A sensitive, precise, accurate and simple in house validated Liquid Chromatography and Tandem mass spectrometric (LC-MS/MS) analytical method was developed for the quantification of acrylamide in potato chips, a probable carcinogenic and neurotoxic compound in fried food items. Acrylamide was extracted and cleaned up by QuEChERS method, then analyzed by triple qu adrupole mass spectrophotometer using positive electrospray-ionization mode and d3-Acrylamide internal standard. The analyte and internal standard were separated on a reversed - phase C18 column (100 × 2.0 mm, 2.5 μm) by gradient elution with water and methanol containing 0.1% formic acid as mobile phase. The analytical method was fully validated by assessment of linearity, specificity, precision, accuracy, selectivity and robustness with relative standard deviation of less than 4%. The calibration curves were found linear with correlation coefficient (r2) of 0.9997 over the concentration range 5 - 500 μg/L. The recoveries of Acrylamide in the concentration of 250, 400, and 500 μg/L were 97% to 104%. Based on the signal to noise ratio, the lower limit of detection (LOD) and lower limit of quantification (LOQ) were achieved 2.0 μg/L and 4.0 μg/L respectively. The proposed method was also applied to other available most popular 24 types of food (265 number of sample) first time in Bangladesh. The highest level of acrylamide has been found in fried products with the range of 197.04 μg/L to 114.63 μg/L, protein - rich food lies between 79.76 to 89.14 μg/L whereas baked food products exhibited less content in the range of 35.23 to 51.17 μg/L.
Food habits are changing rapidly due to variations of lifestyles, economies and global culture. Fried food items have gained its popularity among various age people, especially young generation because of its short time of preparation and consumption. To obtain acceptable food products, it is necessary to maintain nutrients like minerals, carbohydrates, fats, proteins, vitamins, enzymes, etc. But, nutrients may be destroyed or lost when foods are processed because of their sensitivity to heat, light, oxygen, pH of the solvent or a combination of these [
Among different validated analytical methods, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) are found to be prominent for the estimation of acrylamides in various foods [
acrylamide content in various food products. However, no such study has ever been conducted in any food products in Bangladesh. Therefore, regarding the above circumstance the objective of the study was to develop a validated LC-MS/MS method for the determination of acrylamide in potato chips and other locally available foods.
Reference standard of Acrylamide (purity of 99.9%) and isotope labelled internal standard (IS) of Acrylamide d3 were purchased from Scharlu and Sigma-Aldrich, respectively. Methanol (MS Grade, purity of 99.9%), Acetonitrile (MS Grade, purity of 99.9%), Hexane (HPLC grade, purity of 97.09%) and Sodium Chloride (reagent grade, purity of 99.5%) were procured from Sigma-Aldrich. Formic acid (purity of 98%) analytical grade was purchased from BDH England, Magnesium Sulphate (purity of 99%) purchased from Merck, Primary secondary amine (PSA) procured from Agilent Technologies and highest purity demineralized water was used unless mentioned otherwise.
Metler Toledo analytical AL204 balance used from (China), Hwashin sonicator bath power sonic 505 and Vortex mixer 250VM (Korea), Hermle labortechnik GmbH Centrifuge Z206A (Germany), Bibby Scientific Block heater SBH130D/3 (UK), Whatman syringe filter 0.22 µm (UK) were used.
A quaternary liquid chromatographic system (Nexera X2 series) with a triple quadruple mass spectrometer-mass spectrometer equipped with an electrospray interface and lab solution software (LCMS-8050, Shimadzu Corporation, Japan) were used in this study.
For acrylamide analysis Phenomenex C-18 (100 × 2.0 mm, 2.5 µm) column was used which maintained at 40˚C; mobile phase was mixture of two solution with gradient elution mode: B%: 1% (0 to 4.4 min)-80% (4.5 to 5.4 min)-1% (5.5 to 10 min) and delivered at flow rate of 0.2 mL/minutes; chromatographic injection volume was 10 µL and the retention time of acrylamide was about 1.8 minutes.
The MS acquisition parameters were as follows: Run time: 10 minutes; Ion polarity: positive ion mode; ion source: atmospheric pressure electrospray ionisation; Capillary voltage (kV): 4.0; Block temperature: 400˚C; Dessolvation line temperature: 200˚C; CID gas: Argon (270 kPa), Nebulizing gas flow: N2, 1.5 L/min; Drying gas flow: N2, 10.0 L/min; Heating gas flow: 10 L/min; Interface temperature: 300˚C; mass spectrum scan range (m/z): 10 - 200 Da where protonated precursor ions of acrylamide and acrylamide-d3 (IS) was m/z 72.1 and m/z 75.1 respectively. Two multiple reactions monitoring (MRM) transitions of acrylamide and acrylamide-d3 were selected using a dwell time of 100 milli second as quantifier and confirmation ions as shown in below
Water and 0.1% formic acid in methanol were used as mobile phase. Both the solutions were degassed by sonication using ultra sonic bath and filtered through 0.22 µm filter membrane using the Millipore filtration unit.
