Trace-level analysis of geosmin in aquaculture systems by Headspace SPME-GC-MS/MS

Abstract

The persistent challenge of earthy off-flavors in aquatic systems stems largely from geosmin (GSM), a microbial metabolite with an exceptionally low human detection threshold. This compound poses significant organoleptic and economic impacts on both aquaculture operations and municipal water supplies. To enable precise GSM monitoring, we optimized a sensitive headspace solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry (HS-SPME-GC–MS/MS) method. The results indicate that the optimal experimental conditions include the addition of 2.0 g NaCl, magnetic stirring at 1000 rpm, and headspace extraction at 60 °C for 40 min. The ion pairs 112/97 and 112/83 were selected for the qualitative and quantitative analysis of GSM, with optimal collision energies of m/z 97 (CE 10 V) and m/z 83 (CE 10 V). Under these conditions, the method demonstrated excellent precision, accuracy, and sensitivity, facilitating the detection of trace residues of GSM in fishery water environments.

• •GSM was detected by HS-SPME-GC–MS/MS high selectivity, trace detection.
• •The detection limit was 0.31 ng/L, and the recovery rate was from 72.5 % to 111 %.
• •This provides a reliable and effective technical approach to mitigate water odor issues.

Graphical abstract

Specifications table

Subject area:	Environmental Science
More specific subject area:	Environmental pollutant monitoring
Name of your method:	Gas chromatography–tandem mass spectrometry (GC–MS/MS)
Name and reference of original method:	NICOLAS A C,LOANNA K,CHRISTINE J P,et al. Prevalence of Actinobacteria in the production of 2-methylisoborneol and geosmin,over Cyanobacteria in a temperate eutrophic reservoir[J]. Chemical Engineering Journal Advances,2022,9: 100,226. DOI:org/10.1016/j.ceja.2021.100226.
Resource availability:	None.

Background

In recent years, with the development of intensive aquaculture of aquatic products, the production potential of the existing aquaculture area has been further improved [[1], [2], [3], [4]]. The consequent odor problem of aquatic products has gradually become a major obstacle to its further expansion [5]. Geosmin (GSM) and 2-methylisoborneol (2-MIB) are the predominant earthy-musty odor compounds in aquaculture systems, primarily produced by actinomycetes and cyanobacteria. These microbial metabolites accumulate in aquatic organisms, causing off-flavors in farmed fish and crustaceans [[6], [7], [8]]. These hydrophobic and odor compounds quickly accumulate in the adipose tissue of fish even in very low concentrations, bringing a fishy odor. It has been reported to include tilapia (Tilapia), catfish (Clarias macrocephalus), Atlantic salmon (Salmo salar) and bass (Sander luciporca), which is still common in pond farming patterns [[9], [10], [11], [12]]. GSM is a predominant off-flavor compound in freshwater aquaculture systems, exhibits an exceptionally low human olfactory threshold of 4 ng/L in water [8,13]. The matrix of fishery aquaculture water body including ponds, reservoirs and lakes contains a large amount of particulate suspended matter and a complex mixture of organic matter [14,15]. Therefore, a high selectivity and high sensitivity method in order to detect GSM at the trace level is needed. This is not only an essential technical basis for the identification, quantification and removal of GSM in the fishery environment, but also an urgent need to improve the quality of drinking water environment and aquatic products. At present, the earthy smell detection technology is mainly divided into sensory analysis and instrument analysis [[15], [16], [17], [18]], instrument analysis is generally composed of sample pre-processing technology and gas chromatography–mass spectrometry technology (GC–MS), gas chromatography–tandem mass spectrometry technology (GC-MS/MS). Compared with GC–MS method, GC-MS/MS method can select more accurate characteristic ions as qualitative and quantitative ion pairs, which has certain advantages for trace matter detection. Solid phase microextraction technology (SPME) has the advantages of high efficiency, convenient, sensitive, solvent-free and cheap in analyzing volatile and semi-volatile compounds [19], which has been widely used in analyzing of various volatile compounds in food, environment and biomedical fields [[20], [21], [22]]. In this study, the determination of GSM in water by HS-SPME-GC–MS/MS) was systematically analyzed based on the effects of sodium chloride addition, extraction time, extraction temperature and stirring speed. The characteristic ion and collision energy selection is optimized, and the sensitivity and stability of the proposed method are investigated. The preferred highly sensitive GSM detection method is not only the key technical means to solve the problem of odor in fishery aquaculture water and drinking water sources, but also expected to provide new ideas for the identification of volatile odor substances.

