New Fluorescence Derivatization Reagent for the Determination of 7 Unsaturated Fatty Acids in Oil Samples by Pre-Column Derivatization HPLC-FLD

doi:10.21203/rs.3.rs-2837892/v1

4-((2-Methyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)amino)-Ethyl-4-Methyl Benzenesulfonate (MNDMB), was designed and synthesized, and it was first successfully applied to label unsaturated fatty acids and HPLC-FLD determination. MNDMB can quickly complete the derivatization reaction with unsaturated fatty acids within 40 min with K₂CO₃ as the catalyst. The derivatives exhibit high stability and strong fluorescence properties with excitation and emission wavelengths of 285 and 522 nm, respectively. The 7 unsaturated fatty acid derivatives were separated on an Agilent ZORBAX 300SB-C18 column with gradient elution, and good linear correlation coefficients were obtained for all unsaturated fatty acids with correlation coefficients of ≥ 0.9998. The method has low limits of detection (LODs) (4–21 nmol/L) and good precisions ( intraday relative standard deviations [RSDs] were 1.91–3.51%, and interday RSD were 3.33–5.41%). The method was used to analyze two edible oils and successfully detected some unsaturated fatty acids with recoveries ranging from 90.2 to 103.5%. The proposed method has several advantages including specific labeling, simple enrichment operation, and high sensitivity, demonstrating its great utility for monitoring trace unsaturated fatty acids in various complex matrices.

Unsaturated Fatty Acids / Derivatization reagent / HPLC-FLD/ 4-((2-Methyl-1

3-dioxo-2

3-dihydro-1H-benzo[de]isoquinoline-6-yl)amino)-Ethyl-4-Methyl Benzenesulfonate (MNDMB)

Fatty acids are widely distributed in nature and are as important as the nutrients and metabolites in living organisms. A variety of fatty acids play important regulatory roles at the trace level, regulating multiple physiological and biological functions(Ozaki et al. 2019). In nature, these three types of fatty acids are saturated, monounsaturated, and polyunsaturated fatty acids(Nishikiori et al. 2015). The representative long-chain monounsaturated fatty acids include oleic and erucic acids. Oleic acid has a widespread application in daily life, is extremely well absorbed in the body, and has the function of reducing cholesterol, blood pressure, and lipoprotein density(Priore et al. 2017). Erucic acid is not easily absorbed by the body, and large intakes can cause damage such as myocardial fibrosis, stunted growth, and reduced fertility(Kumar and Sharma 2022). Representative polyunsaturated fatty acids include α-linolenic acid (ALA, n-3), eicosapentaenoic acid (EPA, n-3), docosapentaenoic acid (DPA), docosahexaenoic acid ((DHA, n-3), linoleic acid ((LA, n-6), γ-linolenic acid (GLA), and arachidonic acid (ARA, n-6). Research has shown that polyunsaturated fatty acids are important nutrients in human metabolism, and adequate intake of polyunsaturated fatty acids can promote growth and development(Collins et al. 2019), reduce inflammation and cancer(Quiroga et al. 2018), help lower blood lipids, delay aging, and regulate immunity(Hogenkamp et al. 2020), etc. Docosahexaenoic acid (DHA, 22:6n-3) is an abundant fatty acid in animal brains. DHA has been reported to enhance brain functions such as memory and learning(Liput et al. 2021), and the n-6 fatty acid arachidonic acid (ARA, 20:4n-6) has been reported to have inflammatory effects(Allam-Ndoul et al. 2017). In contrast, eicosapentaenoic acid (EPA, 20:5n-3) has been reported to have anti-inflammatory effects(Andreoli Miyake et al. 2020). In addition, γ-linolenic acid (18:3n-6) is known to have anticancer activity, while α-linolenic acid (18:3n-3) has different physiological functions and has been reported to reduce the risk of heart disease(Brouwer et al. 2004). Unsaturated fatty acids (UFAs) have an important physiological function in the human body(Lupette and Benning 2020), and thus the detection of UFAs is of great importance in the study of food science as well as in the field of life sciences. Common methods for the detection of UFAs are based on chromatographic methods, mainly including gas chromatography (GC)(Takahashi and Goto-Yamamoto 2011), gas chromatography-mass spectrometry (GC-MS) (Lv and Yang 2012; Silva et al. 2011; Wang et al. 2020; Yin et al. 2016), high-performance liquid chromatography (HPLC)(Wang et al. 2007) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) (Peng et al. 2017). Although derivatization combined with gas chromatography is widely used for carboxylic acid analysis, it has some drawbacks. For example, long-chain polyunsaturated fatty acids are unstable in gas chromatographic analysis, and thus their accuracy is greatly affected(Schreiner 2005). HPLC analysis of fatty acids can be performed at lower temperatures than GC methods, which can provide simpler processes and faster analysis(Huang et al. 2019). Due to the high price of mass spectrometers, chromatography is generally used more in laboratories, and high-performance liquid chromatography (HPLC) has become an essential tool for the separation of multiple components in complex matrices. Most UFAs do not naturally absorb in the visible or UV region and the fluorescence region, so it is difficult to detect them at trace levels with an absorptiometry. A common method to enhance the sensitivity and selectivity of carboxylic acid detection is the chemical derivatization of the carboxylic acid fraction(Jonnada et al. 2017). It is well known that fluorescence detection is much more sensitive than UV-visible absorption. Therefore the development of fluorescently labeled reagents has been the focus of research in this field. Up to now, many carboxylic acid-responsive fluorescent reagents have been developed for high-performance liquid chromatography. Such as acridone-9-ethyl-p-toluenesulfonate (AETS)(Zhao et al. 2006), 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP)(You et al. 2007), 2-(2-(anthracen-10-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)ethyl 4-methylbenzenesulfonate (APIETS)(Sun et al. 2011), 6-oxy-(acetyl piperazine) flfluorescein (APF)(Du et al. 2007), 9-(2-hydroxyethyl)-carbazole (HEC)(You et al. 2001), 1,3,5,7-Tetramethyl-8-butyrethylenediamine-diflfluoroboradiaza-s-indacene (TMBB-EDAN)(Wang et al. 2013), 9-fluorenylmethyl chloroformate (FMOC-Cl)(Meka and Heagy 2017), 2-(12-benzo[b]acridin-5(12H)-yl)-acetohydrazide (BAAH)(Xie et al. 2012). However, these reagents have limitations in application such as low detection sensitivity, poor stability, cumbersome analytical procedures, and severe interference with the analysis of biological samples(Toyo’oka 2002).

