/mL of USP propylene glycol, 0.050 mg/mL of USP ethylene glycol, 0.050 mg/mL of USP diethylene glycol, and 0.10 mg/mL of 2,2,2-trichloroethanol (as internal standard) in methanol. 

Sample Preparation 

Using 50 mg/mL commercial USP-grade propylene glycol and 0.10 mg/mL of 2,2,2-trichloroethanol (internal standard) in methanol, a sample was prepared. 

Spiked Sample

Ethylene glycol and diethylene glycol standards were used to intentionally spike the commercial USP-grade propylene glycol sample, to mimic a real-life non-USP grade sample under test. Methanol with 50 mg/mL commercial USP-grade propylene glycol, 0.10 mg/mL of 2,2,2-trichloroethanol (internal standard), and approximately 0.050 mg/mL each of USP ethylene glycol and USP diethylene glycol were used to prepare the spiked sample. 

Table 1. Chromatography conditions. Source: PerkinElmer

GC Parameters  
Instrument PerkinElmer GC 2400 System
Column PerkinElmer Elite-624 30 m X 0.53 mm X 3.0 μm (N9316207)
GC Oven Parameters Initial Ramp Final
100° C (4 minutes) 50° C/minute 120° C (10 minutes)
120° C 50° C/minute 220° C (6 minutes)
AS 2400 Liquid Sampler Parameters  
Syringe Size 5 μL (N6402556)
Injection Volume 1.0 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 Capillary Split/Splitless, Septum Flow: 3 mL/minute
Temperature 220° C
Carrier/mode Helium/Constant Flow mode
Flow Rate (mL/ min) 4.5 mL/minute
Split Ratio 10:1
Liner Deactivated glass liner 4mm I.D. with deactivated wool (N6502041)
FID Detector Parameters  
Type FID
Temperature 250° C
Hydrogen 30 mL/minute
Air 400 mL/minute
Data rate 10 pt/second

 

Consumables

Product Description Part Number
Elite 624 30 M X 0.53 mm X 3.0 μm N9316207
4 mm ID Capillary Split /Splitless Deactivated Glass Liners with Deactivated Wool (green), Pkg. 5 N6502041
Advanced Green Inlet Septum, Pkg. 10 N9306218
5 μL Autosampler Syringe, Pkg. 1 N6402556
Graphite Vespel Capillary Column Ferrules 0.8 mm ID, Pkg. 10 09920107
Ceramic Column Cutter, Pkg. 10 N9301376
O-ring for Glass PSS Liner, Pkg. 10 09200714
2 mL Clear Glass 9 mm Screw Top Vial with Write-On Patch, Liquid Autosampler Vials, Pkg. 100 N9307801
9mm blue screw caps with PTFE/SIL Liner (Liquid Autosampler Caps), Pkg. 100 N9306202
Triple Filter (Hydrogen & Nitrogen), Pkg. 1 N9306110
Moisture/Hydrocarbon Trap (Air), Pkg. 1 N9306117
Triple Filter (Helium), Pkg. 1 N9306106
CAP Injector Gold Seal, Pkg. 1 N6400900

 

Data Acquisition 

Streamlined instrument setup, data acquisition, and processing are carried out by performing instrument control and data analysis with PerkinElmer SimplicityChromTM CDS Software (version 2.0). Title 21 of the Code of Federal Regulations (CFR), Part 11, is supported by SimplicityChrom CDS Software

Results and Discussion 

Retention Time and Peak Identification 

A standard chromatogram obtained under the parameters outlined in Table 1 is shown in Figure 1. When using FID retention time (RT), identification is critical because it is a universal detector for hydrocarbon analysis where the response is proportional to the number of carbon atoms.

Standard Chromatogram containing 0.050 mg/mL of USP ethylene glycol (RT 3.854 min), 2.0 mg/mL of USP propylene glycol (RT 4.401 min), 0.10 mg/mL of 2,2,2-trichloroethanol as the internal standard (RT 7.372 min) and 0.050 mg/mL of USP diethylene glycol (RT 10.258 min), in methanol.

