Determination of Phytochemical Compounds in Chicken Breast by Gas Chromatography-tandem mass spectrometry

Volume 29 Issue 2 Article 5

Determination of Phytochemical Compounds in Chicken Breast by Gas Determination of Phytochemical Compounds in Chicken Breast by Gas

Chromatography-tandem mass spectrometry Chromatography-tandem mass spectrometry

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Recommended Citation Recommended Citation

Cozzi, Federico; Eder-Neuhauser, Irene; Teichmann, Klaus; and Zaunschirm, Mathias (2021) "Determination of

Phytochemical Compounds in Chicken Breast by Gas Chromatography-tandem mass spectrometry,"

Journal of Food

and Drug Analysis

: Vol. 29 : Iss. 2 , Article 5.

Available at: https://doi.org/10.38212/2224-6614.3288

This Original Article is brought to you for free and open access by Journal of Food and Drug Analysis. It has been accepted for

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Determination of phytochemical compounds in

chicken breast by gas chromatography-tandem

mass spectrometry

Federico Cozzi*, Irene Eder-Neuhauser, Klaus Teichmann, Mathias Zaunschirm

BIOMIN Research Center, Tulln 3430, Austria

Abstract

A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed for the simultaneous detection

and quantiﬁcation of ﬁve phytogenic compounds (carvone, menthol, thymol, carvacrol and methyl salicylate) in chicken

breast. Chicken breast samples were analyzed using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe)

approach using acetonitrile as extraction solvent, followed by a d-SPE (dispersive-solid phase extraction) clean up step.

The linearities for the 5 substances were studied in the range between 2 and 100 mg/L and the coefﬁcients of determi-

nation (R2) were always > 0.995. Matrix effects were also assessed by comparing the slopes obtained in solvent and

chicken breast matrix. The recoveries for all the substances at 3 different spike levels (5, 10 and 50 mg/kg) were in the

range 80-102% with RSDs < 15%. The instrumental limits of quantiﬁcation were in the range 2.7-4.8 mg/kg, while the

reporting level of the method was 5 mg/kg for all the aforementioned compounds. The method was successfully applied

to 10 chicken breasts samples from the local market.

Keywords: Chicken breast, GC-MS/MS, Phytogenic compounds, QuEChERS

1. Introduction

ntibiotics and antimicrobials are not only

used as veterinary drugs to prevent and

control diseases in animal rearing but also as

growth promoters [1]. For this latter purpose they

are added to animal feed e a practice that can

lead to residues in edible animal products that are

potentially dangerous for human health or induce

antibiotic resistance of microbes even at small

doses [2]. This led to the ban of antibiotics as

growth promoters by the European Union since

2006 [3].

Global meat consumption has seen an increase by

58% in the past two decades and in particular

chicken meat consumption has increased, as it is not

only a source of high-quality protein, important

vitamin and minerals, but also the cheapest of all

livestock meats [4].

The aforementioned ban of antibiotic growth

promoters has fostered the research for alternative

substances: phytogenic feed additives (PFA) are

suggested to be among the most promising ones [5].

PFA are composed of plant-derived natural mate-

rials with positive effects on animal growth and

health, and this deﬁnition applies to preparations of

ground herbs and spices, essential oils (EOs), ex-

tracts an d/or oleoresins. PFA contain secondary

plant metabolites (phytochemicals) and encompass

a wide range of chemical compound classes,

including phenols, terpenes, alkaloids, lectins, al-

dehydes and ketones. The various mechanisms of

action are not yet fully understood but involve

antimicrobial/antiviral, antioxidative and anti-

inﬂammation activity [6].

Several companies offer feed additives containing

PFA in their portfolio and so it is important to

determine their residues in edible animal products

within the scope of consumer safety assessment [7].

We hence developed a method for the quant itative

determination of the following ﬁve phytochemicals

in chicken breast: carvone, menthol, thymol, carva-

crol and methyl salicylate.

Received 9 November 2020; revised 8 January 2021; accepted 8 February 2021.

Available online 15 June 2021.

* Corresponding author: BIOMIN Research Center, Tulln 3430, Austria. Tel: þ43-2272-811660.

