Development of GC-ITQ-MS chromatographic system for the identification of hydroxy derivatives of polycyclic aromatic hydrocarbons in wastewater

The goal of this study was to develop and validate a sensitive and reliable analytical method for determining selected hydroxy derivatives of polycyclic aromatic hydrocarbons (OH-PAHs) in wastewater using gas chromatography-ion trap mass spectrometry (GC-ITQ-MS). Given the toxicological significance of OH-PAHs, the research aimed to optimize each stage of the analytical workflow—from extraction and derivatization to detection.

The method was tailored to environmental samples and validated on real wastewater from the Płaszów treatment plant in Krakow. It enabled accurate quantification of OH-PAHs at nanogram-per-liter levels and also included a stability assessment of these compounds in the aqueous phase, revealing substantial degradation within hours.

The original article

Development of GC-ITQ-MS chromatographic system for the identification of hydroxy derivatives of polycyclic aromatic hydrocarbons in wastewater

Justyna Pamuła, Elżbieta Sochacka-Tatara, Katarzyna Styszko 

Journal of Chromatography A, Volume 1752, 2025, 465969

https://doi.org/10.1016/j.chroma.2025.465969

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Humans have a significant impact on both the animate and inanimate nature, with consequences that include loss of biodiversity, climate change, urbanization, industrial development, and environmental pollution. Human activities continuously contribute to the release of increasing amounts of pollutants into the environment. The growing number of chemical compounds poses a threat to natural ecosystems and, more importantly, to human health and life [1]. Among these pollutants are polycyclic aromatic hydrocarbons (PAHs). Incomplete combustion processes are the primary source of PAHs emissions into the environment [2]. Research by global scientists indicates that the total estimated global emission of PAHs into the atmosphere comes from biofuels (56.7 %), with forest fires contributing 17.0 %. Other significant sources include the use of consumer products (6.9 %), car exhaust fumes (4.8 %), and combustion of household coal (3.7 %). Industrial activities contribute <10 % of global PAH emissions, with coke production being the largest industrial source at 3.6 %. In particular, the volume of PAH emissions is positively correlated with gross domestic product (GDP) and negatively with average income [3]. Globally, biofuel combustion is the main source of PAHs, especially in countries with low GDP per capita such as India and China, where straw and wood are burnt for heat energy in rural areas [4]. In urbanized areas, PAHs are primarily emitted from automobile exhaust fumes and industrial activities [5].

PAHs are present in all elements of the environment: water, soil, air, and food, allowing them to easily enter the human body [2,6]. PAHs have been an important focus in environmental pollution research for many years due to some being highly carcinogenic or mutagenic [7]. According to the International Agency for Research on Cancer (IARC), the most hazardous of the unsubstituted PAHs is benzo(a)pyrene, the only PAH classified as a group 1 human carcinogen [8]. Consequently, benzo(a)pyrene is commonly analyzed in the environment as a marker of carcinogenic risk associated with PAHs.

PAHs are ubiquitous in the environment, leading to human exposure through skin contact, food ingestion, and inhalation [2,9] Once in the body, PAHs are transported through the bloodstream to tissues and organs. Due to their lipophilicity, they accumulate in adipose tissue, with their metabolism occurring mainly in the liver and bile ducts. PAH metabolites in the form of hydroxy derivatives (OH-PAHs) are excreted from the body within a few days [10]. OH-PAHs with lower molecular weight (fewer than four benzene rings) are excreted primarily in urine, while those with higher molecular weight (four or more rings) are mainly excreted primarily through bile and feces [11]. PAH metabolites are used as biomarkers of PAH exposure [12]. There is a need for improved sample preparation techniques that enable more efficient extraction of OH-PAH from the sample matrix. Innovative methods are proposed, including thin-film SPME membranes using polydimethylsiloxane (PDMS) in combination with Ordered Mesoporous Carbon [13], electromembrane extraction [14], or magnetic sorption on Fe₃O₄@HCP-BA, a magnetic composite consisting of magnetite (Fe₃O₄) and hyper-cross-linked boronic acid polymer (HCP-BA) [15].

OH-PAHs have the potential to be more dangerous to living organisms than their parent compounds [12]. Despite the potential risk of OH-PAHs, there is limited research on their occurrence in the environment. Selected OH-PAHs have been determined in lake water [16], surface soil [17], and air particulates [18]. The only available study on OH-PAH concentration levels in wastewater is by Pojan and Marcomini, which used liquid chromatography coupled with a mass spectrometer [19].

This research aimed to develop and validate a methodology for determining selected hydroxy derivatives of PAHs in wastewater using gas chromatography coupled with a mass spectrometer. Using the analytical method, the stability of these compounds in the sewage matrix was assessed, and preliminary tests were conducted on municipal sewage to determine the concentrations of OH-PAHs. Additionally, the research investigated the stability of selected OH-PAHs in wastewater.

2. Experimental

2.3. Instrumentation

The gas chromatograph coupled with a mass spectrometer (GC-ITQ-MS) used was a TRACE 1310 Thermo Scientific gas chromatograph equipped with a Thermo Scientific TG-SQC capillary chromatographic column, 30 m in length, with an internal diameter of 0.25 mm, and a film thickness of 0.25 µm (filled with 5 % phenyl and 95 % methylpolysiloxane). The setup also included an ITQ 900 Thermo Scientific ion trap mass spectrometer and a Tri Plus RSH Thermo Scientific autosampler. The identification of hydroxy derivatives of PAHs was enabled by the mass spectrometer. Destructive ionization of molecules was achieved using an EI energy of 70 eV. A single standard was subjected, after derivatization, to multiple analyzes in the SCAN detector mode, allowing the scanning of the entire mass spectrum range from 50 to 500 m/z. This enabled the determination of spectra for the compounds studied, which had been converted into volatile derivatives using BSTFA and MTBSTFA. Subsequently, optimal column operating conditions were selected to allow for the chromatographic separation of the OH-PAHs mixture.

3. Result and discussion

3.2. Selection of chromatographic separation parameters

The separation of compounds on a chromatographic column is determined by the operating conditions of the column. The selection of the appropriate chromatography temperature depends on the boiling points of the components of the mixture being separated [23]. According to Table 1, the range of boiling points of the compounds is 105 °C. In such cases, isothermal chromatography does not produce the desired results, leading to asymmetric, broad, and asymmetric peaks. Therefore, the column temperature programming is used. This involves maintaining a constant temperature for a certain period, then increasing it at a preset rate, and maintaining the new temperature for a specified duration.

The development of optimal chromatographic separation began with the introduction of GC operating parameters presented in the work of Campo and colleagues [24]. The authors identified 12 hydroxy derivatives of PAHs in urine samples, including all those that were the subjects of the current investigation. However, the obtained chromatogram was not satisfactory, prompting the development of a new separation method for two derivatization reagents, BSTFA and MTBSTFA. The exact operating parameters of the GC are presented in Table A.1., in the appendices.