Acrylamide was extracted from potato chips/crisps, coffee, cocoa and baby foods using a QuEChERS extraction technique. 1 For the QuEChERS extractions, 1 g matrix was combined with 10 mL water, 5 mL n-hexane (defatting) and 10 mL acetonitrile. Internal standard was added at appropriate concentrations. Unbuffered QuEChERS salts were used for the extraction. The acetonitrile layer was cleaned up using a PSA/MgSO 4 dSPE. The cleaned up extract was blown down and reconstituted in 1 mL water. The entire QuEChERS extraction process took about an hour. After about 1500 injections of QuEChERS extracted matrix, the instrument required a lot of maintenance. System back pressure was elevated, despite the removal of the columns. After replacing the tubing between the injector and the column, back pressure returned to normal. Various leaks also occur throughout the system requiring replacement of plunger seals and rotor valves. The ESI source also had become dirty resulting in decreased sensitivity. While QuEChERS was a relatively fast way to process various matrices, it resulted in more time being spent on maintaining the LC-MS/MS. The PGC column consistently lost retention, while the Allure Acrylamide column remained consistent. Multiple matrices were processed using QuEChERS extractions (Figure 2) and semi-quantitative results that correlated well to reference standards where obtained. Acrylamide was extracted from potato chips, and coffee matrices using water and n-hexane (defatting) where appropriate. Internal standard was also added. After 60 min agitation and 20 min centrifugation, (3,600 xg, 10 °C) the extracts were cleaned up with a mixed-mode SPE cartridge, followed by a poly-DVB type SPE cartridge. 2 The final extract was eluted from the second SPE cartridge with 2 mL 40/60 water/methanol that was subsequently blown down to remove the methanol. The final volume was around 0.5 mL. This extract was much cleaner than the QuEChERS extract, but also required much more time to process. Introduction Acrylamide is formed when substances containing asparagine and aldehyde sugars (i.e. glucose, fructose etc.) are roasted, fried or baked at temperatures above 120 °C. The foods with the most acrylamide are roasted coffee and starchy foods such as potato chips, toasted bread, and cereal. Acrylamide is also found in drinking water that has been treated with polyacrylamide as a flocculating agent. Laboratory studies performed on mice show that high level exposure can cause reproductive harm, neurological defects and cancer. Accordingly, many methods have been developed to determine the amount of acrylamide present in foods, tobacco and water. Multiple sample preparation techniques are effective at extracting acrylamide from matrix, but it is important to determine how much time should be invested on sample preparation and what stress the sample will place on your analytical system. QuEChERS-based methods 1 tend to be quicker than SPE-based methods, but typically have more matrix components co-extracted which can lead to worse detection levels and more stress on your detector. SPE-based methods 2-5 , result in a cleaner sample, but take a lot of time to prepare. LC columns used to quantify acrylamide often suffer from irreproducibility and poor column lifetimes. This results in longer turnaround times, less instrument uptime, and poor data quality. The Allure Acrylamide column addresses these disadvantages. This silica-based, aqueous compatible, reversed-phase column is part of a reproducible, retentive, and robust solution. The benefits of this column will be discussed showing examples of acrylamide separation from difficult matrices such as coffee and potato products. Particular emphasis on the role varying degrees of sample preparation will be discussed. QuEChERS Preparation QuEChERS Lifetime Studies Lifetime studies were performed using a QuEChERS coffee extract. A porous graphitic carbon (PGC) column and an Allure Acrylamide column were first tested under isocratic conditions. After only a few injections (fewer than 50), both columns were showing decreased retention times, decreased sensitivity and increased pressure. Efforts to restore these columns failed. A gradient flush was required to maintain the integrity of the columns and preserve lifetime. After incorporating an organic flush into each injection, column lifetime increased greatly. Hundreds of injections were tested on each column (Figure 1). PATENTS & TRADEMARKS Restek patents and trademarks are the property of Restek Corporation. (See www .restek.com/Patents-Trademarks for full list.) Other trademarks in Restek literature or on its website are the property of their respective owners. Restek registered trademarks are registered in the U.S. and may also be registered in other countries. The Analysis of Acrylamide Using an Aqueous Compatible Reversed-Phase Column by LC-MS/MS Detection Landon Wiest, Paul Connolly, Joe Konschnik; Restek Corporation Column: Allure Acrylamide 5 μm, 50 mm x 2.1 mm (Cat.