Analysis of Heterocyclic Amines in Meat by the Quick, Easy, Cheap

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A quick method for analysis of heterocyclic amines in meat by QuEChERS coupled with LC-DAD-MS-MS Han-Yin Hsiao, Bing Huei Chen, and Tsai Hua Kao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03739 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Journal of Agricultural and Food Chemistry

A quick method for analysis of heterocyclic amines in meat by QuEChERS coupled with LC-DAD-MS-MS

Han-Yin Hsiao, Bing-Huei Chen and Tsai-Hua Kao* Department of Food Science, Fu Jen University, Taipei, Taiwan 242 * To whom correspondence should be addressed E-mail: [email protected]; PH: 886-2-29053629; Fax:886-2-29051215

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ABSTRACT

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The traditional way to analyze heterocyclic amines (HAs) is time-consuming and uses large

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amounts of solvents. The objective of this study is to develop a quick and simultaneous analysis

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method for multiple types of HAs contained in meat products. Results showed that 20 HAs and 1

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internal standard (4,7,8-TriMeIQx) can be separated within 30 min using an Inspire C18 column and a

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gradient solvent system containing a 10-mM ammonium acetate (pH 2.9) and acetonitrile. This process

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resulted in a high degree of separation. Using acetonitrile with 1% acetic acid as extraction solvent,

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followed PSA, MgSO4, and C18EC as purified reagent, is highly suitable for extracting HAs using

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QuEChERS. Tandem mass spectrometry with selected reaction monitoring mode were used for

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analysis, which indicated reasonable recovery (58.9-117.4%) for all 20 types of HAs along with limits

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of detection and quantification in the range of 0.003–0.05 ng/g and 0.01–0.05 ng/g, respectively.

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Keyword: heterocyclic amines, QuEChERS, LC-DAD-MS-MS, meat

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INTRODUCTION

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Heterocyclic amines (HAs) are formed by C, H, and N atoms and are cluster compounds with a

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heterocyclic structure. They are commonly found in heat-treated high-protein food products such as

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seafood and meat products. Related literature shows that HAs have mutagenic and carcinogenic

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properties.1 The International Agency for Research on Cancer has listed 12 types of HAs as possible

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human carcinogens (class 2B) and IQ as a probable human carcinogen (class 2A).2

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Since the concentration of HAs in food products is low and the food matrix is complicated, most

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studies have used solvent extraction and solid phase extraction for analysis, wherein, generally,

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diatomaceous earth is used to absorb large particles and then solvents are employed to extract the

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HAs.3-5 The most commonly used solvents are ethyl acetate and dichromathane. A propylsulphonic

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acid silica gel cartridge is then used for weak cation exchange, or a mixed-mode cation exchange

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adsorbent (Oasis MCX type, from Waters Co.) is used for purification, where MeOH/0.1 M HCl

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(40/60, v/v)-based propylsulphonic acid silica gel is used to elute relatively nonpolar HAs. Then, a

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0.5-M ammonium acetate solution (pH 8.0) is used to elute relatively polar HAs. The two solvents are

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then eluted separately using methanol and ammonia (9:1 v/v) in C18 solid phase extraction tubes,3

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whereas MeOH/25% ammonia (19:1 v/v) is used to elute HAs using the Oasis MCX cartridge.4,5 3



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However, the aforementioned traditional method of extraction is extremely time-consuming and

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generally provides a low recovery. Hence, there is a need to develop a faster and more convenient

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method of analysis.

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HAs are generally analyzed using gas chromatography-mass spectrometry (GC-MS) and

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liquid-chromatography-mass spectrometry (LC-MS). GC-MS has high sensitivity, but it induces mild

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pyrolysis in the benzene ring of HAs therefore the derivatization process is necessary for GC analysis.

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However, the derivatization treatment is time-consuming and not all HAs can be derivatized

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appropriately.6 LC has a relatively high sensitivity and can be used with photodiode array,7

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fluorescence,8 and MS9 detectors for the analysis of HAs. Apart from the difference between LC and

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GC systems, the types of columns and mobile phases also affect HAs significantly. The columns used

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for HAs analysis include TSKgel ODS-80, Shim-pack XR-ODS, Acclaim™ 120 C18, and BDS

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Hypersil C18,3,4,10-12 of which TSKgel ODS-80 can separate the most number of HAs (total of 15

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types). This study aimed to develop an effective LC-MS-MS method for separation of HAs

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simultaneously as well as apply the QuEChERS for quick HAs extraction.

