Microextraction-Based Methods for Determination of Sulfonamides in Milk

Microextraction-Based Methods for Determination of Sulfonamides in Milk: Comparison

Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Victoria Samanidou.

Sulfonamides (SAs) represent a significant category of pharmaceutical compounds due to their effective antimicrobial characteristics. SAs were the first antibiotics to be used in clinical medicine to treat a majority of diseases, since the 1900s. In the dairy farming industry, sulfa drugs are administered to prevent infection, in several countries. This increases the possibility that residual drugs could pass through milk consumption even at low levels. These traces of SAs will be detected and quantified in milk.

sulfonamides
determination
extraction
microextraction
milk

1. Introduction

The term “sulfonamide” derives from para-amino-benzene-sulfonamide (sulfanilamide) and it is also known as streptocid. The structure is similar to para-aminobenzoic acid (PABA), which is demanded by microorganisms (such as bacteria) for dihydrofolic and folic acid synthesis. SAs (SAs) or alternative sulfa drugs have been known since the middle of the twentieth century and were proved to have antibacterial properties in 1935, thus they are some of the oldest antimicrobial drugs. Nowadays, SAs are widely used as antibiotics in veterinary medicine either in single formulation or synergistically with other antibiotics (such as tetracyclines (TCs), SAs, quinolones (Qs), fluoroquinolones (FQs) and trimethoprim (TMP). Their use aims to protect the animals from infectious diseases. In addition, their administration as additives to animal feed can promote growth that results in the rise of the productivity of livestock, despite the fact that the use of antibiotics is prohibited in various places around the world. For example, the concentration of SAs in meat produced in Denmark was on average 4.82 mg/kg (pork), 17.2 mg/kg (cattle), 0.033 mg/kg (broilers) and 58.5 mg/kg (fish). Human health can be influenced by the consumption of meat and dairy products (such as eggs, milk and cheese) which contain amounts of SAs. The use of SAs as additives is forbidden, aiming to ensure safety in human health. Furthermore, SAs are widely used as antibacterial drugs because they are of low cost and active against a broad spectrum of microorganisms. SAs can act, despite their bacteriostatic and chemotherapeutic activity, against infections caused by gram-positive and gram-negative bacteria and protozoa. As it is observed, every human is a passive consumer of sulfa-drugs, which are obtained from the treatment of diseases in animals. The systematic and long-term intake of SAs through the food could be characterized as dangerous and in some cases toxic, appearing in the form of allergic reactions, suppression of enzyme activity and alteration of the intestinal microflora ^[1][2].

It should be noted that only 40 of the 10,000 sulfanilamide derivatives that have been synthesized are used in medical and veterinary practice. SAs have similarities in their structure, as all of them carry the same molecule with the addition of diverse radicals at the R position ^[2].

The structures of some of the most commonly used SAs that are mentioned in this entry are presented in Figure 1.

Figure 1.

Chemical structures of sulfonamides (SAs).

Instructions for the withdrawal period have to be followed in order not be found as residues in milk, eggs, meat, tissues and other livestock products after drug administration ^[1][3].

On the other hand, milk is one of the most widely consumed foods and it is a rich source of protein and calcium, especially in children’s growth. With regards to its analysis, milk is considered as a complex matrix, which contains water, proteins, lactose, fats, minerals and vitamins. According to the European Union (EU), the combined total residues of all substances within the sulfonamide group should not exceed 100 μg/kg ^[4]. This is the maximum residue level (MRL) which should be in force for all target tissues (muscle, fat, liver and kidney) and for milk coming from bovine, ovine and caprine. The same MRLs were established in the USA and Canada ^[3]. Also, the regulation of Codex Alimentarius for the MRLs of the above drugs is referred to sulfadimidine and is set as 100 μg/kg for all target tissues, except for milk that is set to 25 μg/L ^[5].

Several methods have been developed and validated for the determination of SAs in different matrices such as environmental, biological or food by applying various techniques including photometry, spectrophotometry, gas chromatography (GC), gas chromatography tandem mass spectrometry (GC-MS/MS), capillary electrophoresis-ultraviolet detection (CE-UV), and high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection, fluorescence detection (FD) and mass spectrometry (MS) ^[6]. All of the developed methods require various sample preparation procedures. Sample preparation is the most demanding step of the analysis in comparison with the other two steps, such as sampling or measurement. At this critical step, the analytes need to be successfully isolated and the sample should reach a capable form for analysis. Sample preparation is crucial to produce accurate results. Thus, this step requires special attention, and it is a time-consuming procedure as well. There are many well-established sample preparation techniques and this field is very interesting for researchers. Therefore, novel techniques are introduced and used in different matrices. In recent years, the trend for every novel technique is to be environmentally friendly according to the principles of Green Analytical Chemistry (GAC) ^[7].