1000 mg/L of acrylamide standard and 1.0 mg/L of IS stock solution were prepared standard in water. Both the solutions were sonicated for 10 minutes and stored at 2˚C - 8˚C for further preparation of calibration solutions.
Intermediated stock solution of 10 mg/L and 1.0 mg/L of acrylamide and IS respectively were prepared by appropriate dilution of stock solutions with water.
| Name | MRM (m/z) | CID Voltage (V) | ||
|---|---|---|---|---|
| Q1 | CE | Q3 | ||
| Acrylamide-d3 | 75.1 > 58.0* | −29 | −15 | −22 |
| 75.1 > 30.1 | −29 | −24 | −30 | |
| Acrylamide | 72.1 > 55.0* | −17 | −16 | −25 |
| 72.1 > 27.1 | −17 | −22 | −30 | |
*MRM transition as quantifier.
These were also undergone sonication for 10 minutes.
1000 µg/L, 800 µg/L, 500 µg/L, 200 µg/L, 100 µg/L, 20 µg/L & 10 µg/L of acrylamide and 100 µg/L of IS standard were prepared by appropriate dilution of intermediated stock solutions. Prepared IS standard solution was added to each acrylamide standards in 50:50 ratio to prepare 500 µg/L, 400 µg/L, 250 µg/L, 100 µg/L, 50 µg/L, 10 µg/L and 5 µg/L acrylamide calibration solutions where 50 µg/L of IS was present in each of those. All the calibration standards were sonicated for 10 minutes in ultrasonic bath and then injected into LC-MS/MS for analysis.
A modified QuEChERS procedure was adopted in acrylamide extraction. Accurately weighed 2 grams of crushed powder sample were weighed accurately and taken into a 50 mL centrifuge tube in which 5 mL hexane, 10 mL water and 10 mL acetonitrile were introduced. Vigorously vortexed and shook for 2 minutes. The purpose of hexane was to defat the samples and to remove oils & non-polar components. Then Q-sep Q 100 Packet extraction salt (containing 4 g MgSO4 and 0.5 g NaCl) and additional 4 g of MgSO4 was added to absorb the water completely. Vortexed and shook again for another 5 minutes. 2 mL extract from the bottom layer was transferred into another 20 mL centrifuge tube were 100 mg primary secondary amine (PSA) was added to remove organic acids. Vortexed for 1 minute and then centrifuged at 11,000 rpm for 10 minutes. 1 mL aliquot was collected and evaporated to dryness under N2 stream. The dried sample was reconstituted with 1 ml of water and filtrated through 0.22 µm syringe filter. Then prepared sample solution and 100 ppb of IS was mixed in 50:50 ratio in a vial. This reconstituted solution and 100 µg/L IS mixed in 50:50 ratio was injected into LC-MS/MS for the determination of acrylamide in the sample.
The validation of LC-MS/MS method is performed using blank sample of potato chips as per guidelines of International Conference on Harmonization [
Selectivity of this method was demonstrated by making 5 µg/L and 250 µg/L acrylamide standard with acetonitrile instead of water and then analyzed by LC-MS/MS. The % RSD of retention time and area from six repeated injections at each concentration was calculated.
Linearity was carried out at seven concentration levels ranging from 5 to 500 µg/L. A calibration curve was obtained by plotting the peak area vs. concentration. The % RSD from three repeated injections at each concentration and % y-intercept bias was calculated.
The accuracy of the method was evaluated at three concentration levels of acrylamide (250, 400 and 500 µg/L) by spiking into samples. The % recoveries reflect an average of six analyses at each concentration and % RSD was reported.
Precision was measured as intra-day precision (repeatability) and inter-day precision (reproducibility) of working standard. The intra-day and inter-day precision of the method was demonstrated by injection (in six analyses) of the standard on two consecutive days and % of RSD was calculated.
The LOD and LOQ of the method were estimated on the basis of signal-to-noise ratio 3:1 and 10:1, respectively by injection series of diluted solution of known concentration. The % RSD from the six repeated injections was calculated.
