Food Chemistry 176 (2015) 342–349

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Analytical Methods

Determination of acrylamide in Sudanese food by high performance liquid chromatography coupled with LTQ Orbitrap mass spectrometry Mei Musa Ali Omar a,b,c, Abdalla Ahmed Elbashir a,b,⇑, Oliver J. Schmitz a,⇑ a

Applied Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany Department of Chemistry, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan c Central Laboratory, Ministry of Sciences & Technology, P.O. Box Office 7099, Khartoum, Sudan b

a r t i c l e

i n f o

Article history: Received 12 August 2014 Received in revised form 20 December 2014 Accepted 20 December 2014 Available online 30 December 2014 Keywords: QuEChERS Acrylamide Dispersive solid phase extraction HPLC LTQ-Orbitrap MS

a b s t r a c t A sample preparation method based on modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) with aluminum oxide (Al2O3) as dispersive solid phase extraction (dSPE) material and high performance liquid chromatography–linear trap quadruple-Orbitrap-mass spectrometry (HPLC LTQ-Orbitrap MS) was established. The performance of two analytical columns namely Kinetex C18 and Rezex ROA-organic acid was compared for acrylamide separation. The method was validated in term of matrix effect, linear range (standard addition method), limit of detection (LOD), limit of quantification (LOQ), precision (RSD%) and recovery. Good linearity (r2 > 0.9979) was achieved using standard addition method in the concentration range 0–200 lg kg 1. The LOD is in the range from 2.91 to 4.04 lg kg 1 and 1.50 to 3.94 lg kg 1 for C18 and ROA columns, respectively. The precision of the method was 67.3% and 5.6% for C18 and ROA columns, respectively. Recoveries of acrylamide ranging from 90% to 97%, (n = 3) were obtained. The proposed Al2O3 dSPE method was successfully applied to the analysis of acrylamide in real food samples. Ó 2015 Published by Elsevier Ltd.

1. Introduction Acrylamide (2-propenamide) is considered as the most actively investigated compound among heat-induced food contaminants (Krska et al., 2012; Wenzl, Lachenmeier, & Gökmen, 2007). Acrylamide naturally forms as a byproduct of cooking process in carbohydrate-rich foods at high temperatures and low moist conditions (Franek, Rubio, Diblikova, & Rubio, 2014; Tareke, Rydberg, Karlsson, Eriksson, & Törnqvist, 2002). Maillard reaction of reducing sugars with asparagine at temperature higher than 120 °C is the most probable route to acrylamide formation during the browning process (Yaylayan & Stadler, 2005; Özer, Kola, Altan, Duran, & Zorlugenç, 2012). Acrylamide is a neurotoxic compound identified as a probable human carcinogen (group 2A) and genotoxicant (IARC, 1994; Liu, Zhao, Yuan, Chen, & Hu, 2008). Acrylamide is a low molecular weight, polar, low volatile and hydrophilic a, b unsaturated amide, which makes it difficult to analyze it using classical analytical techniques, particularly in complex matrixes (DeArmond & DiGoregorio, 2013; Krska et al., 2012; Notardonato, Avino, Centola, Cinelli, & Russo, 2013; Paleologos & Kontominas, 2005). ⇑ Corresponding authors at: Department of Chemistry, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan (A.A. Elbashir). E-mail addresses: [email protected], [email protected] (A.A. Elbashir), [email protected] (O.J. Schmitz). http://dx.doi.org/10.1016/j.foodchem.2014.12.091 0308-8146/Ó 2015 Published by Elsevier Ltd.

