Arabian Journal of Chemistry (2020) 13, 3882–3888

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Fast and simple method for the detection and

quantification of 15 synthetic dyes in sauce, cotton
candy, and pickle by liquid chromatography/tandem
mass spectrometry
Abdullah Al Tamim *, Mohammed AlRabeh, Ahmed Al Tamimi, Abdulaziz AlAjlan,
Abdullah Alowaifeer *

Food Reference Laboratory, Saudi Food and Drug Authority (SFDA), Riyadh 11561, Saudi Arabia

Received 12 July 2018; accepted 29 September 2019

Available online 23 October 2019

KEYWORDS Abstract A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS)
Food additives; method for the simultaneous determination of 15 illegal dyes (Sudan I, Sudan II, Sudan III, Sudan
Synthetic dyes; IV, Sudan Red G, Sudan Orange G, Sudan Red 7B, Para Red, Dimethyl Yellow, Rahodamine B,
Sauce; Sudan Black B, Sudan Red B, Auramine O, Toluidine Red and Orange II) was developed and val-
Cotton candy; idated in sauce, cotton candy, and pickle. The samples were extracted with acetonitrile without the
Pickle; use of solid-phase extraction cartridges. Chromatographic separation was achieved on a Zorbax
LC - MS/MS Eclipse Plus C18 column with a ow rate of 500 mL/min at 45 °C, using a gradient elution with
A (10 mM ammonium formate in water with 0.1% formic acid) and B (10 mM ammonium formate
in acetonitrile (ACN) with 0.1% formic acid) as the mobile phase. The detection was performed on
a AB Sciex 6500 Qtrap mass analyzer under multiple reaction monitoring mode. Limit of detection,
quantication, linearity, and precision were determined during the validation process. Recoveries
ranged from 82% to 119% for all synthetic dyes, in exception to Orange II in cotton candy and
pickle, where signal was suppressed due to high matrix interference and poor ionization. This
method offers a simple and rapid approach to detect and quantify prohibited dyes in foodstuff that
can be utilized in food contaminant laboratories.
Ó 2019 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.
1. Introduction
E-mail addresses: [email protected] (A. Al Tamim), vet.abdullah@
gmail.com (A. Alowaifeer).
Peer review under responsibility of King Saud University. Synthetic dyes are chemical compounds used in food and phar-
maceutical industries for appearance enhancement purposes
due to their effectiveness, stability and low cost (Ma et al.,
2006; Vachirapatama et al., 2008). Azo dyes, such as Sudan
Production and hosting by Elsevier I – IV, are known class 3 carcinogens (IARC, 1975) and their
https://doi.org/10.1016/j.arabjc.2019.09.008
1878-5352 Ó 2019 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Simple detection of 15 dyes using LC – MS/MS 3883