Robustness was studied at seven different concentration of acrylamide with change in some parameters such as column temperature, column type, injection volume and analyst. Percentage (%) RSD from six repeated injections at each concentration under change condition was calculated.
Besides method validation, 24 types of food total 265 items of different batches represent the whole population, manufactured by different companies without revealing their names were collected in mid April 2017 from the local market randomly and prepared according to section 2.6 for quantification of acrylamide.
Chromatographic conditions like mobile phase composition, injection volume, flow rate and column oven temperature were optimized for better quantification of acrylamide in complex food matrix. Acrylamide exhibits both weakly acidic and basic properties. However, solubility varies considerably in polar and unipolar solvents whereas acrylamide is extremely high soluble in water [
Atmospheric pressure electrospray ionization used as ion source for the determination of acrylamide, gave high intensity and good linearity of calibration curve. The parent and product ions were first optimized by injecting a 500 µg/L standard solution of acrylamide in both positive and negative polarity mode. Due to protonation and use of formic acid in mobile phase, the intensity was much higher in positive mode. Quantification of acrylamide was performed by using optimized MRM mode for consistent and adequate response where most abundant product ions m/z 55.0 and m/z 27.1 for the case of acrylamide precursor ion m/z 72.1 were observed by employing 16 eV and 22 eV collision energy, respectively. On the other side product ions at m/z 58.0 and m/z 30.1 was observed in the case of acrylamide-d3 (IS) precursor m/z 75.1 by applying 15 eV and 24 eV collision energy, respectively.
A seven-point calibration curve was constructed by plotting triplicate injected acrylamide concentration versus area over the range of 5 to 500 µg/L with a mean correlation coefficient (r2) was 0.9997. The % RSD values of the repeated injection are <2% within each concentration and the Y-intercept bias is less than 2% which explains excellent linearity of the curve (
The precision was evaluated as the repeatability by calculating the percent relative
standard deviation (% RSD) for six determinations each for seven different solutions which were analyzed by three different analysts on the same day (Intra-day) and on the three different days (Inter-Day) as well (
The accuracy of the method was assessed by spiking different known concentration of acrylamide standard solution into sample. Standard solution of 250 µg/L, 400 µg/L and 500 µg/L was spiked into previously analyzed sample contained 7.05 µg/L acrylamide. The calculated and observed concentration of acrylamide was compared and recoveries of each spiked levels were determined (
| Standard Conc. (µg/L) | Intra-day precision | Inter-day precision | ||||||
|---|---|---|---|---|---|---|---|---|
| Mean observed conc. (µg/L) (n = 6) | % RSD | Days | Analyst | Mean observed conc. (µg/L) (n = 6) | Mean observed conc. (µg/L) (3 days) | % RSD | Mean % RSD | |
| 5 | 5 | 1.73 | Day 1 | Analyst-1 | 4.804 | 4.92 | 3.871 | 2.19 |
| Day 2 | Analyst-2 | 4.95 | 1.977 | |||||
| Day 3 | Analyst-3 | 5.00 | 0.716 | |||||
| 10 | 10.55 | 2.20 | Day 1 | Analyst-1 | 9.95 | 9.87 | 1.169 | 2.23 |
| Day 2 | Analyst-2 | 9.80 | 2.730 | |||||
| Day 3 | Analyst-3 | 9.85 | 2.779 | |||||
| 50 | 50.65 | 0.95 | Day 1 | Analyst-1 | 49.45 | 49.73 | 3.287 | 1.62 |
| Day 2 | Analyst-2 | 49.7 | 1.643 | |||||
| Day 3 | Analyst-3 | 50.05 | 1.984 | |||||
| 100 | 98.25 | 1.84 | Day 1 | Analyst-1 | 99.1 | 99.43 | 1.248 | 2.00 |
| Day 2 | Analyst-2 | 99.2 | 1.587 | |||||
| Day 3 | Analyst-3 | 100 | 3.142 | |||||
| 250 | 247.1 | 1.28 | Day 1 | Analyst-1 | 248.8 | 247.42 | 0.368 | 1.25 |
|---|---|---|---|---|---|---|---|---|
| Day 2 | Analyst-2 | 246.05 | 1.521 | |||||
| Day 3 | Analyst-3 | 247.4 | 1.833 | |||||
| 400 | 395.9 | 0.70 | Day 1 | Analyst-1 | 393.25 | 393.38 | 2.522 | 1.84 |
| Day 2 | Analyst-2 | 391.95 | 2.125 | |||||
| Day 3 | Analyst-3 | 394.95 | 0.890 | |||||
| 500 | 496.4 | 0.54 | Day 1 | Analyst-1 | 486.45 | 480.63 | 1.532 | 1.40 |
| Day 2 | Analyst-2 | 481.55 | 1.338 | |||||
| Day 3 | Analyst-3 | 473.9 | 1.338 |
| Acrylamide standard Conc.(µg/L) | Observed Conc. of Acrylamide standard (µg/L) (n = 3) | Analyte Conc. in sample (µg/L)(n = 6) | Total Conc. (Analyte Conc. in sample + Acrylamide standard Conc.) (µg/L) | Observed Conc. in sample after spiking (µg/L) (n = 6) | % Recovery | % RSD |
|---|---|---|---|---|---|---|
| 250 | 265.9 | 7.05 | 272.95 | 261.55 | 104.36 | 0.96 |
| 400 | 398.45 | 405.5 | 410.7 | 98.73 | 1.08 | |
| 500 | 489.1 | 496.15 | 511.85 | 96.93 | 0.81 |
between the range of 96% - 105% and 0.8 - 1.1, respectively; which is within the acceptable norms signifying the accuracy of the method.