Extraction and cleanup of acrylamide from food matrices is a critical factor for analyte recovery (Elbashir, Omar, Ibrahim, Schmitz, & Aboul-Enein, 2014). Due to the high polarity of acrylamide water is a highly efficient extraction solvent for acrylamide from different food matrices. Because of the low selectivity of water as extractant, tedious and time-consuming clean-up procedures are needed to partially isolate the analyte from other matrix components. Mostly clean-up procedures consist of the combination of two different solid-phase extractions (Fernandes & Soares, 2007; Yusà, Quintas, Pardo, Martí, & Pastor, 2006). The fast analysis of acrylamide in food matrixes became more important when a huge number of food samples need to be investigated. In 2006, the QuEChERS method was introduced by Mastovska and Lehotay as a high throughput extraction method for acrylamide analysis in various food matrices using LC–MS/MS or GC–MS. It involves miniaturized extraction with acetonitrile, liquid–liquid partition by salting out with sodium chloride and magnesium sulfate and clean-up the extract using dispersive solid-phase extraction (dSPE) step with loose sorbent such as primary second_ zelewicz, _ ary amine (PSA) (Oracz, Nebesny, & Zy 2011). Modified QuEChERS cleanup steps with sol–gel hybrid methyltrimethoxysilane–tetraethoxysilane (MTMOS–TEOS) and aluminum oxide as sorbents followed by GC–MS and HPLC–MS respectively were also reported for acrylamide analysis in food matrices (Forstova et al., 2014; Omar, Wan Ibrahim, & Elbashir, 2014). The use of aluminum oxide as dSPE material for the clean-up of acrylamide in food was

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used for only one time and it was not optimized (Forstova et al., 2014). Analytical methods based on liquid or gas chromatography coupled to mass spectrometry (LC–MS/MS or LC–MS, GC–MS) have been used for the determination of acrylamide contents in food samples (Fernandes & Soares, 2007; Hoenicke & Gatermann, 2005; Kaplan, Kaya, Ozcan, Ince, & Yaman, 2009). For GC, a tedious procedure of derivatization is usually required, and the high injection temperature may result in acrylamide production from any co-extracted acrylamide precursors, and thus false results are obtained (Longhua, Limin, Xuguang, Zhixiang, & Jiaming, 2012). The main advantage of the LC–MS/MS based methods is that acrylamide can be analyzed directly without prior derivatization, which considerably simplifies and expedites the analysis (Krska et al., 2012; Riediker & Stadler, 2003). To maintain sample throughput and cost-effectiveness ratio, the development of generic liquid chromatography or ultra-high performance liquid chromatography (UHPLC) coupled to mass spectrometry (MS) screening methods is highly demanded (Gómez-Pérez, Plaza-Bolaños, Romero-González, Martínez-Vidal, & Garrido-Frenich, 2012). The major advantages of UHPLC over classical HPLC utilizing columns packed with 5.0 lm particles include improved resolution within a shorter retention time and higher analytical sensitivity (Zhang, Jiao, Cai, Zhang, & Ren, 2007). Due to the low molecular mass of acrylamide and thus also its low-mass fragment ions, confirmation of the analyte can be achieved with a two-stage mass spectrometer (Riediker & Stadler, 2003). Triple quadrupole presents some limitations for comprehensive analysis, in terms of running time, scan speed and sensitivity. These drawbacks can be overcome by the use of high resolution mass spectrometry (HRMS) instruments, such as Orbitrap, which operates in the full scan mode and provides accurate mass measurements (Gómez-Pérez et al., 2012). Currently the most popular methods for acrylamide quantification include external and internal standard method. However, analyte losses can occur in the course of sample preparation due to incomplete extraction, and cause underestimated results (Viegas, Novo, Pinho, & Ferreira, 2012). There are certain circumstances in which no blanks can be found because most samples tested contain acrylamide (Lehotay et al., 2008). The standard addition method has been used rarely in the determination of acrylamide in food matrices (Zhu et al., 2008). This method implies the use of the analyte as an internal standard, the number of sample preparation operations increase, but the effect of systematic errors decreases, thus it can be a good choice when low levels of analyte are quantified (Viegas et al., 2012). The aim of this work was to optimize the use of Al2O3 as dSPE material combined with the QuEChERS preparation method for the extraction of acrylamide from food samples. The performance of Al2O3 as dSPE sorbent was compared with commercial PSA sorbent in the clean-up of acrylamide. Moreover this work aimed to compare the efficiency of two analytical columns namely Kinetex C18 and Rezex ROA-organic acid for acrylamide separation. Standard addition method was used for quantification of acrylamide content in Sudanese food using HPLC–Orbitrap-mass spectrometry instrument.

