{"id":45282,"date":"2025-10-08T17:56:25","date_gmt":"2025-10-08T17:56:25","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/optimizing-epa-method-1634-for-6ppd-quinone-analysis\/"},"modified":"2025-12-12T19:46:17","modified_gmt":"2025-12-12T19:46:17","slug":"optimizing-epa-method-1634-for-6ppd-quinone-analysis","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/it\/application-notes\/evan4446\/optimizing-epa-method-1634-for-6ppd-quinone-analysis","title":{"rendered":"Optimizing EPA Method 1634 for 6PPD-Quinone Analysis"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Abstract<\/h2>\n\n\n\n<p>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.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Introduction<\/h2>\n\n\n\n<p>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\u2019-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.<\/p>\n\n\n\n<p>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.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Experimental<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Calibration Standards<\/h3>\n\n\n\n<p>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.<\/p>\n\n\n\n<p><strong>Table I:<\/strong> Calibration Standard Concentrations<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table>\n<tbody>\n<tr style=\"background-color: #008eaa;\">\n<td>\u00a0<\/td>\n<td style=\"color: #ffffff;\" colspan=\"7\"><strong>Calibration Standard Concentrations (ng\/mL)<\/strong><\/td>\n<\/tr>\n<tr style=\"background-color: #008eaa;\">\n<td style=\"color: #ffffff;\"><strong>Compound and Intermediate Standard Concentration<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C1<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C2<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C3<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C4<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C5<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C6<\/strong><\/td>\n<td style=\"color: #ffffff;\"><strong>C7<\/strong><\/td>\n<\/tr>\n<tr>\n<td>6PPD-quinone (20 ng\/mL)<\/td>\n<td>0.025<\/td>\n<td>0.050<\/td>\n<td>0.10<\/td>\n<td>0.50<\/td>\n<td>1.0<\/td>\n<td>5.0<\/td>\n<td>10.0<\/td>\n<\/tr>\n<tr>\n<td><sup>13<\/sup>C<sub>6<\/sub>-6PPD-quinone (20 ng\/mL)<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<\/tr>\n<tr>\n<td>D5-6PPD-quinone (20 ng\/mL)<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<td>1.0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<h3 class=\"wp-block-heading\">QC Sample Preparation<\/h3>\n\n\n\n<p>Samples for precision, accuracy, and method detection limit determination were prepared in polypropylene bottles using 250 mL of deionized water spiked with 50 \u00b5L 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 \u00b5L 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.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Water Samples<\/h3>\n\n\n\n<p>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 \u00b0C. All samples were extracted within 14 days of collection. EIS was spiked prior to sample extraction, and NIS was added post-extraction.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Sample Extraction<\/h3>\n\n\n\n<p>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 \u00b5m 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 \u00b5L of the non-extracted internal standards (NIS, Cambridge Isotope Laboratory Inc., cat.# DLM-11616-1.2).<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 1:<\/strong> Sample Preparation Steps (Steps 1-6 are diverted to waste.)<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image45282_59123b-44 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image45282_59123b-44\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"755\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01-1024x755.jpg\" alt=\"\" class=\"kb-img wp-image-19486\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01-1024x755.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01-300x221.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01-768x567.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01-1536x1133.jpg 1536w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-01.jpg 1800w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<p><\/p>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Instrument Method<\/h3>\n\n\n\n<p>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.