# Microplastics' Detection in Honey: Development of Protocols in a Simulation
## 1 Introduction  
Honey is one of the most nutritious natural products, with essential and therapeutic properties for human health [1, 2]. It has been reported that it exhibits an inhibitory effect on many species of pathogenic bacteria [3, 4]. Nowadays, in modern medicine, honey is used for wound healing, including burns, treatment, and prevention of gastrointestinal track diseases, fungal infections, and cardiovascular diseases due to its antioxidant properties [4]. It is a viscous nonhomogeneous colloid substance consisting mostly of sugars, water, and solid particles such as pollen, pollen grains, wax, and propolis [3, 5]. As is already known, plastic pollution has affected bees. Microplastics (MPs) and particles such as syntheticfibers and fragments have been detected on bees' bodies and wings, as well as on the food they eat [6]. MPs are then transferred to the hive from the blossoms and eventually migrate into honey [7; 8]. Different chemical compounds have been detected in honey samples [9], such as styrene, phthalates, and bisphenol A [10]. Also, the detection of MPs in honey samples has been achieved with filtration methods, and identified with Raman and FTIR spectroscopy [11; 12]. The extraction of MPs from honey has not been studied thoroughly because the detection, isolation, and origin determination of MPs in honey samples is challenging for researchers, due to honey composition [13]. Additionally, the interface of food with plastic packaging such as polyethylene terephthalate (PET) and other polymers causes migration of MPs [14]. Their detection is difficult to achieve without any chemical pretreatment to remove the organics and needs to be followed by chromatographic analysis [10; 13]. Food quality assurance is of crucial importance for the protection of consumers' health and, although plenty of work exists in the literature, there is still a gap in developing a protocol for efficient isolation and detection of MPs in honey. In this work, multiple methodologies were developed and tested to achieve the optimum isolation of MPs (with particle dimensions >1 \(\upmu\)m) from real honey samples, based on simulations. The simulations of PET plastic packaging migration into honey involve the introduction of PET powder [15] into honey samples. All the methodologies developed were designed based on equipment found in scientific labs, without the application of any digestion protocol to remove the organics. Raman mapping and optical and fluorescence microscopy were used for PET MP detection. Raman mapping, particularly Raman micro-spectroscopy, has no sample management costs since it is a label-free method, where pretreatment of sample preparation is not obligatory [16; 17]. This is the reason why Raman mapping is the first proposed method for multiple sample measurements [18; 19; 20]. Additionally, optical and fluorescence microscopy [21] is proposed as an alternative economic method for PET MP detection in honey. To enhance MP detection efficiency, pentane, an organic solvent, was used as a density separator [22]. This allowed the distribution of MPs and solid particles of honey in different extraction phases. Then, detection was performed in each phase. Alternatively to density separation, dyed PET MPs can be detected through optical and fluorescence microscopy using 4-dimethylamino-4'-nitrosolibene (DANS) dye [23] and under ultraviolet (UV) radiation. In this case, DANS-dyed MPs presented different fluorescence from the rest of the honey solids, allowing their discrimination through microscopy. Regarding particle sizes of MPs on the scale of nanoplastics (<200 nm), where their detection efficiency is beyond the limits of Raman micro-spectroscopy, mass spectrometry (MS) was employed. MS has been extensively used to identify specific fragments. In combination with other analytical approaches (for example, gas chromatography and liquid chromatography), MS is used for the detection of a limited amount of different polymers in environmental matrices [24] such as water, soil, and food [25].