Method details

Instruments and reagents

The instruments used in this study included the 7000C–7890B gas chromatography – tandem mass spectrometer (Agilent, Santa Clara, USA) equipped with the electron impact (EI) ion source, XS205 Dual Range electronic analytical balance (Mettler Toledo, Zurich Switzerland, with its sensitivity being 0.01 mg), Allegra X-30R high-speed centrifuge (Beckman, Brea, USA), Headspace solid phase microextraction unit (HS / SPME), equipped with SPME fiber needle (PDMS / DVB type coating, film 65 µ m thick, 1 h at 270 °C before first use, ANPEL, Shanghai, China). Type Talboys heated magnetic mixer (speed stability ± 2 %, temperature stability ± 3 °C, Talboys, Trenton, USA), and MilliQ ultrapure water machine (Millipore, Boston, USA).

GSM standard stock solution (100 mg/L, Dr. Ehrenstorfer, Germany); sodium chloride (grade pure, Sinopharm Chemical Reagent Co., Ltd.), baked at 450 °C for 2 h. Methanol was chromatographically pure and was purchased from Merck. The test water is ultrapure water, prepared by MilliQ ultrapure water mechanism with a resistivity of 18.2 MΩ cm.

Preparation of standard solution

The GSM standard solution was initially diluted 100-fold to prepare an intermediate stock solution. Serial dilutions were then performed using isooctane as the solvent to generate a calibration series with final concentrations of 0.2, 0.5, 1, 5, 10, 25, 50, 100, and 200 μg/L. All standard solutions were stored at −20 °C until analysis.

Analysis conditions of instrument

Following salt saturation, sample analytes were concentrated via HS-SPME. The SPME fiber was then thermally desorbed in the GC injection port at 250 °C for 5 min. Compound separation and detection were accomplished using a triple quadrupole GC–MS/MS system operating in multiple reaction monitoring (MRM) mode. Quantification was performed using a nine-point external standard calibration curve (R² > 0.999), with complete instrumental parameters detailed in Table 1.

Table 1.

GC–MS/MS analysis parameters.

Parameter	Instrument conditions
Chromatographic column	DB-5 MS, 30 m × 0.25 mm × 0.25 µm
Temperature programming	At 40 °C for 5 min, 5 °C/min, the rate increased to 80 °C and maintained for 1 min, 20 °C/min, 140 °C and maintained at 1 min, 5 °C/min, 180 °C and maintained for 1 min, 50 °C/min, increased to 280 °C and maintained for 1 min.
Injection port	250 °C, Splitless injection
Carrier gas	He, 1 mL/min
Temperature of mass spectral transmission line	280 °C
SCAN mode	EI, m/z 35 - 200
MRM mode	EI, Multiple-response monitoring mode
Collision gas	N2, 1.5 mL/min
Quenched gas	He, 2.25 mL/min
Target ion pairs	112/97, 112/83
Residence time	100 ms
Ionization voltage	70 ev
Ion source temperature	230 °C
Solvent delay time	8 min

Experiment design

A systematic optimization of HS-SPME parameters was conducted using a 10 mL sample volume. Key variables evaluated included:

• Salt addition: 1.0, 2.0, 3.0, and 4.0 g NaCl

• Extraction time: 15, 20, 30, 40, 50, and 60 min

• Extraction temperature: 30, 40, 50, 60, 70, and 80 °C

• Collision energy: 8, 10, 12, 20, 30, and 40 eV

The parameter space was comprehensively assessed to identify optimal conditions for subsequent analyses.

Statistical significance was determined using a GraphPad Prism 7 plot using one-way ANOVA (analysis of variance, ANOVA), and P < 0.05 was used as the criterion for statistical significance.

