Optimizing EPA Method 1634 for 6PPD-Quinone Analysis
Abstract
Draft EPA Method 1634 is a performance-based LC-MS/MS method that covers the determination of 6PPD-quinone in aqueous matrices, predominantly storm and surface water. In this study, the analytical column and conditions were optimized to improve performance while still meeting all method specifications. In addition to meeting method requirements, the total run time was dropped from 10 to 5.5 minutes, providing labs with an effective way to increase sample throughput and overall lab productivity.
Introduction
In December 2023, in accordance with the Clean Water Act (CWA), EPA released Draft Method 1634, Determination of 6PPD-Quinone in Aqueous Matrices Using Liquid Chromatography with Tandem Mass Spectrometry (LC/MS/MS), in response to concerns about 6-PPD-quinone leeching into runoff water. 6PPD-quinone (6PPD-Q) is formed when the tire additive N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6-PPD) reacts with ozone in the air. Then, after rainfall events, it can be introduced into aquatic ecosystems through stormwater runoff, where it can be lethal to coho salmon and other fish species.
Draft Method 1634 is a performance-based method, meaning changes can be made to it if the results are proven to be equivalent to the established method specifications. In this study, we optimized the analytical column and LC-MS/MS conditions to reduce run times so more samples could be run while still meeting method requirements. In addition, we used high-performance Resprep polymeric SPE cartridges to ensure the sample extracts were free from potential matrix interferences.
Experimental
Calibration Standards
The calibration standards were prepared in acetonitrile at the seven concentrations shown in Table I. Curve fits that minimized the percent relative standard error (%RSE) were chosen in accordance with the method.
Table I: Calibration Standard Concentrations
| Calibration Standard Concentrations (ng/mL) | |||||||
| Compound and Intermediate Standard Concentration | C1 | C2 | C3 | C4 | C5 | C6 | C7 |
| 6PPD-quinone (20 ng/mL) | 0.025 | 0.050 | 0.10 | 0.50 | 1.0 | 5.0 | 10.0 |
| 13C6-6PPD-quinone (20 ng/mL) | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| D5-6PPD-quinone (20 ng/mL) | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
QC Sample Preparation
Samples for precision, accuracy, and method detection limit determination were prepared in polypropylene bottles using 250 mL of deionized water spiked with 50 µL of extracted and non-extracted internal standards (EIS/NIS) procured from Cambridge Isotope Laboratories, Inc. (cat.# CIL-ULM-12288-1.2; CLM-12293-1.2; and DLM-11616-1.2) in accordance with EPA Draft Method 1634, Section 7.2. Four samples for initial precision and recovery (IPR) analysis were spiked with 6PPD-quinone at 40 ng/L. Six MDL samples were spiked with 50 µL of a 5:1 dilution of the native standard, giving a pre-extraction concentration of 20 ng/mL. The MDL samples were prepared, extracted, and analyzed over three days.
Water Samples
Real-world matrix samples were collected from road runoff water on highway 322-East in State College, PA, in 250 mL amber glass jars with PTFE-lined caps. Samples were stored at less than or equal to 6 °C. All samples were extracted within 14 days of collection. EIS was spiked prior to sample extraction, and NIS was added post-extraction.
Sample Extraction
Sample extraction was performed on a Dionex AutoTrace 280 PFAS model system using Resprep polymeric SPE cartridges (6 mL) that contained 200 mg of 60 µm HLB material (cat.# 28264). The samples were extracted over three days following the steps in Figure 1. After extraction, the samples were spiked with 50 µL of the non-extracted internal standards (NIS, Cambridge Isotope Laboratory Inc., cat.# DLM-11616-1.2).
Figure 1: Sample Preparation Steps (Steps 1-6 are diverted to waste.)
Instrument Method
A Waters ACQUITY Premier LC and a Xevo TQ Absolute triple quadrupole MS were used in this study. The original conditions from Draft Method 1634 are given in Table II, and the optimized column and conditions developed in this study are shown in Table III.