<\/p>\n\n\n\n<p><strong>Table II:<\/strong> Draft Method 1634 Suggested HPLC Conditions (Section 10.3.1)<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><tbody><tr><td>Column<\/td><td colspan=\"3\">C18 phase 4.6 x 100 mm, 3.5 \u00b5m<\/td><\/tr><tr><td>Mobile phase A<\/td><td colspan=\"3\">0.2% Formic acid in water<\/td><\/tr><tr><td>Mobile phase B<\/td><td colspan=\"3\">Acetonitrile<\/td><\/tr><tr><td>Column temperature<\/td><td colspan=\"3\">45 \u00b0C<\/td><\/tr><tr><td>Injection volume<\/td><td colspan=\"3\">20 \u00b5L<\/td><\/tr><tr><td>Flow rate<\/td><td colspan=\"3\">0.6 mL\/min<\/td><\/tr><tr><td>Maximum pressure<\/td><td colspan=\"3\">7500 psi (517 bar)<\/td><\/tr><tr><td>Gradient<\/td><td>Time (min)<\/td><td>%A<\/td><td>%B<\/td><\/tr><tr><td>&nbsp;<\/td><td>0<\/td><td>90<\/td><td>10<\/td><\/tr><tr><td>&nbsp;<\/td><td>1<\/td><td>90<\/td><td>10<\/td><\/tr><tr><td>&nbsp;<\/td><td>3<\/td><td>45<\/td><td>55<\/td><\/tr><tr><td>&nbsp;<\/td><td>6<\/td><td>1<\/td><td>99<\/td><\/tr><tr><td>&nbsp;<\/td><td>8<\/td><td>1<\/td><td>99<\/td><\/tr><tr><td>&nbsp;<\/td><td>8.5<\/td><td>90<\/td><td>10<\/td><\/tr><tr><td>&nbsp;<\/td><td>9<\/td><td>90<\/td><td>10<\/td><\/tr><tr><td>Expected 6PPD-quinone elution time<\/td><td colspan=\"3\">7.53 min<\/td><\/tr><tr><td>Total run time<\/td><td colspan=\"3\">10.0 min<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p><strong>Table III: <\/strong>Optimized LC Method Conditions<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><tbody><tr><td>Column<\/td><td colspan=\"3\">Raptor C18 50 x 2.1 mm, 2.7 \u00b5m (cat.# 9304A52)<\/td><\/tr><tr><td>Guard column<\/td><td colspan=\"3\">Raptor C18 EXP guard column cartridge 5 x 2.1 mm, 2.7 \u00b5m (cat.# 9304A0252)<\/td><\/tr><tr><td>Mobile phase A<\/td><td colspan=\"3\">0.1% Formic acid in water<\/td><\/tr><tr><td>Mobile phase B<\/td><td colspan=\"3\">0.1% Formic acid in acetonitrile<\/td><\/tr><tr><td>Diluent<\/td><td colspan=\"3\">Acetonitrile<\/td><\/tr><tr><td>Column temperature<\/td><td colspan=\"3\">40 \u00b0C<\/td><\/tr><tr><td>Injection volume<\/td><td colspan=\"3\">3 \u00b5L<\/td><\/tr><tr><td>Flow rate<\/td><td colspan=\"3\">0.5 mL\/min<\/td><\/tr><tr><td>Maximum pressure<\/td><td colspan=\"3\">3200 psi (220 bar)<\/td><\/tr><tr><td>Gradient<\/td><td>Time (min)<\/td><td>%A<\/td><td>%B<\/td><\/tr><tr><td>&nbsp;<\/td><td>0<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>&nbsp;<\/td><td>3<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>&nbsp;<\/td><td>4<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>&nbsp;<\/td><td>4.01<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>&nbsp;<\/td><td>5.5<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>Expected 6PPD-quinone elution time<\/td><td colspan=\"3\">1.93 min<\/td><\/tr><tr><td>Total run time<\/td><td colspan=\"3\">5.5 min<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\">Results and Discussion<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">LC-MS\/MS Method Optimization and Chromatographic Performance<\/h3>\n\n\n\n<p>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.<\/p>\n\n\n\n<p>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 \u00b5m particle size, a 50 x 2.1 mm, 2.7 \u00b5m 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 \u00b5m fully porous particle column used in the draft method. A Raptor C18 EXP guard column cartridge (cat.#\u00a09304A0252) was also added to remove particulates and protect the analytical column.<\/p>\n\n\n\n<p>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.<\/p>\n\n\n\n<p>Because smaller column dimensions were chosen, the injection volume was scaled down from 20 \u00b5L to 3 \u00b5L. 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.<\/p>\n\n\n\n<p>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.<\/p>\n\n\n\n<p>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.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 2:<\/strong> 6PPD-Quinone in a Runoff Water Sample Analyzed Under Optimized LC-MS\/MS Conditions<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image regular-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_ev0600.png\" alt=\"6PPD-Quinone in Runoff Water on Raptor C18 by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_EV0600<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-12\">\n<thead><tr><th><\/th><th style=\"text-align: left;width: 75px\">Peaks<\/th><th style=\"text-align: center;width: 75px\">t<sub>R<\/sub> (min)<\/th><th style=\"text-align: center;width: 75px\">Conc.