E-mail address: [email protected] (F. Cozzi).

https://doi.org/10.38212/2224-6614.3288

(http://creativecommons.org/lice nses/by-nc-nd/4.0/).

ORIGINAL ARTICLE

Food matrices are still among the most chal-

lenging ones due to their complexity. In addition,

the usual sample preparation methods for these

matrices are labor intensive [8]. A valid alternative

to these methods is the QuEChERS technique [9],

although this method was originally developed for

pesticide analysis in fruits and vege tables its scope

of application has been extended to other analytes

and a grea t variety of different matrices [ 10,11].

Therefore, we decided to apply this approach as a

sample preparation method. A generic QuEChERS

extraction method involves a ﬁrst step in which the

analytes of interest are extracted by using an organic

solvent (normally acetonitrile) in presence of inor-

ganic salts (like MgSO

or NaCl) to ensure a salting

out effect. The extract is then cleaned with disper-

sive sorbents to remove matrix interferences [12 ].

This method involves so, a simple QuEChERS

(Quick, Easy, Cheap, Effective, Rugged and Safe)

sample preparation procedure followed by detec-

tion by gas chromatography coupled with tandem

mass spectrometry (GC-MS/MS).

2. Materials and methods

2.1. Reagents and solutions

Ultra-pure water (18.2 MU cm-1) was obtained in-

house using a Millipore water puriﬁcation system

(Cork, Ireland). Acetonitrile LC-MS grade was

purchased form Chem-Lab NV (Zedelgem,

Belgium).

QuEChERS original method (4 g MgSO

and 1 g

NaCl) and QuEChERS d-SPE (dispersive Solid

Phase Extraction) Animal Origin Food 5982-4950

(50 mg PSA, 150 mg C18EC and 900 mg Na

)

were obtained from Agilent technologies.

2.2. GC-MS/MS instrumentation and settings

The GC-MS/MS analyses were conducted with a

Shimadzu GC 2010 gas chromatograph coupled

with a Shima dzu TQ-8050 tandem mass spectrom-

eter (Shimadzu, Kyoto, Japan). The system was

equipped with a PTV (Programmed Temperature

Vaporization) injection inlet and an AOC-5000

autosampler. GC-MS Real Time Analysis and GC-

MS Postrun Analysis software (Shimadzu, Kyoto,

Japan) were used for instrument control and data

analysis, respectively. The GC analysis was per-

formed on a J&W DB5-MSþDG column (length

30 m, id 0.25 mm, ﬁlm thickness 0.25 mm þ 10 m

Guard Column), the chromatographic conditions

were the following: carrier gas he lium (minimum

purity 99.9995%) in constant linear velocity mode at

30 cm/sec, PTV Injector 60-200



C at 240



C/min

1 min then 330



C for 19 min, septum purge 6 mL/

min, Split 1:1; GC oven temperature program of 40



C for 1 min, ramp 20



C/min to 130



C, hold 7.0 min

then 40



C/min until 280



C (held for 3 min), injec-

tion volume 1 mL.

The mass spectrometer was operated in MRM

mode (MRM transitions of all ﬁve analytes and the

internal standard (IS) in Table 1) with the following

conditions: electron impact ionization at 70 eV, MS

transfer line temperature 280



C, MS source tem-

perature 200



C, solvent delay 9.6 min, dwell time

300 ms, collision gas argon (minimum purity

99.9999%) with a collision cell pressure of 200 kPa

and detector gain ﬁxed at 1.4 kV.

2.3. Phytogenic compounds standards, internal

standard solution preparation

All high purity phytogenic compounds standards

were purchased from Sigma-Aldrich.

Mixed standard solutions of the analytes were

prepared in acetonitrile by diluting the stock solu-

tions to concentrations of 50, 400, 800, 1200, 1600 and

2000 mg/mL. The internal standard (IS; Butyrophe-

none) solution was prepared at a concentration of

200 mg/mL in acetonitrile as well.

The calibration solutions were prepared in tripli-

cate (n ¼ 3) by spiking 240 mL of phytogenics-free

chicken breast extract with 30 mL of the appropriate

standard solutions of the analytes to obtain con-

centrations of 2, 5, 10, 20, 50 and 100 mg/mL. The

concentration of the IS in each sample was main-

tained at 10 ppb by adding 30 mL of the IS stock

solution. In this way each matrix matched standard

solution contained the same amount of matrix an d

pure solvent.