Table II: Draft Method 1634 Suggested HPLC Conditions (Section 10.3.1)
| Column | C18 phase 4.6 x 100 mm, 3.5 µm | ||
| Mobile phase A | 0.2% Formic acid in water | ||
| Mobile phase B | Acetonitrile | ||
| Column temperature | 45 °C | ||
| Injection volume | 20 µL | ||
| Flow rate | 0.6 mL/min | ||
| Maximum pressure | 7500 psi (517 bar) | ||
| Gradient | Time (min) | %A | %B |
| 0 | 90 | 10 | |
| 1 | 90 | 10 | |
| 3 | 45 | 55 | |
| 6 | 1 | 99 | |
| 8 | 1 | 99 | |
| 8.5 | 90 | 10 | |
| 9 | 90 | 10 | |
| Expected 6PPD-quinone elution time | 7.53 min | ||
| Total run time | 10.0 min | ||
Table III: Optimized LC Method Conditions
| Column | Raptor C18 50 x 2.1 mm, 2.7 µm (cat.# 9304A52) | ||
| Guard column | Raptor C18 EXP guard column cartridge 5 x 2.1 mm, 2.7 µm (cat.# 9304A0252) | ||
| Mobile phase A | 0.1% Formic acid in water | ||
| Mobile phase B | 0.1% Formic acid in acetonitrile | ||
| Diluent | Acetonitrile | ||
| Column temperature | 40 °C | ||
| Injection volume | 3 µL | ||
| Flow rate | 0.5 mL/min | ||
| Maximum pressure | 3200 psi (220 bar) | ||
| Gradient | Time (min) | %A | %B |
| 0 | 70 | 30 | |
| 3 | 0 | 100 | |
| 4 | 0 | 100 | |
| 4.01 | 70 | 30 | |
| 5.5 | 70 | 30 | |
| Expected 6PPD-quinone elution time | 1.93 min | ||
| Total run time | 5.5 min | ||
Results and Discussion
LC-MS/MS Method Optimization and Chromatographic Performance
Since Draft Method 1634 is performance based, labs are permitted to modify some method conditions as long as all method performance requirements are still met. In this study, we optimized several parameters in order to speed up run times, improve lab throughput, and create a method that is amendable to both HPLC and UHPLC platforms. Our final optimized method, which is shown in Figure 2, reduced the retention time for 6PPD-quinone from 7.53 minutes to 1.93 minutes, shortened the total cycle time from 10 to 5.5 minutes, and had a maximum back pressure of ~220 bar. A detailed discussion of how each parameter was optimized follows.
Draft Method 1634 suggests that an analytical column with a C18 stationary phase be used, so a Raptor C18 column was chosen for the optimized method. While the draft method used a 100 x 4.6 mm column with a 3.5 µm particle size, a 50 x 2.1 mm, 2.7 µm column was selected for the optimized method in order to speed up the analysis. The Raptor column is a superficially porous particle (SPP) column that provides high efficiency, faster run times, and lower back pressure compared to the 3.5 µm fully porous particle column used in the draft method. A Raptor C18 EXP guard column cartridge (cat.# 9304A0252) was also added to remove particulates and protect the analytical column.
Smaller ID columns allow for faster run times, and they reduce both back pressure and solvent consumption because lower flow rates are used. Even though a faster method on a narrow-bore column was employed here, early eluting matrix interferences were well resolved from the analyte of interest. Additionally, sample preparation using Resprep polymeric SPE cartridges resulted in very clean sample extracts, which further reduced the risk of matrix interferences.
Because smaller column dimensions were chosen, the injection volume was scaled down from 20 µL to 3 µL. Reducing the injection volume has several benefits: it reduces the amount of matrix being introduced onto the analytical column, which can improve chromatography, increase column lifetime, and reduce matrix effects, and it also preserves sample in case reanalysis is necessary.
During method optimization, we compared methanol and acetonitrile, both with 0.1% formic acid, as the organic mobile phase. The results were acceptable for both solvents; however, we selected acetonitrile because it increased sensitivity and improved peak shapes by avoiding solvent mismatch between the sample diluent and the starting mobile phase.
The elution gradient was also altered from the conditions described in Draft Method 1634 to reflect the use of a smaller ID column. The optimized column and gradient conditions provided adequate retention of 6PPD-quinone and prevented interference from early eluting matrix compounds. After 6PPD-quinone eluted, the gradient was run at 100%B from 3-4 minutes to flush any contaminants from the column and prevent carryover. The gradient then returned to the starting conditions, so the column was effectively re-equilibrated between samples. The developed method had a maximum back pressure of 220 bar, making it suitable for use on both HPLC and UHPLC systems.