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MATERIALS AND METHODS

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Materials 4


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Roasted duck was purchased from local market. Pork fiber was self-fried, where 6 kg of pork

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hind leg was cooked in 5 kg of water for 1 h until the meat was soft. After being cooled, the pork was

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beaten into fibrous meat and added with flavoring (30% broth, 20% sugar, 14% lard, 8% soy sauce,

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1.6% salt, 1% MSG). A pork fiber machine was then used to fry the meat fiber (150°C, 40 min). After

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being cooled, the pork fiber was vacuum-packaged and stored at -20°C.

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Chemicals and Reagents

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Twenty HAs standards including 2-amino-1,6-dimethylimidazo[4,5-b]-Pyridine (DMIP),

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2-aminodipyrido-[1,2-a:3',2'-d]imidazole

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(iso-IQ),

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2-amino-3-methyl-imidazo[4,5-f]-quinoline (IQ), 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole

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(Glu-P-1),

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2-amino-3,8-dimethyl-imidazo[4,5-f]-quinoxaline

(8-MeIQx),

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2-amino-1-methyl-imidazo[4,5-b]-quinoline

(IQ[4,5-b]),

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2-amino-1,6-dimethyl-furo[3,2-e]imidazo[4,5-b]-pyridine

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2-amino-3,7,8-trimethyl-imidazo[4,5-f]-quinoxaline

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2-amino-3,4,8-trimethyl-imidazo[4,5-f]-quinoxaline

(Glu-P-2),

2-amino-1-methyl-imidazo[4,5-f]-quinoline

2-amino-3-methyl-imidazo[4,5-f]-quinoxaline

2-amino-3,4-dimethyl-imidazo[4,5-f]-quinoline

(IQx),

(MeIQ),

(IFP),

(7,8-DiMeIQx),

(4,8-DiMeIQx),

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9H-pyrido[3,4-b]indole


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(Norharman), 1-methyl-9H-pyrido[3,4-b]indole (Harman), 2-amino-5-phenylpyridine (Phe-P-1),

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3-amino-1-methyl-5H-pyrido[4,3-b]indole

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2-amino-1-methyl-6-pheny-limidazo[4,5-b]-pyridine

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3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-9H-pyrido[2,3-b]indole (AαC),

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2-amino-3-methyl-9H-pyrido[2,3-b]indole

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2-amino-3,4,7,8-tetramethyl-imidazo[4,5-f]-quinoxaline

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Research Chemicals Co. (Downsview, Ontario, Canada). Deionized water was produced using the

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Milli-Q water purification system manufactured by Millipore (Bedford, MA, USA). Acetic acid and

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ammonium acetate was purchased from Sigma-Aldrich (St, Louis, MO, USA). The QuEChERS

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extraction set, purchased from Uni-Onward Corp. (Taipei, Taiwan), included extraction powder

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(product number UR-EX), purification powder (product number UR-CLEAN-II), ceramic

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homogenizers, and centrifuge tubes. The extraction powder contained 4 g of anhydrous magnesium

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sulfate (MgSO4) and 1 g of anhydrous sodium acetate (NaOAc). The purification powder contained

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300 mg of PSA (primary and secondary amine), 900 mg of MgSO4, and 300 mg of C18EC.

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Instrumentation

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(Trp-P-2),

(MeAαC),

(PhIP),

as

well

as

(4,7,8-TriMeIQx)

1

internal

were

from

standard

Toronto

Inspire C18 and TSKgel ODS-80 (250 mm × 4.6 mm I.D., 5 µm) columns were from Dikma 6


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Technologies (Lake Forest, CA, USA) and TOSOH (Tokyo, Japan), respectively. Gourd columns

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EasyGuardTM and security guard C18 were from Dikma Technologies and Phenomenex (Torrance,

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CA, USA). HPLC-DAD system with PU2089 Plus pump and MD-2010 diode array detector were

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from Jasco (Tokyo, Japan). Dionex UltiMate 3000 Open Sample XRS System UPLC, and TSQ

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Quantiva triple quadrupole tandem mass spectrometer were from Thermo Fisher Scientific (San Jose,

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CA, USA).

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Evaluation of Simultaneous Separation Conditions by LC

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The optimal separation condition was evaluated by using different columns (Inspire C18 and

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TSKgel ODS-80), flow rates (0.6–1 mL/min), and a mobile phase comprising 10 and 50 mM

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ammonium acetate buffer solutions with pH 2.8–3.6.