2. Materials and methods 2.1. Samples Six home-made traditional Sudanese foods, cooked either by frying or baking namely potato (fried), eggplant (fried), gorrasa (baked), minnan (baked), taamia (fried) and Hilumur (âb.rae) (baked) were used as test samples. All food samples were prepared

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by using household food processor, according to our previously reported method (Omar et al., 2014). 2.2. Standards, reagents and materials Acrylamide (purity >99.8%), anhydrous magnesium sulfate MgSO4 (purity >99.5%), potassium hexacyanoferrate(II) (ACS reagent, 98.5–102%) (Carrez I), zinc sulfate heptahydrate (BioReagent) and water with 0.1% formic acid (LC–MS, CHROMASOLV, Fluka) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) (HiperSolvChromanorm for HPLC, 99.9%) and NaCl were from VWR PROLABO chemicals (EC). n-Hexane (for GC) and aluminum oxide (basic) were supplied by Merck (Darmstadt, Germany). PSA was purchased from Supelco (Bellefonte, PA, USA). Water was purified with ariumÒ pro ultrapure water systems from Sartorius Stedim Biotech (Göttingen, Germany). 2.3. Preparation of acrylamide standard solutions Stock solution of acrylamide standard (1000 mg L 1) was prepared by dissolving 0.01 g acrylamide in deionized water in a 10 mL volumetric flask and made up to the mark with deionized water. It was appropriately diluted to prepare working standard solution at 100 lg L 1 and used to prepare the standard addition calibration curve in the range 20–200 lg kg 1. 2.4. Optimized sample preparation procedure The samples were prepared according to our previously reported method with some modifications (Omar et al., 2014). For every sample, five thoroughly homogenized sub-sample (1.0 g of each sample) were weighed into five 50 mL centrifuge tube and 5 mL of n-hexane was added for each tube for defatting process. The centrifuge tubes were shaken vigorously for 1.0 min using a vortex-2 Genie mixer (Bohemia, USA). Then 0, 200, 500, 1000 and 2000 lL of acrylamide working solution (100 lg L 1) was added to each tube, respectively. Then the volumes were completed to 10 mL with deionized water (DW). An aliquot of 10 mL ACN was added to each tube followed by a mixture of 5 g anhydrous magnesium sulfate and 1.0 g sodium chloride. The tubes were vortexed immediately for 1.0 min and then centrifuged in Allegra 25R centrifuge from Beckman Coulter (California, USA) at 6028g (4000 rpm) for 6 min. The n-hexane layer (top) was discarded. An aliquot (3.0 mL) of the ACN layer (middle) containing the acrylamide was transferred into a 10 mL centrifuge tube containing 150 mg basic Al2O3 (equivalent to 50 mg mL 1) or 150 mg PSA and 90 mg anhydrous MgO4 for comparison. The mixture was then vortexed for 30 s and centrifuged at 4000 rpm for 3.0 min. An aliquot (1.0 mL) of upper supernatant was transferred into a glass vial and evaporated to dryness under a gentle stream of nitrogen gas. The residue was reconstituted with 200 lL of DW, vortex for 1.0 min and then filtered through a 0.2 lm Phenex PTFE syringe filter from Phenomenex (Torrance, CA, USA) into a HPLC auto sampler vial for analysis. 2.5. Optimization of dSPE process The parameters affected on dSPE method cleanup efficiency such as Al2O3 amount and contact time were investigated. With an un-spiked extract of potato sample (procedure as in Section 2.4) the effect of different amount of the basic Al2O3 sorbent (10–120 mg) as material for dSPE cleans-up performance was studied. Aliquots of 1.0 mL of ACN extract of the un-spiked potato were separately mixed with different amount (10, 30, 50, 80, 100 and 120 mg) of the basic Al2O3 sorbent. The upper supernatant layer was analyzed to determine the acrylamide content.

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The effect of different contact times of basic Al2O3 (1–10 min) with the acrylamide containing extract was studied using un-spiked extract of potato sample. Aliquots of 1.0 mL of ACN extract of the un-spiked potato were mixed with 50 mg basic Al2O3 sorbent and centrifuged for different periods (1, 2, 3, 5, 7 and 10 min). The upper supernatant layer was analyzed with HPLC LTQ-Orbitrap MS for the determination of acrylamide. 2.6. Quantification Quantification was performed by the method of standard addition as follows: a set of HPLC–MS peak areas of the analyte obtained for each sample (one for un-spiked portions and four for portions spiked with different levels of standard solutions) were plotted as y-axis, while concentrations of acrylamide added in the portions (0, 20, 50, 100 and 200 lg kg 1 for un-spiked and four spiked portions, respectively) were plotted as x-axis. The absolute value for the x-axis obtained from the calibration curve, when the value of the y-axis was equal to zero, was calculated as the amount of acrylamide in un-spiked portion of the sample. 2.7. Acrylamide recovery The recovery of the method was assessed by spiking each food sample at two levels of acrylamide (40 and 150 lg kg 1). Aliquots of 1.0 g sample were spiked with 400 and 1500 lL of acrylamide standard solution (100 lg L 1) after defatting with n-hexane. The procedure was continued as in Section 2.4. Three replicates were performed for each recovery level.

