1. Introduction
Breastfeeding is admitted to present numerous beneficial health effects and it is vastly considered as the best suitable food for the baby [
1]. The World Health Organization recommends to exclusively breastfeed for six months after birth [
2]. However, the European recommendation varies among countries between four and six of exclusive breastfeeding [
3].
In Lebanon and Syria, two Middle Eastern Arab countries sharing almost the same socio-demographic characteristics including religion and language, a percentage of 10% was shown for an exclusive breastfeeding rate at six months [
4]. According to the United Nations Children Fund [
5], the average of breastfed Syrian infants under six months of age was found to be 43%. The absence of support for breastfeeding by social workers and health care providers were considered the main reasons for low prevalence of exclusive breastfeeding. It could be also due to psychological reasons such as mother’s emotional stress and the perceived breast-milk inadequacy [
6].
Breast milk is an important source of energy and nutrients for children’s health [
7]. However, many studies have been conducted to assess the chemical contaminants in breast milk associated with its health effects on the infant and the mother. This contamination could occur due the exposure of the nursing mother on a daily basis to chemical pollution of the environment. These environmental chemicals are released from several basic activities and different sources such as water, air, food and manufactured products [
8].
Few research studies have reported the presence of antibiotic and pesticide residues in breast milk. It is worth mentioning that studies screening antibiotic residues are rare on the international scale. The latest study conducted in Turkey [
9], detected quinolone and beta-lactam residues in human milk samples, with an occurrence of 8/34 (23.5%) and 29/34 (85.2%), respectively. It is to be noted that the lactating mothers enrolled in this study do not have antibiotic history. Antibiotic residues in human milk were thought to derive from the mothers’ food such as chicken, meat and dairy products [
10].
Additionally, some studies have screened the presence of pesticide residues in breast milk especially organochlorine pesticides [
11,
12,
13,
14,
15]. The most important study was carried out in China where a wide range of concentrations for 23 organochlorine pesticide (OCP) compounds were detected in 24 pooled samples of breast milk. The dichlorodiphenyltrichloroethane (DDT) contamination in breast milk was the highest with a mean value 582.8 ± 362.7 ng/g. The explanation for such high contamination was mainly from the mother’s dietary intake since positive correlation was observed between concentration of DDT in human milk and consumption of animal origin food in the Chinese population [
16].
However, to our knowledge no studies have been conducted on Syrian refugee lactating mothers residing in camps especially where the living and environmental conditions are very poor. Moreover, our study is the first conduced in Lebanon, screening antibiotics and pesticide residues in breast milk. Thus, the aim of this study was to assess the occurrence of antibiotic and pesticide residues in the breast milk of Syrian refugee lactating women residing in Lebanon camps. It investigated as well the socio-demographic and nutritional factors associated with milk contamination.
2. Materials and Methods
2.1. Study Population and Area
This cross-sectional study was conducted in Syrian refugee camps located in Lebanon, North Lebanon city—Akkar camps. The participants in this study were lactating Syrian refugee mothers residing in camps. In total, 40 women were randomly recruited and invited to participate in the study by providing mature breast milk samples.
2.2. Ethical Approval
The study protocol and ethics of this study were approved by the institutional review board (IRB) code: 2019H-0087-HS-M-0325 (17 January 2019), of Beirut Arab University prior to approaching the participants and a written, signed informed consent was obtained from all participants. All women who volunteered to participate in this study were informed about the purpose of the study and agreed to be part of it.
2.3. Questionnaire
A questionnaire was used to collect data about the socio-demographic characteristics of participating lactating mothers. The questionnaire was adopted from different studies [
14,
16,
17,
18,
19,
20]. It consisted of eight subsections collected from different sources, addressing general information about the lactating mothers and the new-borns, dietary habits of lactating mothers, smoking status, geographic characteristics, pesticides spraying, medical history and supplements intake as well as the drinking water intake.