Figure 1. Standard Chromatogram containing 0.050 mg/mL of USP ethylene glycol (RT 3.854 minutes), 2.0 mg/mL of USP propylene glycol (RT 4.401 minutes), 0.10 mg/mL of 2,2,2-trichloroethanol as the internal standard (RT 7.372 minutes) and 0.050 mg/mL of USP diethylene glycol (RT 10.258 minutes), in methanol. Image Credit: PerkinElmer

Highly repeatable separations are provided by the PerkinElmer 2400 GC’s advanced Pneumatic Pressure Controller (PPC), allowing reliable identification of compounds by RT.

The RT and relative retention time (RRT) are shown in Table 2, which aligns with those reported in the USP monograph.

Table 2. Retention time (RT) and Relative Retention Time (RRT) for Standard Solution. Source: PerkinElmer

  Ethylene Glycol Propylene Glycol* 2,2,2-trichloroethanol (Internal Standard) Diethylene Glycol
Trial 1 RT (min) 3.855 4.402 7.371 10.261
Trial 2 RT (min) 3.855 4.400 7.370 10.260
Trial 3 RT (min) 3.851 4.399 7.367 10.258
Trial 4 RT (min) 3.854 4.401 7.371 10.261
Trial 5 RT (min) 3.853 4.399 7.367 10.256
Avg. RT (min) 3.854 4.400 7.369 10.259
RSD 0.043% 0.030% 0.028% 0.021%
Trial 1 RRT 0.9 1.0 1.7 2.3
Trial 2 RRT 0.9 1.0 1.7 2.3
Trial 3 RRT 0.9 1.0 1.7 2.3
Trial 4 RRT 0.9 1.0 1.7 2.3
Trial 5 RRT 0.9 1.0 1.7 2.3
Avg. RRT 0.9 1.0 1.7 2.3
USP Literature RRT4 0.8 1.0 1.7 2.4

 

* Propylene glycol is used as a reference peak for RRT calculations.

As shown in Table 2, for each of the components RT % relative standard deviations (%RSD) of 0.02% to 0.04% were obtained. To provide peak identification for the users, the USP monograph also states the RRT for each component against that of propylene glycol. For each component, Table 2 specifies the USP literature values for RRT. Further validating the precise and reproducible temperature and flow control of the PerkinElmer 2400 GC System, good accuracy in terms of RRT to USP literature value were obtained.

System Suitability Requirement for Resolution 

A system suitability (SST) requirement for resolution of not less than (NLT) 5 between ethylene glycol and propylene glycol is stated by USP propylene glycol monograph. SST requirement was exceeded with a reported resolution value of 7 using the Elite 624 column with GC 2400 System. The SST parameters are easy to select when Using SimplicityChrom CDS Software, and more customization flexibility is offered to the user because these calculations can be tailored to individual or group peaks alike. As evidenced by Table 3, resolution values were exceeded, with an average improvement of the resolution by 40%. 

Table 3. Resolution between propylene glycol and ethylene glycol. Source: PerkinElmer

  USP Resolution between Ethylene Glycol and Propylene Glycol
Trial 1 Resolution 7.543
Trial 2 Resolution 7.370
Trial 3 Resolution 7.375
Trial 4 Resolution 7.345
Trial 5 Resolution 7.382
Avg. Resolution 7.403
RSD 1.074%
USP Literature Resolution4 NLT 5

 

Peak Area Ratio 

Along with the peak area ratio for ethylene glycol with respect to 2,2,2-trichloroethanol (Internal Standard) and similarly, peak area ratio for diethylene glycol with respect to 2,2,2-trichloroethanol (Internal Standard), Table 4 presents repeatability for standard injections. As explained later in sample analysis, these peak area ratios relative to the internal standard in the standard solution are used as a limit value to quantitate these impurities in the sample solution.

Sample Analysis 

For a  test sample, commercial USP-grade propylene glycol from Millipore Sigma was used. As per the test conditions specified in Table 1, the sample was prepared as vide supra and then analyzed. 

It is specified by USP that if a peak is present in the sample at the retention time of ethylene glycol or diethylene glycol, it must be quantified based on peak area ratio of these impurities with respect to the internal standard peak area, as in the following equation.