columns are tested in this study because they were previously used in literature for acrylamide separation (Cavalli, Polesello, & Saccani, 2004; Hoenicke & Gatermann, 2005; Jiang, Smith, Ferguson, & Taylor, 2007; Troise, Fiore, & Fogliano, 2014). Good retention time and peak shape was obtained by using ion exclusion Rezex ROA-organic acid column (14.2 min) and a core shell Kinetex C18 columns (3.5 min) (data not shown). This finding is in agreement with work done by Troise et al., 2014; Cavalli et al., 2004. It was observed that the peak area of acrylamide obtained by Rezex ROA-organic acid column is always higher than peak area obtained by Kinetex C18 when acrylamide standard was analyzed at the same concentration (10 lg L 1). But the advantage of Kinetex C18 column is that the retention time of acrylamide is short and this decrease the overall analysis time. Therefore, Rezex ROA-organic acid column and Kinetex C18 columns were used in this study. Different Mobile phase compositions such as; ACN: 0.1% formic acid in water (10:90), sulfuric acid (0.02 M) and 0.1% formic acid were used for acrylamide elution from Rezex ROA-organic acid column. A good peak shape of acrylamide was obtained by using 0.1% formic acid (data not shown), hence it selected as mobile phase for the further study and to compare both columns (Rezex ROA-organic acid and Kinetex C18 column). The retention time of acrylamide obtained by Rezex ROAorganic acid column at room temperature was 19.5 min and to decrease the time of measurement 60 °C was used as separation temperature, which decreases the retention time to 14.2 min. For comparison also the separation with a Kinetex C18 column was done at 60 °C, which decrease the retention time of acrylamide from 3.5 min (RT) to 2.7 min.

2.8. HPLC LTQ-Orbitrap MS analysis The separation of acrylamide was carried out on a UHPLC Accela system (Thermo Fisher Scientific, San Jose, CA, USA) consisting of a degasser, a quaternary pump, a thermostated autosampler, and a column oven. Mobile phase A was 0.1% formic acid, and mobile phase B was 0.1% formic acid in ACN. Four chromatographic columns were tested: Kinetex HILIC 2.6 lm 100 Å (50 * 4.6 mm), Kinetex 2.6 lm C18 100 Å (100 * 3.00 mm), Luna 3.0 lm CN 100 Å (150 * 2.00 mm) and Rezex ROA-organic acid (8% crosslinked sulfonated styrene–divinylbenzene) (150 * 4.6 mm), all from Phenomenex. Mobile phase was 100% (A) for Rezex ROA-organic acid and Kinetex C18 columns, and the flow rate was 0.25 mL min 1 and 60 °C oven temperature. For Kinetex HILIC column mobile phase was 2.0% (A) and 90% (B) and for Luna CN column was 99.5% (A) and 0.5% (B) at flow rate 0.2 mL min 1. Aliquots of 10 lL of the sample extract were injected into the chromatographic system using the autosampler. The HPLC system was coupled to an Exactive LTQ Orbitrap MS (Thermo FisherScientific). The ESI source was operated in positive mode with the spray voltage set at 4 kV, sheath gas (N2) flow rate at 60 arbitrary units, auxiliary gas (N2) flow rate at 35 arbitrary units, capillary voltage and temperature at 30.00 V and 350 °C, respectively. The tube lens was set at 85 V, skimmer voltage at 16 V and mass range m/z was 50–100. Acrylamide was quantified using m/z 71. All data were processed using Xcalibur software version 2.0.7 (Thermo Fisher Scientific). 3. Results and discussions 3.1. Optimization of HPLC conditions To optimize the chromatographic separation, four different columns with different selectivities namely; Kinetex HILIC, Kinetex C18, Luna CN and Rezex ROA-organic acid were tested. These

