The Analysis of Acrylamide Using an Aqueous Compatible ... · LC-MS/MS Detection Landon Wiest, Paul...

Acrylamide was extracted from potato chips/crisps, coffee, cocoa and babyfoods 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 mLacetonitrile. Internal standard was added at appropriate concentrations.Unbuffered QuEChERS salts were used for the extraction. The acetonitrile layerwas cleaned up using a PSA/MgSO4 dSPE. The cleaned up extract was blowndown and reconstituted in 1 mL water. The entire QuEChERS extraction processtook about an hour.

After about 1500 injections of QuEChERS extracted matrix, the instrumentrequired a lot of maintenance. System back pressure was elevated, despite theremoval of the columns. After replacing the tubing between the injector and thecolumn, back pressure returned to normal. Various leaks also occur throughoutthe system requiring replacement of plunger seals and rotor valves. The ESIsource also had become dirty resulting in decreased sensitivity. While QuEChERSwas a relatively fast way to process various matrices, it resulted in more timebeing spent on maintaining the LC-MS/MS.

The PGC column consistently lost retention, while the Allure Acrylamide columnremained consistent. Multiple matrices were processed using QuEChERSextractions (Figure 2) and semi-quantitative results that correlated well toreference standards where obtained.

Acrylamide was extracted from potato chips, and coffee matrices using waterand n-hexane (defatting) where appropriate. Internal standard was also added.After 60 min agitation and 20 min centrifugation, (3,600 xg, 10 °C) the extractswere cleaned up with a mixed-mode SPE cartridge, followed by a poly-DVB typeSPE cartridge.2 The final extract was eluted from the second SPE cartridge with 2mL 40/60 water/methanol that was subsequently blown down to remove themethanol. The final volume was around 0.5 mL. This extract was much cleanerthan the QuEChERS extract, but also required much more time to process.

Introduction

Acrylamide is formed when substances containing asparagine and aldehydesugars (i.e. glucose, fructose etc.) are roasted, fried or baked at temperaturesabove 120 °C. The foods with the most acrylamide are roasted coffee andstarchy foods such as potato chips, toasted bread, and cereal. Acrylamide is alsofound in drinking water that has been treated with polyacrylamide as aflocculating agent. Laboratory studies performed on mice show that high levelexposure can cause reproductive harm, neurological defects and cancer.Accordingly, many methods have been developed to determine the amount ofacrylamide present in foods, tobacco and water. Multiple sample preparationtechniques are effective at extracting acrylamide from matrix, but it is importantto determine how much time should be invested on sample preparation andwhat stress the sample will place on your analytical system. QuEChERS-basedmethods1 tend to be quicker than SPE-based methods, but typically have morematrix components co-extracted which can lead to worse detection levels andmore stress on your detector. SPE-based methods2-5, result in a cleaner sample,but take a lot of time to prepare. LC columns used to quantify acrylamide oftensuffer from irreproducibility and poor column lifetimes. This results in longerturnaround times, less instrument uptime, and poor data quality. The AllureAcrylamide column addresses these disadvantages. This silica-based, aqueouscompatible, reversed-phase column is part of a reproducible, retentive, androbust solution. The benefits of this column will be discussed showing examplesof acrylamide separation from difficult matrices such as coffee and potatoproducts. Particular emphasis on the role varying degrees of sample preparationwill be discussed.

QuEChERS Preparation

QuEChERS Lifetime Studies

Lifetime studies were performed using a QuEChERS coffee extract. A porousgraphitic carbon (PGC) column and an Allure Acrylamide column were firsttested under isocratic conditions. After only a few injections (fewer than 50),both columns were showing decreased retention times, decreased sensitivityand increased pressure. Efforts to restore these columns failed. A gradient flushwas required to maintain the integrity of the columns and preserve lifetime.After incorporating an organic flush into each injection, column lifetimeincreased greatly. Hundreds of injections were tested on each column(Figure 1).

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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 H2O

Mobile Phase B: 0.001% Formic Acid in Acetonitrile

Gradient:

Time (min) %B

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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 bedetermined by the user. Time will either be spent cleaning your LC-MS becauseof matrix contamination, or during the sample preparation process. The AllureAcrylamide 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, theAllure Acrylamide column was equilibrated/restored. These capabilities delivermultiple 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 forcurrent popular PGC columns that can suffer from injection-to-injectionreproducibility, 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 16618:20153. ISO 18862:2016

EN 16618:2015 Preparation

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Figure 2: Acrylamide in QuEChERS Extracted Samples on Allure Acrylamide

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To better preserve the columns and to increase instrument uptime, the EN16618 method was used and guard columns were added to increase thelifetimes of the analytical columns. Lifetime tests were also performed with thiscleaner sample preparation method (Figure 4).

The lifetime tests showed again that the Allure Acrylamide column had betterstability than the PGC column, even with a cleaner matrix. The PGC columnrequired a 7 min runtime for adequate re-equilibration, while the AllureAcrylamide column only required a 5.5 min runtime to fully re-equilibrate. Afterabout 400 injections on the PGC column, acrylamide retention fell below 1.7min, which is a method performance requirement in the EN method. The PGCguard column was replaced and brought the retention time up to 1.89 min, andfurther 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 ofthe carbon phase, which holds on to matrix more readily. The functionalizedsilica stationary phase, used in the Allure Acrylamide, is able to be restoredafter each injection, resulting in longer lifetimes. This phase also separates theacrylamide from potential isobaric interferences from the matrix, has excellentretention for acrylamide and good sensitivity (Figure 5).

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Figure 4: EN 16618 Lifetime Test of PGC and Allure Acrylamide Columns

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Figure 5: Acrylamide Extracted Using EN 16618 Method on Allure Acrylamide

4. J. Agric. Food Chem. 2003, 51, 7547-75545. J. Agric. Food Chem. 2004, 52, 1996-2002