# 9167552) Trident Guard: Allure Acrylamide 5 μm, 10 mm x 2.1 mm (Cat.# 916750212) Pore Size: 60 Å Instrument: Shimadzu LCMS-8045 Mobile Phase A: 0.001% Formic Acid in H 2 O Mobile Phase B: 0.001% Formic Acid in Acetonitrile Gradient: Time (min) %B 0.00 0 1.00 0 2.00 90 2.01 0 5.50 0 Flow Rate: 0.4 mL/min Column Temp.: 22 °C Ion Mode: ESI+ Diluent: Water Injection Volume: 10 μL Table 1: LC Method Conditions for Acrylamide Analysis EN 16618 Lifetime Studies Conclusions The choice between QuEChERS cleanup and the EN 16618 method must be determined by the user. Time will either be spent cleaning your LC-MS because of matrix contamination, or during the sample preparation process. The Allure Acrylamide column performed well separating Acrylamide from both dirty (QuEChERS) and clean (EN 16618) extracts. Over a 1000 injection lifetime test, the retention time was consistent, showing that after a brief gradient flush, the Allure Acrylamide column was equilibrated/restored. These capabilities deliver multiple benefits to the customer: More instrument uptime Better data quality Faster turnaround time The Allure Acrylamide column is capable of being a drop-in replacement for current popular PGC columns that can suffer from injection-to-injection reproducibility, require longer run times and have shorter column lifetimes. Figure 1: QuEChERS Extract Lifetime Test of PGC and Allure Acrylamide Columns References: 1. J. Agric. Food Chem. 2006, 54, 7001-7008 2. EN 16618:2015 3. ISO 18862:2016 EN 16618:2015 Preparation 0% 20% 40% 60% 80% 100% 120% 0 100 200 300 400 500 600 700 % Loss (Retention) PGC-Coffee Extract (QuEChERS) 0% 20% 40% 60% 80% 100% 120% 0 100 200 300 400 500 600 700 % loss (Retention) Injections Allure Acrylamide-Coffee Extract (QuEChERS) Figure 2: Acrylamide in QuEChERS Extracted Samples on Allure Acrylamide 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min 0.0 1.0 2.0 3.0 4.0 5.0 (x10,000) Potato Chips/Crisps 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 2500 5000 7500 10000 12500 15000 17500 20000 1:221.20>85.10(+) 1:72.20>55.10(+) 1:221.20>85.10(+) 1:72.20>55.10(+) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 2500 5000 7500 10000 12500 15000 17500 2:75.20>58.15(+) 2:75.20>58.15(+) Acrylamide Acrylamide-d3 Instant Coffee To better preserve the columns and to increase instrument uptime, the EN 16618 method was used and guard columns were added to increase the lifetimes of the analytical columns. Lifetime tests were also performed with this cleaner sample preparation method (Figure 4). The lifetime tests showed again that the Allure Acrylamide column had better stability than the PGC column, even with a cleaner matrix. The PGC column required a 7 min runtime for adequate re-equilibration, while the Allure Acrylamide column only required a 5.5 min runtime to fully re-equilibrate. After about 400 injections on the PGC column, acrylamide retention fell below 1.7 min, which is a method performance requirement in the EN method. The PGC guard column was replaced and brought the retention time up to 1.89 min, and further flushing with 90% B for 0.5 h increased the retention time to 1.92 min. The loss in retention on the PGC column is likely due to the broad selectivity of the carbon phase, which holds on to matrix more readily. The functionalized silica stationary phase, used in the Allure Acrylamide, is able to be restored after each injection, resulting in longer lifetimes. This phase also separates the acrylamide from potential isobaric interferences from the matrix, has excellent retention for acrylamide and good sensitivity (Figure 5). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min 0 50000 100000 150000 200000 250000 300000 350000 400000 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 0 0.5 1 1.5 2 0 200 400 600 800 1000 Injections Allure Acrylamide- Coffee Extract (EN 16618) Figure 4: EN 16618 Lifetime Test of PGC and Allure Acrylamide Columns 1000th Injection 1st Injection 0 0.5 1 1.5 2 2.5 0 200 400 600 800 1000 Retention Time (min) Injections PGC-Coffee Extract (EN 16618) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min 0 50000 100000 150000 200000 250000 300000 350000 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 2:Acrylamide-d3 TIC(+) 1:Acrylamide TIC(+) 1st Injection 500th Injection Potato Chips/Crisps 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min 0 50000 100000 150000 200000 250000 300000 350000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min 0 50000 100000 150000 200000 250000 300000 350000 Ground Coffee Figure 5: Acrylamide Extracted Using EN 16618 Method on Allure Acrylamide 4. J. Agric. Food Chem. 2003, 51, 7547-7554 5. J. Agric. Food Chem. 2004, 52, 1996-2002