Automated extraction of acrylamide from instant coffee using Biotage® Extrahera™ HV-5000, according to EN 16618:2015.

Figure 1. Structures of analyte and internal standard.

Introduction

Acrylamide is a small polar highly water soluble molecule formed when carbohydrate rich foodstuffs are cooked at high temperatures.

Acrylamide was first discovered in foodstuffs in 2002 causing widespread alarm due to its classification as a probable carcinogen. By 2017, regulatory authorities established bench- mark levels and recommended that companies continue to monitor and minimise the acrylamide levels in heat processed foods. There has since been an increased demand for a reliable and accurate method for acrylamide detection.

This application note describes the extraction of acrylamide from instant coffee using ISOLUTE® Multimode and ISOLUTE® ENV+ SPE cartridges in a two step process according to EN16618:2015 ‘Food analysis — Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS)’.

The sample preparation procedure is automated using Biotage® Extrahera™ HV-5000 delivering clean extracts and analyte recov- eries greater than 90% with RSDs lower than 10%. Linearity of greater than 0.99 is achieved for the concentration range 5-500 ng/mL.

This application note includes optimized conditions for automated processing of the ISOLUTE Multimode and ENV+ cartridges. In addition, an alternative, manual processing conditions using the Biotage® PRESSURE+ 48 positive pressure manifold are included. Data generated using both systems is shown.

Analytes

Acrylamide

Internal standards

Acrylamide-13C3

Sample preparation procedure

Format:

Step 1: ISOLUTE® Multimode 1 g/6 mL cartridge (Tabless), p/n 904-0100-CG

Step 2: ISOLUTE® ENV+ 500 mg/6 mL (Tabless) cartridge, p/n 915-0050-CG

Automated and manual processing:

Automated processing used the Biotage® Extrahera™ HV-5000 system in the 24-position sample batch configuration. The appropriate sample, SPE cartridge and collection racks are detailed in the procedure. Processing conditions are included in the appendix.

Manual processing using Biotage® PRESSURE+ 48 positive pressure manifold: each step below was processed at 1.5 to 3 psi using the adjustable flow setting, providing a flow rate of ~30 drops per minute for all loading and elution steps. Drying steps were processed at 40 psi using the maximum flow setting.

Sample pre-treatment:

Coffee matrix was prepared by adding 250 mL of boiled tap water to 2 g of instant coffee powder. Coffee samples (2000 µL) were spiked with 200 ng internal standard solution.

Note: Internal standard solution consists of a 20 ng/µL aqueous solution. 10 µL of this was added to 2000 µL of whole coffee to give a 100 ng/mL spike concentration.

Step 1: ISOLUTE® multimode

The multifunctional SPE cartridge is used to trap unwanted interferences from the sample matrix, while allowing acrylamide to pass through unretained.

Extrahera HV-5000: Add coffee samples to test tubes (16 x 100 mm, p/n C40708), and place into sample rack (16 x 100 mm, 24 position p/n 414254SP). Place SPE cartridges into column rack (24 x 6 mL tabless p/n 413640SP).

Pressure+ 48: Place SPE cartridges in a 6 mL SPE column rack (p/n PPM-A48-6RCK) above a waste bin rack containing an insert (p/n PPM-A48-WSTRCK).

Conditioning:

Condition each cartridge with methanol (2 mL)

Equilibration:

Equilibrate each cartridge with deionized water (2 x 4 mL)

Sample loading:

Pressure+ 48: Replace the waste bin rack with a 16 x 100 mm collection rack (p/n PPM-A48-16100). Collect into test tubes (16 x 100 mm (p/n C40708)).

Extrahera HV-5000: Load instant coffee samples, controls or QCs spiked with internal standard (2 mL) and collect in test tubes (18 x 75 mm, p/n 414574) held in a collection rack
(18 x 75 mm, 24 positions, p/n 415492 ).

Wash:

Extrahera HV-5000: Wash through residual sample with deionized water (3 mL) and collect into the same test tube.

Pressure+ 48: Wash through residual sample with deionized water (3 mL) and collect into the same test tube.

Part 2: ISOLUTE® ENV+

The highly retentive SPE cartridge is used to trap and concentrate the acrylamide.

Extrahera HV-5000: Transfer the rack containing the extracts from step 1 into sample position.

Place SPE cartridges into column rack (24 x 6 mL tabless, p/n 413640SP).

Pressure+ 48: Place the cartridges in a 6 mL SPE column rack (p/n PPM-A48-6RCK) above a waste bin rack containing an insert (p/n PPM-A48-WSTRCK)

Conditioning:

Condition each cartridge with methanol (5 mL)

Equilibration:

Equilibrate each cartridge with DI water (5 mL)

Sample loading:

Load each cartridge with the combined volume of sample and wash from Step 1 above (5 mL).

Wash:

Elute interferences with deionized water (4 mL)

Dry:

Dry cartridges for 5 mins under positive pressure (40 psi)

Elution:

Extrahera HV-5000: Collect final extract into test tubes (18 x 75 mm, p/n 414574) held in collection rack (18 x 75 mm, 24 positions, p/n 415492).