During recent decades, several extraction techniques have been used for sample pre-treatment. For the analysis of solid samples, the most applied techniques are Soxhlet (SOX) and pressurized solvent extraction techniques (e.g., supercritical fluid extraction (SFE), accelerated solvent extraction (ASE) and subcritical water extraction (SWE)), and the well-known liquid–liquid extraction (LLE) for the analysis of liquid samples. However most of the conventional techniques (SOX, SPE and LLE) seem to have significant drawbacks. They are time-consuming and complicated, consume large amounts of sample and organic solvents and are difficult to be automated.

In 1990, a novel technique, known as solid phase microextraction (SPME), was introduced by Pawliszyn and co-workers. SPME uses a fused silica fiber, which is coated with a sorbent (the fiber is incorporated in a chromatographic syringe) to extract the target analytes which subsequently are directly transferred into GC or HPLC. The technique has significant advantages. It is fast, simple, solvent free and it is compatible with analyte separation and detection by a chromatographic system (directly in gas chromatography, or via an interface in high-performance liquid chromatography) ^[8]. Due to the plethora of advantages, the SPME was extensively used for sample preparation of environmental and food samples. The environmental samples include water, air, soil and sediments, whereas food applications are based on fruits, vegetables, fats, oils, wine, meat and dairy products ^[9].

However, the use of SPME fibers involves some drawbacks such as:

their maximum operating temperatures are in the range between 240–280 °C
they are not stable with the organic solvents due to swelling
they break easily
the possibility of stripping of coatings due to analyst’s handling errors.

A modification of SPME was introduced in 1999 by Baltussen. This novel technique is called stir bar sorptive extraction (SBSE) and uses a stir bar consisting of a magnet covered with glass which in turn is coated by a layer (typically 0.5–1 mm) of sorptive material (usually polydimethylsiloxane- PDMS) for the extraction. Furthermore, microextraction by packed sorbent (MEPS) was developed and introduced as a further miniaturization version of SPE. In MEPS, SPE’s conventional polymeric cartridge was replaced by a stainless steel, miniaturized version termed the barrel insert and needle (BIN), which could hold any of a great number of sorbents, such as those used in SPE ^[7][10].

In the meantime, liquid phase microextraction (LPME) was introduced in order to overcome significant drawbacks of liquid phase extraction modes. In LPME, the amount of solvents is smaller in comparison with LLE; only some μL are required, whereas LLE consumes hundreds of mL. It is simple and cheap, as well as adaptable with capillary electrophoresis (CE), HPLC and GC.

Three modes of LPME can be applied. These include single drop microextraction (SDME), hollow fiber liquid-phase microextraction (HF-LPME) and dispersive liquid–liquid microextraction (DLLME). Extraction occurs into a small amount (usually 1–100 μL) of organic solvent (acceptor phase) from an aqueous matrix containing the analytes (donor phase) ^[11].

The essential feature of the above extraction techniques is the elimination of large amounts of organic solvents due to the fact that organic solvents are toxic and hazardous for the environment and human health. This complies with the principles of GAC, following the trend of using solvent-less or better described as solvent-free extraction methods.

The introduction of new sorbent materials in sample preparation is also of significant importance and has been widely investigated in order to prepare materials with higher adsorption capacity and selectivity, as well as to expand the availability of cheaper, more easily synthesized sorbents. The combination of microextraction techniques with the new sorbent materials is also base to the GAC demands ^[12][13].

2. The Demand of Microextraction Techniques

As already mentioned, sample preparation is the most demanding step in any analytical workflow. The main purpose of sample preparation is to transfer the analytes of interest from a complex matrix to a compatible medium for further determination by an analytical technique. In addition, sample preparation often includes procedures such as clean up, analyte enrichment and derivatization. So, it is clear that this step is time consuming and usually requires the use of large organic solvent volumes and the waste of reagents and consumables. For these reasons, the trend is the introduction of more “green” and micro-techniques in sample preparation. The idea of sustainable ecological development was introduced in 1987 in a report of the World Commission on Environment and Development. The term green chemistry was mentioned by P. Anastas in 1991 at the US Environmental Protection Agency (EPA). As a result, in 1993, the comprehensive US Green Chemistry Program was established, which included cooperation among many governmental agencies and research institutions. While Anastas and co-workers were elaborating the ideas of green chemistry, the first paradigms of green analytical chemistry were introduced. GAC, introduced in 1999, became a whole part of chemical nomenclature, and numerous reviews and original studies have been published in this topic. The principles of green chemistry and by extension of GAC are presented in Table 1 ^[14].

Table 1.

The twelve principles of Green Analytical Chemistry (GAC).