<br \/>(ng\/mL)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion 1<\/th><th style=\"text-align: center;width: 75px\">Product Ion 2<\/th><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for 6PPD-Quinone (6PPD-Q)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/2754428-18-5\/6PPD-Quinone\" target=\"_blank\" rel=\"noopener\">6PPD-Quinone (6PPD-Q)<\/a><\/td><td class=\"oth\">1.93<\/td><td class=\"oth\">2.8<\/td><td class=\"oth\">299.22<\/td><td class=\"oth\">215.07<\/td><td class=\"oth\">241.08<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\">6PPD-Quinone-D5 (6PPD-Q-D5)<\/td><td class=\"oth\">1.93<\/td><td class=\"oth\">1<\/td><td class=\"oth\">304.28<\/td><td class=\"oth\">220.08<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\">6PPD-Quinone-<sup>13<\/sup>C<sub>6<\/sub> (6PPD-Q-<sup>13<\/sup>C<sub>6<\/sub>)<\/td><td class=\"oth\">1.93<\/td><td class=\"oth\">1<\/td><td class=\"oth\">305.28<\/td><td class=\"oth\">221.19<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<\/tbody><\/table><div class=\"peaknotes\">The concentration listed is the final concentration following sample preparation.<\/div><\/div><div class=\"chromatogram-conditions\"><h4>Conditions<\/h4><div class=\"conditions-container container-fluid\"><div class=\"row\"><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Column<\/th><td>Raptor C18  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9304A52?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_EV0600\" rel=\"noopener\">cat.# 9304A52<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>50 mm x 2.1 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>2.7 \u00b5m<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Pore Size:<\/th><td>90 \u00c5<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Guard Column:<\/th><td>Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9304A0252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_EV0600\" rel=\"noopener\">cat.# 9304A0252<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>40 \u00b0C<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Standard\/Sample<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Diluent:<\/th><td>Acetonitrile<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>3 \u00b5L <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Mobile Phase<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">A:<\/th><td>0.1% Formic acid, water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>0.1% Formic acid, acetonitrile <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%A<\/th><th>%B<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.5<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>3.00<\/td><td>0.5<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>4.00<\/td><td>0.5<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>4.01<\/td><td>0.5<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>5.50<\/td><td>0.5<\/td><td>70<\/td><td>30<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>220 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>Waters Xevo TQ-S<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Ion Mode:<\/th><td>ESI+ <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>Waters ACQUITY Premier<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>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-<sup>13<\/sup>C<sub>6<\/sub>) was added to 250 mL of sample. Resprep Polymeric SPE cartridges, HLB (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/28264?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_EV0600\" rel=\"noopener\">cat.# 28264<\/a>), and a Thermo AutoTrace PFAS instrument were used for the following sample preparation procedure (steps 1-6 were diverted to waste):<br \/>1. Wash each cartridge with 5 mL of acetonitrile.<br \/>2. Wash each cartridge with 5 mL of reagent water. <br \/>3. Add 5 mL of reagent water to each cartridge. <br \/>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. <br \/>5. Rinse the sample bottle with 5 mL of 50:50 methanol:reagent water (v\/v) and then pour onto the column reservoir.<br \/>6. Once the rinse has completely passed through the cartridge, dry the cartridge under nitrogen for at least 5 minutes. <br \/>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. <br \/>8. Repeat step 7 with a second 4-5 mL aliquot of acetonitrile. The total volume collected should be ~9-10 mL. <br \/>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.