Results and discussion

Optimization of the extraction conditions

The effects of NaCl concentration, extraction temperature, extraction time and stirring speed on GSM extraction in water were studied, comparing the extraction effect under different conditions using the ion response intensity of GSM on the mass spectrometer. During the NaCl optimization tests, sample saturation was achieved by adding 4.0 g NaCl to 10 mL water, which represented the maximum salt concentration for complete phase separation. As shown in Fig. 1a, while GSM extraction efficiency exhibited a modest increase with NaCl addition, no statistically significant difference (p > 0.05) was observed in analyte response intensities between 2–4 g salt additions. In general, NaCl can improve the ionic strength of the solution through the salting out effect, which may reduce the solubility of organic compounds and improve the extraction efficiency of these compounds [[23], [24]]. Therefore, choosing the addition amount of 2.0 g NaCl can not only meet the test requirements, but also save the test cost relatively, the consumption of sodium chloride was reduced by 33 % [19,23]. Fig. 1b demonstrates that the adsorption capacity of the carbon fiber probe for GSM varies with water activity levels, reaching maximum adsorption at 70 °C. This temperature-dependent behavior arises from changes in the partition coefficient between aqueous and headspace phases. The CAR/PDMS coating exhibits optimal performance for small molecule adsorption due to its non-polar characteristics [23,25]. When the extraction temperature rises to more than 70 °C, the molecular activity in the sample is more intense, and with more water vapor rising to the gas phase, GSM is soluble in water in this environment, and the number of molecules combined with carbon fiber is less, which reduces the adsorption of GSM. With the extraction temperature, GSM can be detected during the test, indicating that the increase in temperature will lead to the volatilization and dispersion of the target. In addition, it can be seen from Fig. 1b that the response intensity of the target is comparable at 60 °C −70 °C, the standard deviation of the test results at 70 °C −80 °C is increased, and the higher extraction temperature (80 °C) leads to the loss of the target, which is similar to the rule obtained by Moralesvalle H et al. [26] in the analysis of GSM in grape juice. From the stability and repeatability of the test, 60 °C was selected as the optimal extraction temperature. Magnetic stirring is performed to efficiently transfer the analyte from the aqueous phase to the headspace phase, while also uniformly dissolving sodium chloride in the sample. Research shows that [27], when the stirring speed is 1000 r/min, can meet the test needs, faster stirring is often uncontrollable, may lead to a decrease in measurement accuracy and stability. Therefore, the magnetic stirring rate was determined to be 1000 r/min. The impact of sample extraction time on extraction efficiency is illustrated in Fig. 1c. The data reveal that an extraction time of less than 40 min may be insufficient for complete adsorption of the target analyte onto the SPME fibers, as indicated by a relatively low GSM response value. Typically, extended extraction durations lead to enhanced response levels until the system achieves dynamic equilibrium between the extractant and analyte. Analysis of the extraction period from 40 to 60 min demonstrated that the target's response intensity plateaued, with no statistically significant differences observed (p > 0.05). This stabilization suggests that the extraction process reached equilibrium at approximately 40 min. Consequently, 40 min was established as the optimal extraction duration for this protocol.

Fig. 1.

GSM extraction efficiency optimization test (n = 3)

Note: a. Effect of NaCl concentration (0–4 g/10 mL) on GSM extraction efficiency, b. Effect of extraction temperature on GSM extraction efficiency, c. Effect of extraction time on GSM extraction efficiency.

Feature ion selection

To ensure accurate quantification, a high-concentration GSM standard solution (1.00 mg/L) was subjected to full-scan analysis (m/z 50–300) under the instrument conditions specified in Table 1. The characteristic peak of GSM was observed at a retention time (RT) of 27.258 min under the specified chromatographic conditions(Fig. 2a), and the precursor ions with the highest response intensity and large mass: 112,125,149 and 182 (the relative molecular mass of GSM is 182) were selected as characteristic ions (Fig. 2b).

Fig. 2.

GSM scan spectrogram

Note: a. Total ion flow map, b. Full scan mass spectrogram.

The GC–MS method generally selects 112 as quantitative ions and 125 or 182 as qualitative ions [19,23], but in the case of complex sample matrix and low target content, the influence of ion interference and matrix effect can not be ignored. Therefore, the qualitative and quantitative GSM should be further determined by GC-MS/MS. Product ion scans of the selected characteristic ions at the same collision energy (30 V), as shown in Fig. 3. Peak-to-peak signal-to-noise ratios (S/N) were automatically calculated by measuring peak heights and detecting noise regions, yielding the following values for characteristic ions: m/z 182 (S/N = 42.3), m/z 149 (S/N = 744.2), m/z 125 (S/N = 394.1), m/z 112 (S/N = 1103.0) (Fig. S1). As illustrated in Fig. 3, although m/z 149 and m/z 125 demonstrated excellent S/N ratios, they were accompanied by significant interfering ions. This observation suggests that neither m/z 125 nor m/z 149 can generate stable product ion fragments when selected as precursor ions. In contrast, m/z 112 exhibited both superior S/N characteristics (1103.0) and exceptional interference resistance, making it the optimal quantitative ion [19]. Furthermore, comparative analysis of ion intensities revealed that the product ions at m/z 97 and m/z 83, generated from the precursor ion at m/z 112, exhibited the highest ion abundances among all detected fragments. Therefore, 112 / 97,112 / 83 were identified as qualitative, quantitative ion pairs of GSM.

Fig. 3.

Product ion scanning analysis of GSM by GC–MS/MS.