Selectivity of this method was demonstrated in the presence of acetonitrile instead of water. Six repeated injections of 5 µg/L and 250 µg/L of acrylamide standard prepared in acetonitrile along with 50 µg/L IS were analyzed by LC-MS/MS. A slight change at elution time (1.7 minutes) of acrylamide was observed with % RSD of only 0.17 to 0.23, which demonstrates that the method is selective and interferences free (
LOD (Limit of Detection) is the lowest detectable but not quantifiable target analyte concentration and LOQ (Limit of Quantitation) is the lowest quantifiable concentration of target analyte with acceptable precision; was determined by considering signal to noise ratio (S/N) of the strongest mass transition with respect to background noise obtained from the blank (
By analyzing seven different concentration of acrylamide with change in some parameters such as column temperature, column type, injection volume and different analyst; the robustness of method was determined (
| Acrylamide standard concentration (µg/L) | Developed method | Change in operational method | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Mean Conc. (µg/L) (n = 6) | RSD % | Column (Shim-pack XR-ODS II, C18, 2.0 mm × 100 mm, 2.2 µ) | Column temparature (35˚C) | Injection volume (10 µl) | Analyst II | |||||
| Mean Conc. (µg/L) (n = 3) | RSD % | Mean Conc. (µg/L) (n = 3) | RSD % | Mean Conc. (µg/L) (n = 3) | RSD % | Mean Conc. (µg/L) (n = 3) | RSD % | |||
| 5 | 5.00 | 1.73 | 4.65 | 2.75 | 5.0 | 1.49 | 5.0 | 1.49 | 5.0 | 1.49 |
| 10 | 10.55 | 2.20 | 9.65 | 3.82 | 9.85 | 3.80 | 9.85 | 3.80 | 9.85 | 3.80 |
| 50 | 50.65 | 0.95 | 50.8 | 1.16 | 47.15 | 2.39 | 47.15 | 2.39 | 47.15 | 2.39 |
| 100 | 98.25 | 1.84 | 100.7 | 1.15 | 98.4 | 3.32 | 98.4 | 3.32 | 98.4 | 3.32 |
| 250 | 247.1 | 1.28 | 249.7 | 1.92 | 254.35 | 1.00 | 254.35 | 1.00 | 254.35 | 1.00 |
| 400 | 395.9 | 0.70 | 401.7 | 0.09 | 399.55 | 1.17 | 399.55 | 1.17 | 399.55 | 1.17 |
| 500 | 496.4 | 0.54 | 480.3 | 4.55 | 497.45 | 1.46 | 497.45 | 1.46 | 497.45 | 1.46 |
acrylamide content obtained by the developed method and that obtained under the mentioned change conditions, remained almost the same with % RSD less than 4.0 in every case.