Figure 2: 6PPD-Quinone in a Runoff Water Sample Analyzed Under Optimized LC-MS/MS Conditions
| Peaks | tR (min) | Conc. (ng/mL) | Precursor Ion | Product Ion 1 | Product Ion 2 | |
|---|---|---|---|---|---|---|
| 1. | 6PPD-Quinone (6PPD-Q) | 1.93 | 2.8 | 299.22 | 215.07 | 241.08 |
| 2. | 6PPD-Quinone-D5 (6PPD-Q-D5) | 1.93 | 1 | 304.28 | 220.08 | - |
| 3. | 6PPD-Quinone-13C6 (6PPD-Q-13C6) | 1.93 | 1 | 305.28 | 221.19 | - |
| Column | Raptor C18 (cat.# 9304A52) | ||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 50 mm x 2.1 mm ID | ||||||||||||||||||||||||
| Particle Size: | 2.7 µm | ||||||||||||||||||||||||
| Pore Size: | 90 Å | ||||||||||||||||||||||||
| Guard Column: | Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252) | ||||||||||||||||||||||||
| Temp.: | 40 °C | ||||||||||||||||||||||||
| Standard/Sample | |||||||||||||||||||||||||
| Diluent: | Acetonitrile | ||||||||||||||||||||||||
| Inj. Vol.: | 3 µL | ||||||||||||||||||||||||
| Mobile Phase | |||||||||||||||||||||||||
| A: | 0.1% Formic acid, water | ||||||||||||||||||||||||
| B: | 0.1% Formic acid, acetonitrile | ||||||||||||||||||||||||
| |||||||||||||||||||||||||
| Max Pressure: | 220 bar |
| Detector | Waters Xevo TQ-S |
|---|---|
| Ion Mode: | ESI+ |
| Instrument | Waters ACQUITY Premier |
| Sample Preparation | Runoff water was collected from roadway 322-East, located in State College, PA, U.S. One-half milliliter of an extracted internal standard (EIS) solution (20 ng/mL of 6PPD-Q-13C6) was added to 250 mL of sample. Resprep Polymeric SPE cartridges, HLB (cat.# 28264), and a Thermo AutoTrace PFAS instrument were used for the following sample preparation procedure (steps 1-6 were diverted to waste): 1. Wash each cartridge with 5 mL of acetonitrile. 2. Wash each cartridge with 5 mL of reagent water. 3. Add 5 mL of reagent water to each cartridge. 4. Add the sample to the SPE column (250 mL) at a flow rate of 10-15 mL/min until the entire sample has been loaded onto the column. 5. Rinse the sample bottle with 5 mL of 50:50 methanol:reagent water (v/v) and then pour onto the column reservoir. 6. Once the rinse has completely passed through the cartridge, dry the cartridge under nitrogen for at least 5 minutes. 7. Rinse the sample bottles with 5 mL of acetonitrile and collect the SPE eluent using 15 mL polypropylene tubes. Samples should be pulled through using a low vacuum such that the solvent exits the cartridge in a dropwise fashion. 8. Repeat step 7 with a second 4-5 mL aliquot of acetonitrile. The total volume collected should be ~9-10 mL. 9. Add 0.5 mL of the non-extracted internal standard (NIS) solution (20 ng/mL of 6PPD-Q-D5) to the sample extract and vortex. 10. Transfer an aliquot of the sample to a screw-thread polypropylene autosampler vial (cat.# 23243) and cap with a polypropylene screw-thread cap (cat.# 24486). |
Linearity
As shown in Figure 3, the optimized method produced good linearity across the 0.025–10 ng/mL calibration range with a %RSE = 3.77%, which was well within the method specification of <20%. RSE is evaluated here instead of r2 because Draft Method 1634 states: “The correlation coefficient, r, and the coefficient of determination, r2, are no longer considered appropriate metrics for linearity and shall not be used in conjunction with this method [1].”