2.4. Samples

Samples of chicken breast were purchased from

the local market in Austria. Each sample was ho-

mogenized with the use of a Retsch Mixer Mill MM

400 and stored at 20



C before analysis.

2.5. Sample preparation

One g of homogenized chicken breast was

weighed in a Retsch stainless steel jar, 1 mL of ul-

trapure water, 2 mL of acetonitrile and 0.7 g of

Agilent QuEChERS original method salts were

added; the jar was closed and the sample was

extracted using a Retsch Mixer Mill MM 400 for

2 min at 30 Hz. Hereafter 1.5 mL of the solution were

taken, transferred in a 1.5 mL glass vial and

256 JOURNAL OF FOOD AND DRUG ANALYSIS 2021;29:255e261

ORIGINAL ARTICLE

centrifuged for 5 min at 3234 rcf. An aliquot of

700 mL of the supernatant was transferred in a

1.5 mL glass vial containing 105 mg of QuEChERS

Dispersive SPE, the content of the vial was vortexed

for 1 min and then centrifuged for 5 min at 14000 rcf.

The sample was then transferred into a GC vial and

only at this point the IS was added, to avoid losses

during the sample prepar ation. The IS was used to

compensate e.g. for detector and injection volume

ﬂuctuations [ 13 ].

2.6. Method validation

The following performance characteristics were

evaluated for the validation of the method: selec-

tivity, identiﬁcation, linearity, matrix effects, limits

of quantiﬁcation (LOQ), recoveries and precision.

2.7. Selectivity

The selectivity was veriﬁed by analyzing chicken

breast samples that were free of the compounds of

interest and the presence of peaks that could inter-

fere with those substances were assessed.

2.8. Identiﬁcation

The criteria used for the identiﬁcation of analytes

were retention time (Rt) and the presence and the

relative intensities of three MRM transitions (one

quantiﬁer and two qualiﬁers). According to SANTE

2019 criteria, the Rt of a compound of interest in a

sample should not vary more than ± 0.1 min

compared to a calibration standard and, the relative

intensities for the samples should be within ± 30%

(relative) of average of calibration standards from

the same sequence [14].

2.9. Linearity

Linearity was assessed by measuring six points

calibration curves in triplicate (2, 5, 10, 20, 50 and

100 ppb e corresponding to a range of 4 to 200 mg/kg

in chicken breast) prepared in acetonitrile and in

blank chicken breast extracts as well.

2.10. Matrix effects

In order to assess the suppression or enhance-

ment of the signal of the analytes due to the matrix,

the slopes obtained from the linearity study were

used to calculate the percentage of the matrix effect

(%ME), according to the following formula:

Table 1. MRM Transitions of the investigated phytogenic compounds and an internal standard.

Compound Retention

Time (mins)

MRM Transition

1 (Quantiﬁer)

Collision

Energy (eV)

MRM Transition

2 (Qualiﬁer)

Collision

Energy (eV)

MRM Transition

3 (Qualiﬁer)

Collision

Energy (eV)

Menthol 9.202 95 > 55.1 16 95 > 67.1 10 81 > 79.1 12

Methyl salicylate 9.545 105 > 77.1 16 105 > 51.1 24 77 > 51.1 16

Carvone 10.721 120 > 92.1 12 152 > 120.1 8 120 > 64.1 22

Butyrophenone (IS) 10.910 82 > 54.1 6 108 > 93.1 10 93 > 77.1 16

Thymol 11.793 135 > 91.1 16 150 > 135.1 12 135 > 115.1 16

Carvacrol 12.113 135 > 91.1 16 150 > 135.1 12 135 > 115.1 16

JOURNAL OF FOOD AND DRUG ANALYSIS 2021;29:255e261 257

ORIGINAL ARTICLE

%ME¼



Slope Matrix matched standards

Slope Standards in solvent



 1



 100

Negative values indicate signal suppression

while positive values indicate signal enhancement:

matrix effects in the range /þ 20% are permi ssible

[15].

2.11. LOQ

The instrumental LOQ were determined

following [16]: the standard deviation of a number of

samples at a low concentration was determined.