<br \/>10. Transfer an aliquot of the sample to a screw-thread polypropylene autosampler vial (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/23243?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_EV0600\" rel=\"noopener\">cat.# 23243<\/a>) and cap with a polypropylene screw-thread cap (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/24486?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_EV0600\" rel=\"noopener\">cat.# 24486<\/a>). <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_ev0600.pdf\" target=\"_blank\" rel=\"noopener noreferrer\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"18\" height=\"18\" viewBox=\"0 0 18 18\"><g data-name=\"Group 2996\"><path data-name=\"Rectangle 1246\" d=\"M0 0h18v18H0z\" style=\"fill: none;\"><\/path><\/g><g data-name=\"Group 2997\"><path data-name=\"Path 729\" d=\"M13.412 11.4v2.017H5.345V11.4H4v2.017a1.349 1.349 0 0 0 1.345 1.345h8.068a1.349 1.349 0 0 0 1.345-1.345V11.4zm-.672-2.694-.948-.948-1.741 1.735V4H8.706v5.493L6.965 7.758l-.948.948 3.361 3.361z\" transform=\"translate(-.437 -.414)\" style=\"fill: rgb(13, 123, 196);\"><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n<p><\/p>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Linearity<\/h3>\n\n\n\n<p>As shown in Figure 3, the optimized method produced good linearity across the 0.025\u201310 ng\/mL calibration range with a %RSE = 3.77%, which was well within the method specification of &lt;20%. RSE is evaluated here instead of r<sup>2<\/sup> because Draft Method 1634 states: \u201cThe correlation coefficient, r, and the coefficient of determination, r<sup>2<\/sup>, are no longer considered appropriate metrics for linearity and shall not be used in conjunction with this method [1].\u201d<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 3:<\/strong> Calibration Curve (Quadratic 1\/X Fit; Ignore 0)<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image45282_ab3817-9f .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image45282_ab3817-9f\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"600\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03-1024x600.jpg\" alt=\"\" class=\"kb-img wp-image-19492\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03-1024x600.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03-300x176.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03-768x450.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03-1536x900.jpg 1536w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-evan4446-03.jpg 1800w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<p><\/p>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">IPR<\/h3>\n\n\n\n<p>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 &lt;20%, so these results were well within the specifications given in the method.<\/p>\n\n\n\n<p><strong>Table IV:<\/strong> IPR Performance Data (40 ng\/L spike [n = 4])<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table style=\"border-color: #ced4d9;\">\n<tbody>\n<tr style=\"background-color: #008eaa; color: #ffffff;\">\n<td><strong>Sample #<\/strong><\/td>\n<td><strong>Recovery (ng\/L)<\/strong><\/td>\n<td><strong>Recovery (%)<\/strong><\/td>\n<\/tr>\n<tr>\n<td>1<\/td>\n<td>40.1<\/td>\n<td>100%<\/td>\n<\/tr>\n<tr>\n<td>2<\/td>\n<td>38.3<\/td>\n<td>95.80%<\/td>\n<\/tr>\n<tr>\n<td>3<\/td>\n<td>35.8<\/td>\n<td>89.50%<\/td>\n<\/tr>\n<tr>\n<td>4<\/td>\n<td>37.8<\/td>\n<td>94.50%<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">MDL and ML<\/h3>\n\n\n\n<p>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\u2019s 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 \u201cML 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].\u201d In our study, the MDL (unpooled) was 0.285 ng\/L, resulting in an ML of 1 ng\/L (Table V).<\/p>\n\n\n\n<p><strong>Table V:<\/strong> MDL and ML Values for 6-PPD-quinone in DI Water<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table class=\"has-fixed-layout\" style=\"border-color: #ced4d9;\">\n<tbody>\n<tr style=\"background-color: #008eaa; color: #ffffff;\">\n<td><strong>MDL (ng\/L)<\/strong><\/td>\n<td><strong>ML (ng\/L)<\/strong><\/td>\n<\/tr>\n<tr>\n<td>0.285<\/td>\n<td>1<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">EIS and NIS<\/h3>\n\n\n\n<p>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%.