use as a food coloring agent is mostly illegally worldwide (Zhu 2.2. Instrument
et al., 2014). However, these colorants are still commonly
reported in many imported food products (Abdelmigid, The LC-MS/MS system consisted of an ACQUITY UPLC I-
2009). Following the detection of several Sudan dyes in chilli Class System (Waters Technologies USA) equipped with a
powder, spice mixtures and sauces imported from India, Pak- Binary Solvent Manager, Sample Manager - Flow-Through-
istan, Thailand, Lebanon and Turkey, the European Union Needle and a column heater with active pre-heating coupled
(EU) adopted emergency measures regarding the application to a 6500 Qtrap mass analyzer (AB Sciex, Canada). Nitrogen
of synthetic dyes in foodstuff (Directive, 2003/460/EC). Regu- was used as curtain gas, nebulizer gas, and collision gas on
lations require that all food products exported to the EU be the MS. Separation was carried out on a Zorbax Eclipse Plus
certied as fraudulent dyes free. C18 column (2.1  50 mm. 1.8 mm) with a C18 guard column
Several methods have been developed to detect synthetic (Agilent, USA).
dyes in multiple food matrices. Among the reported methods,
high performance liquid chromatography (HPLC) coupled
2.3. Standard solutions
with ultraviolet (UV) or diode array detection (DAD) is the
most common (Zheng et al., 2007; He et al., 2007). In addition,
thin layer chromatography (TLC) and spectrophotometry are The stock standard solutions were prepared individually at a
also popular approaches considering their low cost (Oka et al., concentration of 100 mg/mL. Sudan I, Sudan II, Sudan Red
1994; Zalacain et al., 2005). However, these methods usually G, Sudan Orange G, Sudan Red 7B, Dimethyl Yellow, Rho-
introduce complications due to their low sensitivity and high damine B, Sudan Black B, Sudan Red B and Auramine O were
interferences. Liquid chromatography/tandem mass spectrom- prepared in ACN. Sudan III, Sudan IV, Para Red and Tolu-
etry (LC-MS/MS) and gas chromatography/mass spectrome- idine Red were dissolved in DCM, and Orange II was prepared
try (GC–MS) methods have shown to be reliable, but the in ethanol. An intermediate standard solution of the dyes at a
solid- phase extraction (SPE) involved in the sample prepara- concentration of 1000 ng /mL was prepared by dissolving a
tion is time-consuming (Calbiani et al., 2004; Wanget al., suitable volume of stock standard solutions in ACN. Standard
2008). solutions were stored in the in the dark at 4 °C.
To the authors knowledge there is only one published
method to detect synthetic dyes by LC-MS/MS that does not 2.4. Sample preparation and extraction
require SPE (Tsai et al., 2015). Tsai et al. (2015) method uses
10 g of sample and the analysis time exceeds 20 min, which Tomato sauce, cotton candy, and cucumber pickle were
are considered disadvantages when accommodating large obtained from the local market. A 1 g of homogenized sample
surveillance programs. Also, the method did not include aura- was weighed into a 15 mL polypropylene centrifuge tube and
mine O, Sudan black, Rhodamine B, Toluidine Red and extracted with 10 mL of ACN. Sample was then vortexed for
Orange II, which are prohibited additives of great signicance 1 min and placed in an ultrasonic bath for 30 min. After son-
to food safety. ication, sample was centrifuged at 7500 rpm (10 min, 15 °C)
Saudi Arabia imports large quantities of food from south and 1 mL of supernatant was transferred to an LC autosam-
east Asia; therefore, it was crucial to develop a simple and pler vial. Sample vials were kept in the dark at 4 °C prior to
practical method to ensure consumers’ safety against fraudu- analysis by LC-MS/ MS.
lent colorants. In this study, a rapid analytical method for
the detection and quantifying of 15 illegal dyes in sauce, cotton 2.5. LC-MS/MS analysis
candy, and Pickle by LC MS/MS was developed. The pro-
posed method offers a single-step extraction and a 5 min anal-
ysis run time. The mobile phase system consisted of A (10 mM ammonium
formate in HPLC grade water with 0.1% formic acid) and B
(10 mM ammonium formate in ACN with 0.1% formic acid).
2. Material and methods The linear gradient elution was as follows: 75% B (initial),
75% B (from 0 to 2 min), 98% B (2 to 3.50 min), 75% B
2.1. Reagents and chemicals (3.50 to 5 min). The ow rate was set at 500 mL/min, while
the column temperature was maintained at 45 °C and the injec-
HPLC grade acetonitrile (ACN), HPLC grade water, Dichlor- tion volume was 5 mL. Electrospray ionization mass spectra
omethane (DCM), Ethanol, ammonium formate (99.0%), (ESI-MS) were acquired in the positive (ES+) and then nega-
and formic acid (98%) were purchased from Merck (Darm- tive (ESI) ion mode. The Turbo Ion source operated at
stadt, Germany). Sudan I, Sudan II, Sudan III, Sudan IV, 500 °C with the electrode voltage at 5500 V. Other MS param-
Sudan Red G, Sudan Orange G, Sudan Red 7B, Para Red, eters were as follows: turbo gas, 50 psi; curtain gas, 25 psi; col-
Dimethyl Yellow, Rahodamine B and Orange II were obtained lision gas, 12 psi; entrance potential, 10 V. A total of 14 dyes –
from the Institute of Leather Industry (Łódź, Poland). Sudan Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Red G, Sudan
Black B and Sudan Red B were purchased from Dr. Ehrenstor- Orange G, Sudan Red 7B, Para Red, Dimethyl Yellow, Raho-
fer GmbH Company (Augsburg, Germany), and Auramine O damine B, Sudan Black B, Sudan Red B, Auramine O and
and Toluidine Red were obtained from Sigma Aldrich (St. Toluidine Red – were detected by positive ESI, and Orange
Louis, MO, USA) (Fig. 1). II was detected by negative ESI. Dyes were identied by reten-
tion time and multiple reaction monitoring of each standard.