Multiplying this standard deviation (SD) with 3.3*ta

(ta th e Student's t correlating to the number of

samples the standard deviation is based on) gives

the LOQ.

LOQ ¼ SD*3.3*ta

The reporting level of the method was set at the

spike level (see Recoveries and precision) in which

the SANTE 2019 criteria (70-120% recoveries range

with a 20% RSD) were fulﬁlled [14].

2.12. Recoveries and precision

Recoveries and precision were evaluated by

spiking chicken breast samples that were free of the

compounds of interest and preparing them accord-

ingly to the procedure outlined in the Sample

preparation. The recoveries were determined for six

replicates prepared on three different days (day 1,

day 2 ¼ day 1 þ 24h and day 3 ¼ day 1 þ 144h) at

three different spike levels: 5, 10 and 50 mg/kg. The

average intraday and interday recoveries and the

relative standard deviation (RSD) were calculated as

the ratio of the analyte-to-IS peak area an d the re-

sults were evaluated for compliance to the SAN TE

2019 criteria, according to which the average re-

covery should be in the range 70-120% with an RSD

less or equal 20%.

3. Results and discussion

3.1. Method development

3.1.1. GC-MS/MS conditions

The GC parameters (initial PTV injector temper-

ature and ramp rate, initial and ﬁnal column tem-

peratures, as well as the column temperature ramp

rate and carrier gas ﬂow rate) were optimized to

achieve the best sensitivity and chromatographic

separation. It was possible to achieve chromato-

graphic separation for all the compounds of interest

and the IS, see Fig. 1.

In order to ﬁnd the best MRM transitions the

Shimadzu MRM Optimization Tool 1.14 (Shim adzu,

Kyoto, Japa n) softwar e was used: this software au-

tomates the process by collecting product ion scan

data and ﬁnding the optimum collision energy for

each trans ition. Three MRM transitions per analyte

were chosen: one quanti ﬁer and two qualiﬁers.

3.2. Sample preparation

The selection of the right sorbent is critical in

order to minimize matrix interferences and achieve

good and consistent analytes recove ries. Due to the

nature of our matrix we decided to use an animal

origin food sorbent. We evaluated the efﬁciency of

the dSPE step by measuring extracts which have

been treated or non-treated with the dSPE sorbent

in Full Scan mode, and then comparing the sum of

the areas of the chromatographic peaks in the TIC

chromatograms (m/z 50-600) [17]. The reduction for

the treated was in the range of 60%.

3.3. Method validation

3.3.1. Selectivi ty

No interfering peaks that could prevent the

identiﬁcation or the quantiﬁcation of the com-

pounds of interest were observed in 12 different

chicken breast samples that were analyzed. In

Fig. 1. MRM chromatogram of the chicken breast spiked with target analytes at the concentration of 10 mg/kg with the assigned peaks.

258 JOURNAL OF FOOD AND DRUG ANALYSIS 2021;29:255e261

ORIGINAL ARTICLE

addition, 10 procedural blanks were prepared as

well [18]. Also, in this case no interfering peaks were

detected.

3.3.2. Identiﬁcation

The SANTE 2019 criteria mentioned before were

fulﬁlled by the matrix matched standards and all the

reals samples as well. In Fig. 2 a chromatogram of a

real sample is shown.

3.3.3. Linearity

Good linearity was achieved for all the com-

pounds of interest in the measured calibration range

with a coefﬁcient of determination (R

) al-

ways > 0.995 in pure solvent and matrix as well.

3.3.4. Matrix effects

The matrix effect results, expressed as %ME are

shown in Table 2. Signal enhancement was

observed for all investigated substances, this is the

most common behavior for GC where the matrix

components block the active sites of the column [19].

Only for menthol the %ME is below ± 20% so we

decided to validate the method with matrix matched

calibration. Not surprisingly the %ME for the two

isomers thymol and carvacrol are very similar.

Fig. 2. MRM chromatogram of a real chicken breast sample with the assigned peaks.

Table 2. %ME of investigated analytes, LOQ (mg/kg), accuracy and precision. Conc. in mg/kg and precision data are given in parenthesis as %

coefﬁcient of variation.