No.	Principle of GAC
1	Direct analytical approaches should be preferred in order to avoid sample preparation

^15], diode-array ^[16], or mass spectroscopy ^[17]. A significant number of contributions can be found in literature with regards to sample preparation of milk for the determination of SAs. These include either traditional techniques or modern ones. Solid phase extraction is widely used either in the classical approach or in an alternative way, based on the use of modern adsorbent materials. The use of the commercial SPE presents some disadvantages:

Although there is a wide range of chemistries, many choices for manipulating solvent and pH conditions, optimization is time consuming. In many cases, several steps are required.
The cost per sample is higher than that of simple liquid–liquid extraction (LLE).

Novel microexraction techniques were introduced to overcome these drawbacks. The new techniques require less time and labor than the multi-step procedures of SPE. These include SPME, SBSE, magnetic solid phase microextraction (MSPE), and other greener approaches. For example, a new in-tube solid-phase microextraction technique was introduced by Wen Y. et al. in 2005. The aim for this sample preparation technique is the determination of five SA residues in milk with HPLC-UV. The on-line in-tube SPME used poly-(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary as extraction media. The method is easily applied and environmentally friendly, following the demands of GAC ^[18].

3. Microextraction Techniques for the Determination of SAs in Milk

The reported techniques are summarized in Table 2.

Table 2.

Microextraction techniques for the determination of SAs in milk.

Analyte	Extraction Type	Determination	LOD	Recovery	Reference

sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfamethoxypyridazine, sulfaquinoxaline, sulfathiazole, sulfisoxazole	MSPE	HPLC-DAD	7–14 μg/L	81.88%–114.9%	^[16]
2	Minimum sample size and minimum number of samples are the main goals of this principle

sulfadiazine, sulfamerazine, sulfamethazine sulfamethizole, sulfamethoxazole, sulfadimethoxine	SBSE	HPLC-MS/MS	0.9–10.5 μg/L	68%–115%	^[17]	3
	In situ measurements are considered necessary
sulfamerazine, sulfamethizole, sulfadoxine, sulfamethoxazole, sulfisoxazole	MSPE	HPLC-UV	1.16–1.59 μg/L	62.0%–104.3%	^[19]

9	The use of energy should be reduced
10	Reagents ensured from renewable sources should be preferred
11

Concerning green analytical methods, the goals to be achieved include:

elimination or reduction of the use of chemical substances

(such as solvents, reagents, preservatives, additives for pH adjustment)

Toxic reagents should be minimized or replaced

elimination of energy consumption


4

sulfadiazine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfaquinoxaline	Energy saving and reagent reduction is achieved by process integration
	SBSE	HPLC-DAD	4.29–26.3 μg/L	54.8%–126%	^[20]	5	Automated and miniaturized methods should be developed and applied
sulfapyridine, sulfadiazine, sulfachloropyridazine, sulfadoxin, sulfamethoxazole, sulfadimethoxin, sulfamethizol, sulfameter, sulfamethazine	DLLME	HPLC-FL	0.60–1.21 μg/L	90.8%–104.7%	6	Derivatization is not preferable
	^[	²¹	^]
(same analytes with the above)	QuEChERS	HPLC-FL	1.15–2.73 μg/L	83.6%–104.8%	^[21]	7	Production of a large volume of analytical waste should be avoided and proper management of analytical waste should be provided
sulfamethazine	MIP-silica column	HPLC-UV	7.9 μg/L	79.3%–87.4%	^[22]	8	Multi-analyte or multi-parameter methods are preferred versus methods using single-analyte

sulfamethazine, sulfamethoxazole, sulfadizaine, sulfaquinoxaline, sulfametoxydiazine, sulfadimethoxine, sulfamethizole	RA-MIP	HPLC-UV	0.8–2.7 μg/L	93%–107%	^[23]		The operator should work under safe conditions

proper management of analytical waste
increased safety for the operator

The demands of GAC are automatization, no derivatization, and no sample treatment in the step of sample preparation. The latter is not possible in most cases, so sample preparation by a microextraction technique is the next best choice. Microextraction techniques arose as a development of conventional extraction techniques. The term microextraction means that all modes of these techniques require small volumes of extraction, which becomes under described conditions ^[13][14]. With a quick review of the literature, it is obvious that various methods have been developed for the determination of the SAs in several food matrices. The most often applied techniques for the determination of SAs in milk are HPLC coupled with different detectors, such as ultraviolet ^[


sulfamethazine, sulfisoxazole, sulfadimethoxine


FPSE

	HPLC-UV		-	22.98%–49.5%	^[24]
sulfadimidine, sulfachloropyridazine, sulfamonomethoxine, sulfachloropyrazine	M-G-PTE	LC-UV	0.004–0.012 μg/L	90.1%–113.5%	^[25]
sulfamethazine , sulfamethoxypyridazine, sulfamethoxydiazine, sulfamethoxazole, sulfadimethoxine, sulfaphenazole	IL-based MADLLME	HPLC-FL	0.018–0.031 μg/L	97.3%–107.9%	^[26]

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