The questionnaire was translated into Arabic to ensure accuracy of the translation, back translation from Arabic to English was conducted by another translator. Both English versions were the same. The Arabic version of the questionnaire was pilot-tested on a sample of 30 mothers. The questionnaire was administered twice within a period of two weeks between each sitting. To determine test–retest reliability, a paired
t-test was performed to compare mean scores at T
1 (before) and T
2 (after two weeks). Pearson’s correlation was calculated between T
1 and T
2, with a consistency interclass correlation (ICC) [
21]. The values of ICC were considered excellent if >0.75 [
22].
2.4. Sample Collection
During December 2018 and February 2019, 50 nursing mothers were contacted but only 45 agreed to answer the survey and participated in our study. The 45 nursing mothers that were visited for the survey were contacted again and only 40 of them agreed to donate breast milk by triplicate over three weeks.
Breast milk collection was done in the morning an hour after the previous breastfeeding. Samples (25–40 mL) were collected using a sterile, single-use electrical pump (Chico, Mod.06836, China). A polypropylene pump container was sterilized with 70% alcohol solution to prevent cross contamination between samples. Each sample was placed into a separate 100 mL dark glass (to preserve the samples from light), numbered then transported on ice (at 4 °C) to the laboratory, where they were transferred into urine cups, numbered and stored at −20 °C until analysis.
2.5. Chemicals and Reagents
The reagents used were of analytical grade. The antibiotic standards belonged to three different families: sulphonamides (sulfamethazine), tetracyclines (tetracycline, oxytetracycline) and beta-lactam antibiotics (ampicillin); in addition, 161 kinds of pesticide were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standards used present high purity grades (>99%). Individual stock solutions were prepared at 1000 µg/L in acetonitrile and stored at −20 °C.
The working standard solutions of a concentration of 10 µg/L each were prepared as dilution of the stock solution in water/methanol (50:50, v/v). The working standard solutions were stored at −20 °C. HPLC-grade water, HPLC-grade acetonitrile, and magnesium sulphate (MgSO4) were also supplied by Sigma-Aldrich.
2.6. Sample Extraction for Antibiotics Residues Analysis
Using liquid chromatography–mass spectrometry, a multi-class method was developed for identifying and quantifying three antibiotics belonging to two different chemical classes (tetracyclines and beta-lactams). Afterwards, this method was optimized for the detection of antibiotics in breast milk.
100 μL from 1 % formic acid was added to a 2 mL of breast milk then vortexed for 60 s and kept in the dark for 10 min. Then 500 μL of EDTA (0.5%) was added and shaken vigorously for 1 min. Next, 8 mL TCA (5%) was added then shaken for 10 min before subjecting the tubes to centrifugation at 5000 rpm at 4 °C for 10 min. The supernatant obtained was then subjected to filtration through a 0.45 μm polyvinylidene fluoride (PVDF) filter for further liquid chromatography tandem mass spectroscopy (LC-MS/MS) analysis.
2.7. Sample Extraction for Pesticide Residues Analysis: QuEChERS Extraction
To ensure a quick and easy sample treatment, a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) approach, originally developed as a powerful sample preparation, was used to analyse hundreds of pesticides in different kinds of food.
Acetonitrile (10 mL) was added to 10 mL of breast milk and shaken vigorously before adding magnesium sulphate MgSO4 (4 g), sodium chloride NaCl (1 g), sodium dibasic citrate (1 g) and sodium tribasic citrate (0.5 g) and then shaken for 3 min. The tubes were then subjected to centrifugation at 5000 rpm at 4 °C for 5 min.
The aqueous supernatant (5 mL) was mixed with dispersive Enhanced Matrix Removal (EMR) dissolved in 5 mL water then shaken vigorously for 5 min. Another centrifugation was conducted at 5000 rpm at 4 °C for 5 min.
The aqueous supernatant (5 mL) obtained was transferred in a new Polish EMR Lipid (MgSO4 and NaCl tube, then shaken vigorously for 3 min before being centrifuged at 5000 rpm at 4 °C for 5 min. Afterwards, a filtration was made through a 0.22 μm polyvinylidene fluoride (PVDF) filter for further LC-MS/MS and GC/MSMS analysis.