<\/p>\n\n\n\n<p><strong>Table VI:<\/strong> Internal Standard Recoveries (n = 17)<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table style=\"border-color: #ced4d9;\">\n<tbody>\n<tr style=\"background-color: #008eaa; color: #ffffff;\">\n<td><strong>Compound<\/strong><\/td>\n<td><strong>Avg. % Recovery<\/strong><\/td>\n<td><strong>%RSD<\/strong><\/td>\n<\/tr>\n<tr>\n<td>EIS<\/td>\n<td>82.2%<\/td>\n<td>5.6%<\/td>\n<\/tr>\n<tr>\n<td>NIS<\/td>\n<td>117.8%<\/td>\n<td>1.9%<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Runoff Water<\/h3>\n\n\n\n<p>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.<\/p>\n\n\n\n<p><strong>Table VII:<\/strong> Matrix Recovery (40 ng\/L spike)<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table style=\"border-color: #ced4d9;\">\n<tbody>\n<tr style=\"background-color: #008eaa; color: #ffffff;\">\n<td><strong>Matrix<\/strong><\/td>\n<td><strong>%Recovery<\/strong><\/td>\n<\/tr>\n<tr>\n<td>DI water<\/td>\n<td>100.70%<\/td>\n<\/tr>\n<tr>\n<td>Runoff water<\/td>\n<td>95.50%<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Conclusion<\/h2>\n\n\n\n<p>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\u201310 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\u2019s 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.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Acknowledgments<\/h2>\n\n\n\n<p>The authors thank Grayson Ritch, Diego L\u00f3pez, and Colton Myers for their contributions to this work.<\/p>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">References<\/h2>\n\n\n\n<p>1. &nbsp; &nbsp;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.&nbsp;<a href=\"https:\/\/www.epa.gov\/system\/files\/documents\/2025-02\/draft-method-1634_1-24-24_508.pdf\" target=\"_blank\" rel=\"noreferrer noopener\">https:\/\/www.epa.gov\/system\/files\/documents\/2025-02\/draft-method-1634_1-24-24_508.pdf<\/a><\/p>\n\n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n        <div class=\"cpb\">\n            <h3 class=\"cpb-heading\">Products Mentioned<\/h3>\n            <hr class=\"cpb-heading-underline\" \/>\n            <div class=\"cpb-list\">\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304A0252\">                                Catalog No. 9304A0252                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Cartuccia per precolonna Raptor C18 EXP, 2,7 \u00b5m, 5 x 2,1 mm, 3 pz.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304A0252\">Esplora Prodotto<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304A52\">                                Catalog No. 9304A52                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Colonna HPLC Raptor C18, 2,7 \u00b5m, 50 x 2,1 mm<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304A52\">Esplora Prodotto<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/28264\">                                Catalog No. 28264                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Cartuccia SPE Resprep Polimerica, HLB, 6 mL\/200 mg, 60 \u00b5m, 30-conf.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/28264\">Esplora Prodotto<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/25808\">                                Catalog No. 25808                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Holder EXP a connessione diretta per cartucce per precolonne EXP, comprende raccordo e ferrule<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/25808\">Esplora Prodotto<\/a>\n                                                    <\/div>\n                    <\/div>\n                            <\/div>\n        <\/div>\n        \n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>Based on EPA Method 1634, this optimized LC-MS\/MS analysis of 6PPD-quinone reduced run times from 10 to 5.5 minutes while still meeting all method requirements.<\/p>\n","protected":false},"author":36,"featured_media":6583,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kadence_starter_templates_imported_post":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[13],"tags":[],"industries-application":[2161],"post-badge":[],"resource-type":[],"product-library":[2391,2373],"resource-technique":[2299,2302],"hf_cat_post":[651],"ppma_author":[456,447,432],"class_list":["post-45282","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-application-notes","industries-application-environmental","product-library-lc-columns","product-library-liquid-chromatography-products","resource-technique-liquid-chromatography","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":13,"label":"Application 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