 Equations

Limit for Ethylene Glycol 

The acceptance criteria for an ethylene glycol peak, if present in a sample, is required by USP to be calculated as the peak response ratio of ethylene glycol relative to the internal standard (2,2,2-trichloroethanol) in a sample solution must not exceed (NMT) the peak response ratio of ethylene glycol relative to the internal standard (2,2,2-trichloroethanol) in the standard solution. 

Limit for Diethylene Glycol 

USP states that the acceptance criteria for a diethylene glycol peak (if present in a sample) needs to be calculated as the peak response ratio of diethylene glycol relative to the internal standard (2,2,2-trichloroethanol) in the sample solution should not be more than (NMT) the peak response ratio of diethylene glycol relative to the internal standard (2,2,2-trichloroethanol) within the standard solution. 

Sample Analysis 

For ethylene glycol and diethylene glycol, the analyzed propylene glycol commercial USP-grade sample did not present any peak at the RT. Therefore, it surpasses the USP criteria for the limit of ethylene glycol and diethylene glycol, and can be categorized as USP grade propylene glycol as marketed by the supplier.

Figure 2 presents an overlay chromatogram of propylene glycol commercial USP-grade sample and standard injection. There are no peaks detected at the RT of either of the impurities in question, as can be seen. Based on the RT of the propylene glycol peak in the sample solution corresponds to that of the standard solution, the identity of propylene glycol is confirmed. 

Overlay Chromatogram of Standard solution and commercial USP-grade propylene glycol sample solution in methanol. Analyte Peak in standard solution ethylene glycol (RT 3.854 min), propylene glycol (RT 4.401 min), 2,2,2-trichloroethanol as the internal standard (RT 7.372min) and diethylene glycol (RT 10.258 min). Analyte Peak in commercial USP-grade propylene glycol Sample Solution propylene glycol (RT 4.449 min) and 2,2,2-trichloroethanol as the internal standard (RT 7.379 min).

Figure 2. Overlay Chromatogram of Standard solution and commercial USP-grade propylene glycol sample solution in methanol. Analyte Peak in standard solution ethylene glycol (RT 3.854 minutes), propylene glycol (RT 4.401 min), 2,2,2-trichloroethanol as the internal standard (RT 7.372 minutes) and diethylene glycol (RT 10.258 minutes). Analyte Peak in commercial USP-grade propylene glycol Sample Solution propylene glycol (RT 4.449 minutes) and 2,2,2-trichloroethanol as the internal standard (RT 7.379 minutes). Image Credit: PerkinElmer

Spiked Sample Analysis 

The commercial USP-grade propylene glycol sample was intentionally spiked with ethylene glycol and diethylene glycol standards to mimic a real-life non-USP grade sample under test to test the validity of the method.

As per the test conditions in Table 1, this spiked sample was analyzed, and the peak area ratio method was used to perform the quantification of ethylene glycol and diethylene glycol as specified by USP and as explained prior. 

Overlay Chromatogram of Standard solution and Spiked Sample solution in methanol. Analyte Peak in standard solution ethylene glycol (RT 3.854 min), propylene glycol (RT 4.401 min), 2,2,2-trichloroethanol as the internal standard (RT 7.372min) and diethylene glycol (RT 10.258 min). Analyte Peak in Spiked Sample solution ethylene glycol (RT 3.873 min), propylene glycol (RT 4.498 min), 2,2,2-trichloroethanol as the internal standard (RT 7.382min) and diethylene glycol (RT 10.273 min).

Figure 3. Overlay Chromatogram of Standard solution and Spiked Sample solution in methanol. Analyte Peak in standard solution ethylene glycol (RT 3.854 minutes), propylene glycol (RT 4.401 minutes), 2,2,2-trichloroethanol as the internal standard (RT 7.372 minutes) and diethylene glycol (RT 10.258 min). Analyte Peak in Spiked Sample solution ethylene glycol (RT 3.873 minutes), propylene glycol (RT 4.498 minutes), 2,2,2-trichloroethanol as the internal standard (RT 7.382 minutes) and diethylene glycol (RT 10.273 minutes). Image Credit: PerkinElmer

