Report
Since October 2012, I have worked in a special project about Elimination of pharmaceuticals and organic compounds: Development of concepts and novel, cheap cleaning procedures – Formation of metabolites by ozonoation. It was a kind of training for me and a special key for my PhD work. We are interested in screening of non target compounds in wastewater samples. Wastewater samples are collected from different wastewater treatment plants at different treatment points. For example: before ozonation, after ozonation and after biological treatment steps. These compounds are extracted by solid phase extraction (SPE) method using certain solid material (OASIS HLB). This kind of SPE is suitable to extract hydrophilic and lipophilic analytes. We did several steps to extract those compounds using different procedures and different analyzing tools. We used HPLC-UV, LC-MS and GC-MS to analyze the extracted compounds. The toxicological tests are achieved for each sample in order to discover the efficiency of wastewater treatment and the effect of treatment to obtain new unknown transformation products.
In this project, I learned so many things like:

How to validate and develop a method to extract polar and non polar compounds from wastewater matrixes using certain SPE steps.
How to choose the best solid phase materials to extract a wide range polarity of chemical compounds in wastewater matrixes.
Getting the principles for handling of different analytical instruments to validate specific methods and to analyze the extracted compounds.
Taking enough time to review the statistical principles which are necessary to find the optimum ways for treatment.
Achieving the possibilities to analyze the output data and comparing different procedures to find out the best one for analysis.
Combining chemistry with biology in order to evaluate the toxicological estimates and to know the best wastewater treatment step.

Now, I am working on developing a method to extract the following six compounds from water matrix:
Catechol, trans,trans-Muconic acid, p-Benzoquinone, 1H-Benzotriazole, p-Nitrophenol and 2,6-Dichloraniline.
Catechol, trans,trans-Muconic acid and p-Benzoquinone are transformation products generated after treatment of the parent compound (Bisphenol A) with ozone in aqueous solution (water). Bisphenol A (BPA) belongs to the numerous anthropogenic compounds considered as endocrine disruptors. Although less estrogenic toward aquatic organisms than natural hormones, BPA has been reported to be able to induce feminization phenomena in various species of animals. BPA is commonly used for the production of polycarbonate plastics and epoxy resins. It is also used as stabilizer or antioxidant for many types of plastics such as polyvinyl chloride (PVC). BPA is detected with a high frequency in surface waters.
1H-Benzotriazole has been found in wastewater and the subsequently impacted surface water. This compound is employed to prevent metal corrosion and as UV-inhibitors, and found to use in applications such as airplane deicing fluids, dishwashing detergents, and plastic formulations.
p-Nitrophenol is degradation product generated throughout the ozonation process of the antibiotic sulfamethoxazole. Sulfamethoxazole in combination with trimethoprim is used to treat a wide range of human diseases, such as urinary and respiratory tract infections. There are many publications that have reported about the presence of this antibiotic drug in sewage treatment plants, hospital effluents and rivers. Besides, this compound might be expected to be present in groundwater, because the antibiotic sulfamethoxazole is also applied to animals as growth promoter.

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2,6-Dichloraniline is a metabolite produced after ozonational step of the drug diclofenac in aqueous solution. Diclofenac is one of the most commonly used pain killers. It is used to treat painful conditions such as arthritis, sprains and strains, gout, migraine, dental pain, and pain after surgical operations. There are many articles that have reported about the presence of this drug in sewage treatment plants.
First step, I prepared different standard solutions for each compound, then validated and developed a method to analyze these compounds by using HPLC-UV instrument. A mixture of methanol and water (pH=2) are used at certain progress time and different ratios to obtain the best LC chromatogram with good separation and resolution for each compound in the mixture. The maximum wavelength (λ) for each compound is known by using UV-spectrophotometer.
Table (1) summarizes the information obtained after analyzing my target compounds by HPLC-UV.
Table 1

Substance

Retention time
tR (min)

λ (nm)

Catechol (1)

26.35

278

t,t-Muconic acid (2)

28.43

278

p-Benzoquinone (3)

30.46

278

1H-Benzotriazole (4)

34.42

278

p-Nitrophenol (5)

39.89

228

2,6-Dichloraniline (6)

48.04

228

Figure (1) shows the HPLC-UV chromatogram (relative response in mV vs. tR) for a mixture of six compounds in one prepared stock solution.
5 6
Figure 1
 
2
4
1
3
In order to develop a method to analyze the target compounds; the limit of detection (LOD) and limit of quantification (LOQ) should be calculated in order to know the sensitivity of HPLC-UV instrument towards each compound.
The LOD value for each compound was calculated from a chromatogram on the basis of signal-to-noise (S/N) ratio of 3.
The LOQ value for each compound was calculated from a chromatogram on the basis of signal-to-noise (S/N) ratio of 10.
Table (2) shows the values of LOD and LOQ for each compound.
Table 2

Substance

LOD
(ng/L)

LOQ
(ng/L)

Catechol (1)

8.8

29.3

t,t-Muconic acid (2)

1.2

4.0

p-Benzoquinone (3)

6.3

21.0

1H-Benzotriazole (4)

8.5

28.3

p-Nitrophenol (5)

27.4

91.3

2,6-Dichloraniline (6)

18.0

60.0

For linearity and statistical evaluation, I prepared mixture solutions of six compounds at concentrations of 0.1, 0.25, 0.4, 0.55, 0.7, 0.85 and 1.0 mg/L.
Table (3) shows the statistical data for the prepared stock solution of 0.7 mg/L.
Table 3

Substance

% Relative response

Correlation coefficient (r)

Catechol (1)

100

0.999

t,t-Muconic acid (2)

101

0.999

p-Benzoquinone (3)

106

0.999

1H-Benzotriazole (4)

100

0.999

p-Nitrophenol (5)

100

0.999

2,6-Dichloraniline (6)

104

0.999

The linearity at my working range is shown as follows:

Now I am working in developing a preparation method to extract these compounds from water matrix by using different solid phase materials. Up to now, I tried two types of solid phase cartridges: OASIS HLB and SPEED DISK cartridges. It’s noticed that OASIS HLB gave higher recovery than SPEED DISK.
In order to find out the best solid phase material, I will try many types of SPE.
After finishing this step, I will develop a method to extract and analyze about 30 chosen chemical compounds which are produced after the ozonation process of pharmaceutical and parent compounds in water matrix. These compounds will be:
Diclofenac, 2,6-Dichloroaniline, Metoprolol, Sulfamethoxazole, 4-nitrophenol, Bisphenol A, Catechol, trans,trans-Muconic acid, cis,cis-Muconic acid, p-Benzoquinone, Ciprofloxacin, Paracetamol, 1,2,4-Benzenetriol, Oxalic acid, Succinic acid, Malic acid, Malonic acid, Oxaloacetic acid, Hydroquinone, Maleic acid, EDTA, Glyoxylic acid, Formaldehyde, Carbamazepine, Glyoxylic acid, Oxamic acid and 1H-Benzotriazole.