Pressure+ 48: Replace the waste bin rack with a collection rack (16 x 100 mm, p/n PPM-A48-16100). Elute acrylamide with methanol:H2O (60:40, v/v, 2 mL) into test tubes (16 x 100 mm, p/n C40708)

Post elution & reconstitution:

Add 10 µL of ethylene glycol to each tube to prevent complete evaporation of analytes. Transfer elution solvent tubes to the evaporation system*. Dry the extract in a stream of air using a TurboVap® LV until only the ethylene glycol remains (see Table 1 for recommended evaporation settings). Evaporation for a batch of 24 samples is complete in ~70 minutes.

* If using alternative TurboVap® EH or TurboVap® P+ systems for evaporation, racks containing the final extracts for evapora- tion can be transferred directly from the processing system (Pressure +48 or Extrahera HV-5000 respectively) into the evaporator eliminating the need for sample transfer.

Table 1: TuboVap® LV conditions

Reconstitute evaporated samples with deionized water (500 µL). Vortex mix, transfer to 96-well 2 mL collection plate and then to the LC-MS/MS for analysis.

HPLC conditions

Instrument

Waters Acquity UPLC (Waters Assoc., Milford, MA, USA)

Column

Restek Allure Acrylamide 5 µm 50 mm (Thames Restek, Saunderton, UK)

Mobile phase

A – deionized water containing 0.1% Formic Acid
B – methanol containing 0.1% Formic Acid

Flow rate

0.3 mL/min

Injection volume

10 μL

Column temperature:

40 ° C

Sample temperature:

20 ° C

Table 2: UPLC gradient conditions

Curve 6: Linear Gradient

MS conditions

Instrument:

Quattro Premier XE triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis

Desolvation Temperature: 450 °C
Ion Source Temperature: 120 °C
Collision Cell Pressure: 3.33 e-3 mbar

Positive ions acquired in the multiple reaction monitoring (MRM) mode:

Table 3. MS conditions for target analytes in positive mode.

Results

High (> 90% total recovery) and reproducible (RSD < 10%) recoveries were achieved for all analytes using the method described in this application note with the ISOLUTE® Multimode and ISOLUTE® ENV+ cartridge formats processed using the Extrahera™ HV-5000.
Figure 2. shows analyte % recoveries from three sets of four samples spiked at low, medium and high concentrations (5, 50 and 200 ng/mL respectively) using the optimized ISOLUTE Multimode and ENV+ protocol described in this application note.

Calibration curves were generated for instant coffee spiked with acrylamide over the concentration range 5-500 ng/mL . Good linearity was observed delivering r2 values greater than 0.99. Figure 3 details linearity performance for each method of extraction.

Calibration curves

a)

b)

Figure 3. Calibration curves for acrylamide in instant coffee using (a) Extrahera™ HV-5000 and (b) PRESSURE +48 using the ISOLUTE® Multimode and ENV+ cartridges

Table 4. Analyte calibration curve r2 for Acrylamide extraction

Accuracy and precision

Accuracy and precision for the automated and manual processing procedures were investigated, see tables 5 and 6 below.

Table 5. Accuracy and precision using Extrahera HV-5000

Table 6. Accuracy and precision using Pressure+ 48

Throughput

For a batch size of 24 samples, automated and manual processing times were comparable:

Table 7. Processing times for each extraction method

Conclusion

ISOLUTE® Multimode and ENV+ solid phase extraction cartridges provided robust automated extraction of acrylamide from instant coffee samples using the 24 position Biotage® Extrahera™ HV-5000 automated sample preparation system.

Good, reproducible recoveries were achieved, with a processing time of approximately 70 minutes. Including ~70 minutes for concentration and reconstitution, total sample preparation time is ~140 mins for a batch of 24 samples.

Chemicals and reagents

• Methanol (LC-MS grade) was purchased from Rathburn Chemicals Ltd (Walkerburn, UK).
• Instant coffee used was Nescafé Azera Americano (Nestle, York, UK) (other instant coffees are available)
• All analyte standards and formic acid were purchased from Sigma- Aldrich Company Ltd. (Gillingham, UK).
• Deionized water used was 18.2 MOhm-cm, drawn daily from a Direct-Q5 water purifier.
• Internal standard (20 ng/mL) was prepared from a 1 mg/ mL stock solution by adding 20 µL to 980 µL of deionized water. 10 µL of this solution was then added to each sample.
• Elution solvent (methanol:H2O (60:40, v/v)) was made up by measuring out 60 mL of methanol and 40 mL of deionized water.

Additional information

Mobile phase preparation

• Deionized water containing 0.1% formic acid: Add 1 mL of formic acid to 1 L of H2O.
• Methanol containing 0.1% formic acid: Add 1 mL of formic acid to 1 L of methanol.

Ordering information

Appendix

Biotage® Extrahera™ HV-5000 settings

The method described in this application note was automated on the Biotage® Extrahera™ HV- 5000 Automated Sample Preparation Workstation using ISOLUTE® Multimode 1 g/6 mL and ISOLUTE® ENV+ 500 mg/6 mL SPE cartridges. Two separate Extrahera methods were created due to the 2 step method requiring transfer of the eluent from the clean-up (Multimode) stage to the load position of the concentration (ENV+) stage. This appendix contains the software settings required to configure Extrahera™ to run these methods. Screenshots may or may not match those here depending upon the instrument software version.

ISOLUTE® multimode method

                         Screenshot                                                         Settings

Solvent properties

                          Screenshot                                                         Settings

ISOLUTE® ENV+ method

                            Screenshot                                                         Settings

Solvent properties

                                   Screenshot                                                         Settings

Literature Number: AN986