3.2. Optimization of Al2O3 added amount and contact time for dSPE The principle of QuEChERS method briefly involves an initial extraction with organic solvent, liquid–liquid-partitioning after addition of a mixture of MgSO4 and NaCl, followed by dispersivesolid-phase extraction (dSPE) clean-up step with loose sorbent (Mastovska & Lehotay, 2006). All critical parameters affected on QuEChERS and dSPE extraction and cleanup efficiency such as partitioning solvent, amount of NaCl and anhydrous MgSO4, sorbent type and amount of sorbent are optimized in our previously work (Omar et al., 2014). In the present work basic Al2O3 was used as dSPE sorbent instead of sol–gel hybrid MTMOS–TEOS sorbent. It was observed that acrylamide peak was decreased when an acrylamide containing extract mixed with Al2O3 centrifuged longer than 10 min. Hence the Al2O3 sorbent added amount and centrifugation time with the sample extract were studied. In order to evaluate the effect of the basic Al2O3 sorbent amount on the dSPE efficiency, un-spike fried potato sample was extracted following Section 2.4. An aliquot of 1 mL ACN sample extract is mixed separately with different amounts of basic Al2O3 sorbent (10, 30, 50, 80, 100 and 120 mg) and vortexed for 30 s and centrifuged. A 0.5 mL aliquot of the upper layer was transferred into glass vial and dried by N2 stream and then reconstituted with 0.5 mL DW and measured. It was found that 50 mg sorbent per 1.0 mL fried potato extract gave the highest acrylamide peak area and this may be due to that highest amount of sorbent has absorbed acrylamide resulting in lower recoveries. It was thus selected as the optimum sorbent mass for every mL of acrylamide extract. To investigate the effect of centrifugation time of sample extract mixed with Al2O3 on acrylamide peak area response, one milliliter of fried potato ACN extract was centrifuged with 50 mg Al2O3 at different centrifugation time (0–10 min). The optimal centrifugation time was 3 min.

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Fig. 1. Extracted ion chromatogram of fried potato ACN extract (A) without sorbent cleanup (B) cleanup with PSA (C) cleanup with Al2O3. Column Kinetex C18, mobile phase: 0.1% formic acid, m/z = 71.5–72.5.

3.3. Al2O3 cleanup procedure compared with PSA The clean-up efficiency of PSA and Al2O3 was compared by extracting fried potato sample using QuEChERS method. The two sorbents were mixed separately with 1.0 mL fried potato ACN extract and followed the procedure as in Section 2.4. The extracts cleaned by using the two different sorbents were analyzed with both analytical columns, Rezex ROA-organic acid and Kinetex C18. The suitable sorbent for dSPE should easily remove the matrix

components but let the analytes remained (Yan et al., 2013). The separation with Kinetex C18 column show for both sorbents, PSA and basic Al2O3 (Fig. 1B and C), a decrease of interfering peaks at the chromatogram of fried potato ACN extract in comparison without sorbent clean-up (Fig. 1A) but Al2O3 is more effective in removing the matrix. The peak areas of acrylamide obtained by using the two sorbents are comparable. Basically, PSA is the most commonly used adsorbent in the dSPE cleanup of QuEChERS. PSA is a weak anion exchanger which can remove various organic acids, fatty

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Fig. 2. Extracted ion chromatogram of fried potato (A) extracted by QuEChERS method followed by dSPE with (A) Al2O3 (B) PSA. Column Rezex ROA-organic acid, mobile phase: 0.1% formic acid, m/z = 71.5–72.5.

acids and some sugars. As demonstrated by Sßenyuva and Gökmen (2006), the most matrix compounds in analysis of acrylamide in food using HPLC–MS are the amino acids, which might be adsorbed at the surface of basic Al2O3 as mentioned by Lopes, Piao, Stievano, and Lambert (2009). Fig. 2A and B shows the EIC chromatogram of fried potato sample separated on Rezex ROA-organic acid and extracted by QuEChERS method followed by dSPE with Al2O3 and PSA sorbents, respectively. Only one peak is appeared on the chromatogram for all food matrices when Rezex ROA-organic acid used as separation column with both clean-up sorbents.