Figure 3: Calibration Curve (Quadratic 1/X Fit; Ignore 0)
IPR
Accuracy and precision for IPR evaluation were determined by analyzing four 40 ng/L spike replicates, and the results are presented in Table IV. The average %recovery was 95.00%, and the %RSD was 4.00%. Draft Method 1634 specifies that recoveries must be within 70-130%, and the RSD must be <20%, so these results were well within the specifications given in the method.
Table IV: IPR Performance Data (40 ng/L spike [n = 4])
| Sample # | Recovery (ng/L) | Recovery (%) |
| 1 | 40.1 | 100% |
| 2 | 38.3 | 95.80% |
| 3 | 35.8 | 89.50% |
| 4 | 37.8 | 94.50% |
MDL and ML
To establish the MDL and ML, six replicate samples were extracted over three days in accordance with the draft method. The standard deviations of the spike and blank results were multiplied by the Student’s t-value of 3.143, and the higher of the results between the spikes and blanks was selected as the MDL. In Section 20.2, Draft Method 1634 states “ML may be established by multiplying the MDL (pooled or unpooled, as appropriate) by 3.18 and rounding the result to the number nearest to 1, 2, or 5 x 10n, where n is zero or an integer [1].” In our study, the MDL (unpooled) was 0.285 ng/L, resulting in an ML of 1 ng/L (Table V).
Table V: MDL and ML Values for 6-PPD-quinone in DI Water
| MDL (ng/L) | ML (ng/L) |
| 0.285 | 1 |
EIS and NIS
As shown in Table VI, the EIS average %recovery was 82.2% and the RSD was 5.6%. For the NIS, the average recovery was 117.8% with an RSD of 1.9%.
Table VI: Internal Standard Recoveries (n = 17)
| Compound | Avg. % Recovery | %RSD |
| EIS | 82.2% | 5.6% |
| NIS | 117.8% | 1.9% |
Runoff Water
The data shown in Table VII demonstrate that recoveries for fortified DI water and runoff water were within the specifications of the method (70-130%). In addition, the unfortified runoff water had ~30% higher response for 6PPD-quinone compared to unfortified DI water, indicating that 6-PPD-quinone is present in the location where the matrix sample was collected but at a level below the limit of quantitation.
Table VII: Matrix Recovery (40 ng/L spike)
| Matrix | %Recovery |
| DI water | 100.70% |
| Runoff water | 95.50% |
Conclusion
Draft EPA Method 1634 outlines the analysis of 6PPD-quinone in aqueous matrices, primarily stormwater and surface water, using LC/MS/MS. Since it is a performance-based method, changes can be made as long as method requirements are still met. This study focused on developing an optimized LC-MS/MS method to speed up analysis and allow labs to increase sample throughput. Following sample preparation and extraction with a Dionex AutoTrace 280 PFAS system in accordance with EPA Draft Method 1634 and employing an optimized LC-MS/MS method, the study achieved the following results. The calibration range was 0.025–10 ng/mL with a %RSE of 3.77%. The EIS (13C6-PPD-quinone) showed an average recovery of 82.2% (n = 17) with an RSD of 5.6%, while the NIS (D5-6-PPD-quinone) had an average recovery of 117.8% with an RSD of 1.9%. The method detection limit was determined to be 0.285 ng/L, corresponding to an ML of 1 ng/L. For an IPR spike level of 40 ng/L, the average recovery was 95.0% with an RSD of 4.0%. Matrix samples spiked at 40 ng/L achieved a recovery of 95.5%, while DI water recovery was 100.7%. All results met or exceeded the method’s defined specifications. In addition to the aforementioned results, the optimized HPLC method was able to reduce the total run time from 10 to 5.5 minutes, taking the elution time of 6PPD-quinone from 7.5 minutes to 1.9 minutes. The optimized method also reduced the injection volume and maintained a low back pressure, so it can be run on both HPLC and UHPLC instruments.
Acknowledgments
The authors thank Grayson Ritch, Diego López, and Colton Myers for their contributions to this work.
References
1. U.S. Environmental Protection Agency, Draft Method 1634, Determination of 6PPD-quinone in aqueous matrices using liquid chromatography with tandem mass spectrometry (LC MS/MS), December 2023. https://www.epa.gov/system/files/documents/2025-02/draft-method-1634_1-24-24_508.pdf