2.8. LC-MS/MS Equipment
LC-MS/MS analyses was conducted using an Agilent 6430 LC/MS (Agilent technologies, Santa Clara, USA) for antibiotic and pesticide residues detection, and LC-MS/MS analyses were performed on LC-NexeraX2 Shimadzu 8045 LC/MS (Kyoto, Japan). The mass spectrometer was operated with an electron spray ionisation (ESI) in multiple reaction monitoring (MRM) mode, ionspray voltage 4 kV, nitrogen for desolvation and dried gas 11 L/min.
The quantification of the three antibiotics families and 161 kinds of pesticide in 120 breast milk samples was done through measuring peak areas in the multiple reaction monitoring chromatogram, and comparing them with the relevant matrix-matched calibration curves. A calibration curve ranging between 1 μg/L and 500 µg/L was carried out to verify linearity.
The performance of the analytical method was evaluated by checking the identification criteria for the presence of two transitions at the same retention time, the signal to noise ratio ≥ 10, the relative retention time of the analyte within a tolerance of 2.5% and the relative ion intensities ratio within a tolerance defined by the EU commission decision 2002/657/EC. The MRM parameters are shown in
Table 1.
2.9. LC/MS/MS Parameters
The separation of the pesticide residues was performed using a C18 analytical column (zorbax 2.1 mm inner diameter × 150 mm length, 3.5 μm particle size; Agilent technologies, Santa Clara, CA, USA) and a C18 analytical column (Shim-pack GIST 2.1 mm inner diameter × 100 mm length, 3 μm particle size; Japan) for antibiotic residues separation. The separation of pesticides was accomplished at 40 °C. The flow rate and injection volume were 0.4 mL/min and 10 µL, respectively. The mobile phases used were (A) 5 mM ammonium acetate and 0.1% formic acid in water, and (B) 5 mM ammonium acetate in methanol. The gradient elution program started with 100% A for 5 min, then decreased to 50% for 13 min, then to 0% for 2 min and returned back to the initial conditions for 5 min. The final run time of the method was 29 min.
The separation of sulfamides, tetracyclines and beta-lactam was accomplished at 40 °C. The flow rate and injection volume were 0.3 mL/min and 10 µL, respectively. The mobile phases used were (A) 5 mM ammonium acetate and 0.1% formic acid in water, and (B) 5 mM ammonium acetate in methanol. The gradient elution program was as follow: B (40%) (2 min), B (90%) (9 min), B (10%) (9 min and 1 s), the final run time of the method was 13 min.
2.10. GC/MSMS Parameters
GC-MS/MS analyses were carried out with an Agilent 7890A GC equipped with 7693 Agilent auto-sampler and 7000C triple quadrupole GC/MS system. The separation was performed on a HP-5MS Agilent technologies (Santa Clara, CA, USA), 0.25 mm × 30 m, 0.25 μm, and helium (purity 99.996%) was used as a carrier gas at a constant pressure of 11 psi. The inlet temperature was set at 250 °C; the mode of inlet was splitless; the injection volume was 1 μL. The column temperature program was as follows: the initial temperature was maintained at 70 °C for 1 min, increased to 160 °C at a rate of 50 °C/min, raised to 200 °C at 2 °C/min, and then at 16 °C/min up to 280 °C, and held there for 7.2 min. The total run time was 35 min. The mass spectrometer was operated with an electron impact (EI) source in multiple reaction monitoring (MRM) mode. The electron energy was 70 eV, and the source, transfer line and quadrupole temperatures were set at 280 °C and 150 °C, respectively. In order to prevent instrument damage, the solvent delay was set at 4.5 min.
2.11. Recovery Test
In-house validation was performed by fortifying the blank matrix at a level of 50 µg/L. The extraction was performed by the methods described in the
Section 2.6 and 2.7. The spiked and blank samples were then analysed by LCMS/MS. Recovery was calculated by comparing the analysed concentrations with spiked concentrations.