3.4. Method performance characteristics To ensure that these modified methods were applicable to real samples, several basic analytical parameters were evaluated, including matrix effect, linear range (standard addition method), limit of detection (LOD), limit of quantification (LOQ), precision (repeatability) (RSD%) and accuracy (recovery) were evaluated. All these parameters are determined for the QuEChERS extraction procedure using Al2O3 and PSA as dSPE sorbents and for both analytical columns used in this work (Rezex ROA-organic acid and Kinetex C18).

3.4.1. Matrix effect The matrix effect by using ESI source is a main problem in LC–MS analysis. Because of interactions of co-eluting matrix components with target compounds in the ion source, ion suppression of the analytes were often observed (Yan et al., 2013; Ying et al., 2013). To evaluate the matrix effect, boiled potato (matrix matched) and fried potato (standard addition method) samples were spiked with 10 lg L 1 acrylamide standard and was extracted using optimized QuEChERS method. It was found that the peak area of 10 lg L 1 acrylamide, spiked into fried potato, is less than the peak area of 10 lg L 1 acrylamide, spiked into boiled potato. This means, that fried potato induce more ion suppression. To

obtain more accurate results standard addition method was used as a calibration method. 3.4.2. Linearity and quantification with standard addition method Food samples were spiked with acrylamide at concentration range (0–200 lg kg 1) and then extracted using QuEChERS method. The calibration curves were constructed by plotting acrylamide peak area against added concentration of acrylamide. Good linearity was obtained by using both sorbents and columns (Rezex ROA-organic acid and Kinetex C18) with coefficient of determination, (r2) > 0.9979 for all food matrices (Table 1). Acrylamide concentrations were determined in six Sudanese food samples (fried potato, fried eggplant, minnan, gorrasa, taamia and hilumur/âb.rae) with standard addition method using QuEChERS method followed by PSA or Al2O3 clean-up and analyzed on the two analytical columns for comparison. The amount of the analyte in un-spiked portion of the sample was calculated from the calibration curve. The results obtained that the different sorbents and the two columns are comparable. The results showed that all the analyzed Sudanese food samples contained detectable levels of acrylamides, ranging from 16 to 341 lg kg 1. The highest amount of acrylamide was found in a fried eggplant sample (325 ± 9.15–341 ± 7.60 lg kg 1) and the lowest amount in a minnan sample (16 ± 0.91–18 ± 0.78 lg kg 1). In minnan sample no acrylamide was found in crumbs but it was detected in minnan crust. The preparation recipe of minnan sample is relatively similar to bread. During baking process the temperature inside the loaf does not exceed 100 °C and moisture content is around 40%. Therefore, Maillard reaction originating acrylamide may be does not occur (Forstova et al., 2014). 3.4.3. Limit of detection and limit of quantification The limit of detection (LOD) and the limit of the quantification (LOQ) were calculated using the standard addition method calibration curve for all food samples. The LOD was established using LOD = 3.3  (s/S) and the LOQ = 10  (s/S), where s is the standard deviation of the intercept and S is the slope of the curve

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Table 1 Equations for standard addition calibration curves, regression coefficient, limit of detection (LOD) and limit of quantification (LOQ) for acrylamide in different food matrices using Kinetex C18 and Rezex ROA-organic acid analytical columns and the two dSPE clean-up procedures with PSA and Al2O3. Matrix

Fried potato Fried eggplant Minnan Abri Gorrasa Taamia

Cleanup sorbent

PSA Al2O3 PSA Al2O3 PSA Al2O3 PSA Al2O3 PSA Al2O3 PSA Al2O3

C18 analytical column Equation Y = 1624.3x + 36,6143 Y = 1647.4x + 34,7162 Y = 5987.8x + 2E+06 Y = 5857.1x + 2E+06 Y = 24,312x + 83,468 Y = 16,050x + 1425.8 Y = 2486.2x + 13,5046 Y = 2490.1x + 148,005 Y = 6717.4x + 163,521 Y = 5915.9x + 112,378 Y = 1489.5x + 110,318 Y = 966.75x + 64,250

ROA analytical column Regression coefficient (r2)

LOD (lg kg

0.9979 0.9987 0.9987 0.9988 0.9998 0.9990 0.9989 0.9993 0.9992 0.9990 0.9987 0.9988

3.23 3.18 3.50 3.45 3.39 3.68 3.88 2.91 3.06 3.20 4.04 3.88

1

)