Analyte ME% LOQ (mg/kg) Conc. Intraday Interday

Day 1 Day 2 Day 3

Menthol 12 3.8 5 106 (11) 106 (10) 94 (13) 102 (12)

10 97 (8) 89 (10) 88 (7) 91 (10)

50 78 (8) 82 (6) 79 (6) 80 (7)

MES 70 4.3 5 89 (10) 98 (14) 86 (13) 91 (14)

10 88 (15) 95 (9) 88 (9) 90 (12)

50 89 (14) 84 (12) 79 (6) 84 (12)

Carvone 26 2.9 5 87 (16) 91 (6) 87 (12) 89 (12)

10 83 (10) 76 (9) 76 (18) 87 (12)

50 89 (9) 82 (10) 81 (6) 84 (10)

Thymol 34 2.7 5 99 (10) 103 (11) 100 (14) 100 (12)

10 87 (7) 99 (7) 99 (9) 95 (11)

50 81 (10) 92 (12) 95 (6) 89 (12)

Carvacrol 38 4.8 5 97 (9) 82 (11) 94 (14) 91 (14)

10 88 (9) 91 (14) 112 (4) 100 (13)

50 81 (11) 89 (14) 100 (8) 90 (14)

Table 3. Concentration of phytogenic compounds in real samples in mg/kg.

Conc. Chicken Breast (mg/kg) Menthol Methyl salicylate Carvone Thymol Carvacrol

Sample 1 15,9 104,2 57,1 3,8 6,3

Sample 2 4,9 53,8 18,8 4,2 6,8

Sample 3 7,6 43,8 22,1 6,4 9,0

Sample 4 9,4 90,1 33,8 6,1 8,7

Sample 5 12,6 47,7 24,3 7,5 10,1

Sample 6 5,0 28,0 9,9 6,6 9,2

Sample 7 7,1 81,1 31,0 7,3 9,9

Sample 8 18,8 74,6 36,0 6,1 8,7

Sample 9 15,9 98,8 61,9 7,8 10,5

Sample 10 10,2 100,7 45,8 3,6 6,1

JOURNAL OF FOOD AND DRUG ANALYSIS 2021;29:255e261 259

ORIGINAL ARTICLE

3.3.5. LOQ

Instrumental LOQ were determined by injecting

10 times the lowest matrix matched standard at

4 mg/kg, the instrumental LOQs were always below

the lowest calibration level and are reported in

Table 2.

The SANTE 2019 criteria were fulﬁlled at the

lowest spike level of 5 mg/kg and so the reporting

level for the method was set at this concentration.

3.3.6. Recoveries and precision

The recoveries and the associated accuracy and

precision values were within the acceptable in-

terval of the SANTE 2019 criteria for all the three

spike levels and both for intraday and interday

measurements. The overall results are shown in

Table 2.

3.4. Application to real samples

The validated method was applied to 10 samples

of chicken breast bought in the local market. As

shown in table 3 all the substa nces of interest were

detected in the samples in amounts above the LOQ

and with a quite variable range among the different

samples, from just above the LOQ for thymol to

104 mg/kg for methyl salicylate.

From a consumer safety point of view, all the

analyzed substances have been assess ed and

currently authorized for food [20] and feed [21] uses.

For menthol and carvone an acceptable daily intake

(ADI) is established, and it is equal to 4 mg/kg body

weight for menthol [22] and 60 mg/kg body weight

for carvone [23]. Even taking the highest concen-

trations found in the real samples the chronic

exposure is more than 5 orders of magnitude below

the aforementioned ADIs.

4. Conclusions

A simple analytical method involving a QuECh-

ERS extraction followed by a dSPE cleanup step

coupled with GC-MS/MS determination was

demonstrated to be suitable for the quantiﬁcat ion of

phytochemical residues in chicken breast, a com-

plex food matrix. The method has been validate d

according to the SANTE 2019 criteria for accuracy

and precision. It was applied to real chicken breast

samples and revealed the presence of the com-

pounds of interest in all of them, however well

below any level of concern for the consumer safety.

This is possibly an indication of the widespread use

of phytogenic feed additives after the ban of anti-

biotics as growth promoters by the European Union

in 2006.

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