Sponsored by PerkinElmerReviewed by Olivia FrostSep 15 2022
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. 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.
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.
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.
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.
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
- McMartin, K. Propylene Glycol. In Encyclopedia of Toxicology, 3rd ed.; Wexler, P., Ed.; Academic Press: Oxford, UK, 2014; pp. 1113–1116
- 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
- World Health Organization (WHO), “Report of the Diethylene Glycol Contamination Prevention Workshop, 1997, p. xi
- USP-NF Propylene Glycol Monograph: USP43-NF38 P. 3753
- 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.