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Development of the QuEChERS Method for HAs Extraction

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Two gram of homogeneous roasted duck meat and 10 mL of deionized water were placed into a

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50-mL centrifuge tube. After shaken for 10 min, 10 mL of different extraction solvents (methanol,

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ethyl acetate, acetone, and acetonitrile, which contains 1% formic acid and 1% acetic acid acetonitrile)

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was added. After the mixture was shaken for 10 min, a ceramic homogenizer and a packet of extraction

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powder were added, which were vigorously shaken manually for 1 min before being centrifuged at 7



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3200 g and 4°C for 5 min. Then, 6 mL of supernatant was collected and transferred into a 15-mL

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centrifuge tube along with the purification powder. After vigorous manual shaking for 1 min, the

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mixture was centrifuged (3200 g, 4°C, 5 min), 1 mL of supernatant was collected and solvent was

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removed by dryness with nitrogen. Finally, 200 µL methanol contained 1 ppb of internal standard was

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used to dissolve the extract and then filtered using a 0.22-µm PVDF syringe filter prior to LC analyses.

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Identification of HAs

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The UV–Vis spectra, retention time, and MS-MS spectra of the samples were compared with

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those of the standards. The MS-MS spectra were detected with electrospray ionization in positive

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mode with the following condition: 3000 V spray voltage for ion source, sweep gas flow rate 2

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arbitrary units, sheath gas flow rate 50 arbitrary units, auxiliary gas flow rate 20 arbitrary units, ion

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transfer tube temperature 368°C, and vaporizer temperature 315°C.

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Method Validation

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Precision

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HAs extract with 20 HA standards (0.2 ng/mL) and internal standard (1 ng/mL) was prepared

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and injected five times on the same day, with the relative standard deviation (RSD%) being calculated

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to obtain the intra-day variability. Similarly, the HAs extract containing standards and internal standard 8


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was injected three times on three non-continuous days, and the inter-day variability was measured

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based on RSD%.

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Limits of Detection and Quantification

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The confirmation ion signal (Sc) and quantitation product ion signal (Sq) of 20 HAs were

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detected by LC-MS-MS. The detection limit (LOD) was determined based on Sc/N ≥ 3 and Sq/N ≥ 3.

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The limit of quantification (LOQ) of harman and norharman was obtained by multiplying the LOD

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with 3.3. The LOQ for the other HAs was measured based on Sc/N ≥ 3 and Sq/N ≥ 10.

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Recovery

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Two levels of HAs standards including 10 ng/g and 1 ng/g of DMIP, Glu-P-2, iso-IQ, IQ, IQx,

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MeIQ, Glu-P-1, 8-MeIQx, IQ[4,5-b], IFP, 7,8-DiMeIQx, 4,8-DiMeIQx, Phe-P-1, Trp-P-2, PhIP,

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Trp-P-1, AαC, MeAαC; 20 ng/g and 40 ng/g of norharman, and 400 ng/g and 800 ng/g of Harman,

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were mixed with 2 g of fried pork fiber. Following extraction by QuEChERS and HPLC analysis, the

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recovery of each HAs was obtained based on the following formula:

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Recovery (%) = [(spiked amount plus original amount) - (original amount)] / spiked amount

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Matrix Effect

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The 20 HAs standards were separately dissolved in methanol (sample solvent) and fried pork 9



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fiber extract (matrix) to prepare solutions with 0.05, 0.1, 0.5, 1, 2, 2.5, and 10 ng/mL concentrations.

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These were then analyzed using LC-MS-MS to obtain standard calibration curves (SCC) and matrix

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matched calibration curves (MCC). The matrix effect was obtained using the following formula:

matrix effect =

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(slope of MCC − slope of SCC) × 100% slope of SCC

Quantification of HAs

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HAs standards were mixed and prepared in fried pork fiber extract with concentration of 0.05,

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0.1, 0.5, 1, 2 and 2.5 ng/mL. Then each standard solution was mixed with internal standard with a

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fixed concentration at 1 ng /mL. After analyzing by LC-MS-MS based on selective reaction

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monitoring (SRM), each standard curve was prepared by plotting concentration ratio (standard vs

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internal standard) against its area ratio, and the regression equation and correlation coefficient (r2) were

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automatically obtained. Each HA was quantified using the following formula:

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Concentration of HAs (ng/g) =

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{[(A/RRF)/Ai] x Ci x volume of extract x dilution factor /recovery} / weight of sample (g)

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where A is the area of each HAs; RRF is relative response factor = (A/Ai) / (C/Ci); Ai is the area of

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internal standard; Ci is the concentration of the internal standard.

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Statistical Analysis 10


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Both LC analysis and QuEChERS were performed twice. The data were analyzed by ANOVA

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and Duncan’s multiple rang test for significant difference (P

Analysis of Heterocyclic Amines in Meat by the Quick, Easy, Cheap

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