LOQ (lg kg

Equation 1

)

9.72 9.55 10.51 10.38 10.16 11.03 11.65 8.73 9.18 9.59 12.12 11.65

Y = 4166.2x + 90,1505 Y = 3774.8x + 85,8797 Y = 15,363x + 5E+06 Y = 15,155x + 5E+06 Y = 54,181x + 153,601 Y = 35,316x + 3763.3 Y = 4664.6x + 229,835 Y = 4710.6x + 242,568 Y = 14,998x + 337,449 Y = 12,760x + 254,290 Y = 3341.9x + 239,301 Y = 2126.9x + 144,506

Coefficient of determination (r2)

LOD (lg kg

0.9987 0.9986 0.9986 0.9990 0.9994 0.9993 0.9990 0.9990 0.9996 0.9993 0.9993 0.9989

2.68 2.73 2.95 2.81 3.11 3.53 3.08 2.58 1.50 2.80 3.06 3.24

1

)

LOQ (lg kg

1

)

8.04 8.19 8.87 8.43 9.35 9.77 9.27 7.73 4.49 8.50 9.20 9.72

Table 2 Recovery and precision (RSD%) for food samples using Kinetex C18 and Rezex ROA-organic acid column and the two dSPE clean-up procedures. Matrix

Fried potato

Cleanup sorbent

Spiked level (lg kg

PSA

0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150 0 40 150

Al2O3

Fried eggplant

PSA

Al2O3

Minnan

PSA

Al2O3

Abri

PSA

Al2O3

Gorrasa

PSA

Al2O3

Taamia

PSA

Al2O3

a

1

)

C18 analytical column

ROA analytical column

Native AA amount (lg kg 1) ± SDa

Recovery (%, n = 3)

RSD% (%, n = 3)

Native AA amount (lg kg 1) ± SDa

Recovery (%, n = 3)

RSD% (%, n = 3)

225 ± 7.11 – – 210 ± 8.19 – – 334 ± 6.80 – – 341 ± 7.60 – – 18.00 ± 0.78 – – 17.20 ± 1.26 – – 54.32 ± 1.76 – – 59.43 ± 2.03 – – 24.34 ± 0.43 – – 19.00 ± 0.54 – – 74.06 ± 1.90 – – 66.64 ± 2.32 – –

– 96 93

3.16 – – 3.90 – – 2.04 – – 2.22 – – 4.5 – – 7.3 – – 3.25 – – 3.41 – – 1.76 – – 2.88 – – 2.57 – – 3.50 – –

216 ± 6.04 – – 227 ± 9.93 – – 325 ± 9.15 – – 330 ± 6.82 – – 16.50 ± 0.62 – – 16.00 ± 0.91 – – 49.30 ± 0.90 – – 51.50 ± 1.50 – – 22.49 ± 0.32 – – 20.00 ± 0.45 – – 71.60 ± 3.08 – – 68.00 ± 3.61 – –

– 94 93 – 93 95 – 90 93 – 92 93 – 92 97 – 90 91 – 92 95 – 93 94 – 92 97 – 90 95 – 92 97 – 85 89

2.80 – – 4.36 – – – 2.81 – 2.06 – – – 3.8

92 94 – 90 94 – 91 95 – 96 94 – 91 93 – 90 91 – 92 93 – 95 99 – 90 95 – 88 93 – 86 93

5.6 – – 2.00 – – 3.10 – – 1.42 – – 2.24 – – 4.30 – – 5.30 – –

n = 3.

(Fernandes & Soares, 2007). The LODs and LOQ obtained using Al2O3 and PSA as sorbent material are comparable and shown in Table 1. In all measurements the obtained LODs with Rezex ROA-organic acid column are less than the LODs obtained by Kinetex C18 column (Table 1). This finding may be due to adsorption of some amount of acrylamide onto C18 column and the mobile phase used in this work (0.1% formic acid) is not effective enough to elute all acrylamide from C18 column. To achieve good sensitivity organic modifier should added to mobile phase to

insure complete elution of acrylamide from C18 column (Longhua et al., 2012). 3.4.4. Precision and recovery The precision (RSD%) and recovery (%) data for QuEChERS method with Al2O3 and PSA as dSPE sorbents for Sudanese food samples were determined by using two different analytical columns. The precision of this method was tested by intra-day repeatability. Three aliquots (1.0 g) of the same sample were