The developed validate method of acrylamide quantification in potato chips by LC-MS/MS was also applied to analyze in other 265 food items like french fry, beef tikka, peyaju, chicken grill, flour chips, potato Biscuits, chanachur, cheese, puffs, coffee candy, chicken fry, puri, shik kabab, fuska, bread, brinjal fry, nut without shell, nut with shell, chicken nuggets etc. as shown in
| Item | Number of Sample | Samples positive | Level of Acrylamide (µg/L) | |||
|---|---|---|---|---|---|---|
| Min. | Max. | Mean | SD | |||
| Potato chips | 52 | 44 | 44.97 | 252.79 | 131.48 | 62.78 |
| French fry | 43 | 43 | 162.63 | 244.67 | 197.04 | 22.92 |
| Beef tikka | 10 | 10 | 41.27 | 87.71 | 60.33 | 15.58 |
| Peyaju | 13 | 13 | 51.09 | 113.17 | 82.47 | 16.30 |
| Chicken grill | 11 | 11 | 142.72 | 169.98 | 156.29 | 9.45 |
| Flour chips | 5 | 5 | 18.12 | 25.17 | 21.19 | 2.82 |
| Potato Biscuits | 5 | 5 | 43.28 | 57.32 | 51.17 | 5.22 |
| Chanachur | 10 | 10 | 13.66 | 23.65 | 19.33 | 3.16 |
| Cheese puffs | 1 | 0 | ||||
| Coffee candy | 1 | 0 | ||||
| Chicken fry | 8 | 8 | 104.32 | 127.50 | 114.63 | 8.02 |
| Puri | 10 | 0 | ||||
| Shik kabab | 5 | 5 | 151.21 | 187.22 | 172.87 | 13.74 |
| Fuska | 7 | 7 | 17.77 | 23.29 | 20.43 | 1.92 |
| Bread | 9 | 0 | ||||
| Brinjal fry | 10 | 10 | 20.79 | 31.49 | 25.53 | 3.61 |
| Nut without shell | 5 | 0 | ||||
| Nut with shell | 7 | 0 | ||||
| Chicken nuggets | 5 | 0 | ||||
| Fish fry | 10 | 10 | 74.17 | 85.27 | 79.76 | 3.67 |
| Egg fry | 10 | 10 | 83.21 | 93.33 | 89.14 | 3.49 |
| Boiled egg | 5 | 0 | ||||
| Cake | 8 | 0 | ||||
| Biscuit | 15 | 14 | 10.72 | 57.73 | 35.23 | 16.80 |
from 19.33 to 197.04 μg/L, among which 54% were found to be more than 100 μg/L. Average acrylamide content in Chanachur was found to be lowest (19.33 μg/L) while in processed and cooked food like potato chips, french fries, Beef tikka, Peyaju, Chicken grill, Flour chips, Chicken fry, Shik kabab, fuska, Fish fry, Egg fry, Brinjal it was highest with average values of 20.43 - 197.04 μg/kg.
Acrylamide contents in baked products except for biscuit and other food items like cheese puffs, coffee candy, nut without shell, nut with shell, chicken nuggets and boiled egg were found to be below the detection limit. Levels of acrylamide in protein-rich food (79.76 - 89.14 µg/L) were higher than that of baked food products (35.23 - 51.17 µg/kg) indicating the occurrence of the acrylamide contamination due to cooking procedures.
Previous research also supports the above statement, that acrylamide may be
| Product | Acrylamide Range (µg∙Kg−1) |
|---|---|
| Potato chips | 117 - 2762 |
| French Fries | 20 - 1352 |
| Breads & bakery products | 10 - 364 |
| Chocolate products | 0 - 909 |
| Nuts & nut butters | 28 - 457 |
| Protein foods | 0 - 116 |
| Canned fruit & vegetables | 0 - 83 |
formed due to the differences in cooking and processing parameters, methods, type and quality of raw materials, as well as formulations [
Though there is a declaration regarding daily allowable limit of acrylamide uptake (0.3 - 0.8 µg/kg body weight) [
The developed LC-MS/MS method for the determination of acrylamide is simple, rapid, sensitive accurate and precise and can be applicable for routine analysis. The method is mainly applicable for starch-based products but can also be adopted for other sample matrices as well. Participation in Proficiency Testing might help to get a clear view about the applicability of this method. Besides, the highest acrylamide levels were found in fried products, such as potato chips, french fries, chicken grill and sheikh kabab, which indicated the general risk on public health due to toxicity of acrylamide from foods. This investigation supplied important information on acrylamide in food for public health experts, food policy makers and consumers. Further research is suggested to find out the correlation among temperature, sugar and asparagine, and also to minimize the acrylamide formation during the processing of food.
We are great full to all employee of Designated Reference Institute for Chemical Measurements (DRiCM) for their cordial help during analysis. Additionally, M. Bruno, Mohammad Mizanur Rahman and Abdullah-al-Mamun are acknowledged for reviewing the paper.
All of the authors are employees of Designated Reference Institute for Chemical Measurements. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Khan, M., Moniruzzaman, Md. and Razu, M.H. (2019) Method Development and Validation for the Quantification of Acrylamide in Potato Chips and Other Locally Available Food by LC-MS/MS in Bangladesh. Food and Nutrition Sciences, 10, 876-892. https://doi.org/10.4236/fns.2019.107063