An overlay of chromatograms of the spiked propylene glycol sample and standard solution is represented in Figure 3. The results for the limit of ethylene glycol and diethylene glycol by peak area ratio calculations are presented in Figure 5. The peak area ratio of ethylene glycol and diethylene glycol (with respect to the internal standard in the standard solution) are shown in Table 4, and the spiked propylene glycol sample meets the specifications for the limit of ethylene glycol, based on the results in Table 5, but failed to meet the specifications for diethylene glycol. Nonetheless, it is shown by the analysis that the GC 2400 System performs the USP propylene glycol analysis for the limit of ethylene glycol and diethylene glycol. 

Table 4. Peak area repeatability of standard injections along with peak area ratio for ethylene glycol and diethylene glycol. Source: PerkinElmer

  Ethylene Glycol Peak Area Propylene Glycol Peak Area 2,2,2-trichloroethanol (Internal Standard) Peak Area Diethylene Glycol Peak Area Peak Area Ratio of Ethylene Glycol w.r.t (Internal Standard) Peak Area Ratio of Diethylene Glycol w.r.t (Internal Standard)
Trial 1 12.136 665.988 13.522 12.670 0.898 0.937
Trial 2 12.211 666.075 13.511 12.643 0.904 0.936
Trial 3 12.269 659.284 13.448 12.644 0.912 0.940
Trial 4 12.553 670.391 13.534 12.470 0.928 0.921
Trial 5 12.410 663.500 13.423 12.404 0.925 0.924
Avg 12.316 665.047 13.488 12.566 0.913 0.932
RSD 1.350% 0.611% 0.363% 0.960% 1.422% 0.913%

 

Table 5. Peak Area Ratio results for Ethylene Glycol and Diethylene Glycol, in spiked sample. Source: PerkinElmer

Sample # Ethylene Glycol Peak Area Diethylene Glycol Peak Area Internal Standard (2,2,2-trichloroethanol) Peak Area Peak Area Ratio (Ethylene Glycol/Internal Standard) Peak Area Ratio (Diethylene Glycol/ Internal Standard)
Sample 1 9.795 15.591 13.631 0.72 1.14
Sample 2 9.605 14.996 13.222 0.73 1.13
Sample 3 9.641 14.754 13.275 0.73 1.11
Avg 9.680 15.114 13.376 0.72 1.13
RSD 1.039% 2.850% 1.663% 0.62% 1.47%
Avg Peak Area Ratio in Standard Solution (Refer Table 4) 0.91 0.93

 

Conclusion 

The PerkinElmer GC 2400 System meets the USP propylene glycol monograph requirements for the limit of ethylene glycol and diethylene glycol. With an average %RSD of 0.02% to 0.04% and average peak area reproducibility of around 1% or less, the retention time reproducibility for this analysis was reasonably precise, demonstrating superior performance. Furthermore, an improvement of 40% of that specified under USP monograph for propylene glycol: limit of ethylene glycol and diethylene glycol took place regarding the resolution requirement of NLT 5 between ethylene glycol and propylene glycol.

Compliance with 21 CFR Part 11 data requirements is supported by SimplicityChrom CDS Software supports, which provide a customizable and highly practical user experience, adapting to different user-proficiency levels. Moreover, versatility and portability is provided by the use of a detachable touchscreen, offering time optimization for busy lab environments. 

References 

  1. McMartin, K. Propylene Glycol. In Encyclopedia of Toxicology, 3rd ed.; Wexler, P., Ed.; Academic Press: Oxford, UK, 2014; pp. 1113–1116 
  2. European Medicines Agency, “Propylene glycol used as an excipient”, Report published in support of the questions and answers on propylene glycol used as an excipient in medicinal products for human use, EMA/ CHMP/704195/2013, 9 October 2017 
  3. World Health Organization (WHO), “Report of the Diethylene Glycol Contamination Prevention Workshop, 1997, p. xi 
  4. USP-NF Propylene Glycol Monograph: USP43-NF38 P. 3753 
  5. The USP Excipients Stakeholder Forum, “Excipient Monograph Modernization, Lawrence H. Block”, Meeting 1, 7 June 2013

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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