Note: a. The product ion mass spectrum derived from the precursor ion at m/z 182, b. The product ion mass spectrum derived from the precursor ion at m/z 149, the product ion mass spectrum derived from the precursor ion at m/z 125, and d. The product ion mass spectrum derived from the precursor ion at m/z 112.

Collision energy test

The systematic optimization of collision energy (CE) represents a critical parameter in GC–MS/MS method development, as it fundamentally determines the method's analytical performance characteristics including sensitivity, selectivity, and accuracy. The m / z 112 was selected as the precursor ion of GSM, m / z 97 and m / z 83 as the product ion. As shown in Fig. 4, the precursor ion intensity exhibited a gradual decrease with increasing collision energy (CE). Optimal product ion responses were observed at 8–12 V, with the most efficient fragmentation occurring at m / z 97 (CE 10 V) and m / z 83 (CE 10 V). Through collision energy optimization, we obtained more than 5-fold increase in product ion signals while simultaneously reducing false-positive risks - a combination rarely achieved in prior research [19,23,28]. These advances not only enhanced the method's interference resistance but also yielded critical technical references for establishing future analytical standards.

Fig. 4.

Collision energy optimization for GSM product ions

Note: 1–1 to 1–6 successively display the ion intensity of m/z 83 at collision energies of 8, 10, 12, 20, 30, and 40 V, 2–1 to 2–6 successively display the ion intensity of m/z 97 at collision energies of 8, 10, 12, 20, 30, and 40 V.

Method validation

Quality assurance/quality control (QA/QC)

The quantitative ion pairs of GSM were m / z 112 / 97 and 112 / 83 and monitored alternately at a residence time of 100 ms. The calibration curve of GSM demonstrated excellent linearity (R² = 0.9999) across a concentration range of 0.2–200 ng/L (Fig. 5). Method precision was validated through seven replicate analyses.

Fig. 5.

GSM standard curve.

The limit of detection (LOD) was determined following HJ 168–2020 Technical Guidelines for Environmental Monitoring Method Development. Briefly, seven replicate 10.00 mL aliquots of blank matrix water were spiked with GSM at 1.00 ng/L. Based on the Student's t-distribution (t = 3.143, 99 % confidence level), the calculated LOD was 0.31 ng/L, with the corresponding limit of quantification (LOQ) determined to be 0.98 ng/L. The developed method demonstrated superior sensitivity and linearity compared to existing approaches [[28], [29], [30]], enabling accurate quantification of trace-level GSM residues in aquaculture environments.

Method validation was performed through triplicate recovery tests at three concentration levels (10, 50, and 100 ng/L), including parallel analysis of reagent blanks and sample blanks. The method exhibited excellent precision (RSD 4.12–11.1 %) and satisfactory accuracy (recovery rate 72.5–111 %), meeting international validation criteria for environmental analysis.

Additional information

GC–MS/MS demonstrates substantial analytical advantages compared to GC-MS, particularly in handling complex matrices, trace-level analysis, and interference resistance. While conventional GC–MS relies exclusively on chromatographic separation and single-stage mass spectrometric fragmentation - an approach particularly vulnerable to matrix effects that necessitates rigorous sample preparation and chromatographic optimization (currently the primary research focus in GSM detection) - our study has developed superior analytical solutions. The LOD of this method (0.31 ng/L) surpasses most reported values for GSM analysis in water (0.30–1.5 ng/L), while maintaining R² > 0.9999 over the range of 0.2–100 ng/L, the consumption of sodium chloride was reduced by 33 % [23,28]. Using the optimized analytical protocol, we quantified GSM concentrations in 14 freshwater aquaculture ponds across Anhui and Liaoning provinces, China. The measured concentrations ranged from ND - 54.7 ± 5.8 ng/L. Among them, the maximum value appeared in a farm in Wuhu city, Anhui Province, the main breed is herring (Mylopharyngodon piceus). The measured GSM concentrations (ND - 54.7 ± 5.8 ng/L) exceeded those reported by Saito et al. [28] for pond waters (ND - 18.5 ng/L) and were comparable to levels observed by Zou et al. [8] in tilapia ponds (40.78 ± 20.39 ng/L) from Wuxi, Jiangsu Province.

Limitations

None.

Ethics statements

None.

CRediT authorship contribution statement

Shi-Zhan Tang: Conceptualization, Methodology, Software, Validation, Data curation, Writing – original draft, Writing – review & editing. Zhong-Xiang Chen: Validation, Data curation, Writing – original draft, Visualization, Investigation, Writing – review & editing. Li Huang: Visualization, Investigation. Chen-Hui Li: Supervision. Ning-Ning Du: Software, Validation. Jing Ren: Supervision. Dong-Li Qin: Supervision. Peng Wang: Software, Validation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.