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Table 3 Comparison of LOD and acrylamide concentrations detected in Sudanese foods in the present work using HPLC LTQ-Orbitrap MS detection and Al2O3 dSPE compared to our previously work using GC–MS detection and sol–gel organic–inorganic hybrid MTMOS–TEOS dSPE. Sample

Fried potato Fried eggplant Minnan Âb.rae Gorrasa Taamia

Present work, Al2O3 dSPE (ROA column)

Present work, Al2O3 dSPE (C18 column)

Previous work, MTMOS–TEOS dSPE and GC–MS analysis

LOD (lg/kg)

Acrylamide conc. (lg/kg)

LOD (lg/kg)

Acrylamide conc. (lg/kg)

LOD (lg/kg)

Acrylamide conc. (lg/kg)

2.73 2.81 3.53 2.58 2.80 3.24

227 ± 9.93 325 ± 9.15 16.00 ± 091 51.50 ± 1.50 20.00 ± 0.45 68.00 ± 3.61

3.18 3.45 3.68 2.91 3.20 3.88

225 ± 7.11 334 ± 6.80 17.20 ± 0.78 59.43 ± 2.03 19.00 ± 0.54 66.64 ± 2.32

9.50 12.80 9.20 9.60 9.20 9.10

750 ± 8.1 338 ± 8.0 nd nd nd nd

nd: not detected.

prepared simultaneously and submitted to the overall method and injected in triplicate at the same day. Intra-day precision results obtained are summarized in Table 2. The recovery test was studied by carrying out by spiking six food samples (fried potato, fried eggplant, minnan, gorrasa, taamia and hilumur/âb.rae) with acrylamide at two concentration levels (40 and 150 lg kg 1). The percentage recoveries for QuEChERS method ranged from 90% to 97% as shown in Table 2. 3.5. Comparison of performance of present work with our previous QuEChERS methods with sol–gel hybrid MTMOS–TEOS In comparison with our previously work with QuEChERS and sol–gel hybrid MTMOS–TEOS as dSPE sorbent material and GC– MS detection, this optimized QuEChERS with Al2O3 as dSPE sorbent and HPLC LTQ-Orbitrap MS detection is more sensitive as shown in Table 3. The detection limits for analyzed Sudanese food samples were ranged from 9.1 to 12.8 lg kg 1 for GC–MS and sol–gel hybrid MTMOS–TEOS as dSPE sorbent and from 2.58 to 3.24 lg kg 1 for the present work by using HPLC LTQ-Orbitrap MS and Al2O3 as dSPE sorbent. The use of HPLC LTQ-Orbitrap MS analysis results in excellent selectivity and sensitivity better than GC–MS for direct determination of acrylamide in food. 4. Conclusion In this work, the performance of traditional QuEChERS method with PSA was compared to modified QuEChERS method with Al2O3 as dSPE material for determination of acrylamide in Sudanese food samples and analysis with HPLC LTQ-Orbitrap MS. It was found that Al2O3 improves the performance of dSPE cleanup. From the results obtained in this work Al2O3 can be used as a cheaper alternative to PSA as a dSPE sorbent material for determination of acrylamide in food. The sensitivity with HPLC LTQ-Orbitrap MS analysis is better than our previously work using GC–MS analysis. HRMS analyzers can improve the detection/identification process with the information provided by accurate mass measurements. HPLC–Orbitrap-MS allows efficient performance and adequate quantification/identification values of acrylamide. The LODs obtained by using Rezex ROA-organic acid column are lower than that obtained by using Kinetex C18 column for all analyzed food samples. However the total analysis time obtained by C18 column is shorter than that obtained by ROA-organic acid column. Hence Kinetex C18 column can be used for routine analysis of acrylamide in a huge number of food samples and ROA-organic acid column can be used for analysis of samples containing low contents of acrylamide. Acknowledgments The financial support from Deutscher Akademischer Austausch dienst (DAAD) is gratefully acknowledged and the authors thank Phenomenex for providing columns.

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Determination of acrylamide in Sudanese food by high performance liquid chromatography coupled with LTQ Orbitrap mass spectrometry.

A sample preparation method based on modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) with aluminum oxide (Al2O3) as dispersive solid...
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