In this paper, 4-((2-methyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)amino)-ethyl-4-methyl benzenesulfonate (MNDMB) was selected as the pre-column derivatization reagent to establish a rapid, efficient, and accurate high-performance liquid chromatographic fluorescence detection method for the determination of UFAs. The derivatization reagent is simple and rapid to synthesize, with good fluorescence performance. After the completion of the derivatization reaction with UFAs, the derivatives emit strong fluorescence, and the derivatives have good stability. The use of the chemical derivatization method greatly improved the detection limit and accuracy of the method, which was successfully applied to the determination of 7 UFAs in corn oil and soybean oil.

Eicosapentaenoic acid (EPA), linolenic acid (LNA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA), oleic acid(OA), and erucic acid(EA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methylamine aqueous solution (30%-33%), chromatographically pure acetonitrile, ethanol, and pyridine were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). 4-Methylbenzenesulfonyl chloride, 4-bromo-1,8-naphthalenedicarboxylic anhydride were purchased from Shanghai Maclean Biochemical Technology Co.(Shanghai, China). 2-Methoxyethanol, ethanolamine were purchased from Shanghai Aladdin Biochemical Technology Co.(Shanghai, China). Purified water was purchased from Watson's (Guangzhou, China). Unless otherwise noted, all other reagents used were of analytical grade.

Instrument

The samples of unsaturated fatty acids were performed on an Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an online degasser, binary pump, autosampler, thermostatic column compartment, and fluorescence detector. A fluorescence detector (model G1321B, Agilent, USA) was set to excitation and emission wavelengths of 285 nm and 522 nm, respectively. Chromatographic separation was achieved on an Agilent ZORBAX 300SB-C18 column (4.6 mm × 200 mm, 5 μm inner diameter, China). Solvent A was 30% acetonitrile in water and B was acetonitrile. The flow rate was constant at 1.0 mL/min and the column temperature was kept at 30 °C. The gradient conditions for the mobile phase were as follows: 60-80% B from 0 to 25 min; 80-100% B from 25 to 30 min and then held for 10 min. The column was equilibrated with the initial mobile phase for 5 min before each injection. The injection volume was 10 μL. The fluorescence excitation and emission spectra of the derivatives were obtained using an F7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at room temperature.

Synthesis of 4-((2-methyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)amino)-ethyl-4-methyl benzenesulfonate (MNDMB)

Synthesis of 4-Bromo-N-methyl-1,8-naphthalimide

In a 250 mL round bottom flask, ethanol (150 mL), 4-Bromo-1,8- naphthalimide (2.77 g, 10 mmol), and methylamine-methanol solution (30-33%, 4.00 g, 39 mmol) were successively added. The mixture was heated and stirred under reflux for 6 h. After the reaction was completed, the mixture was cooled and allowed to stand overnight to obtain complete precipitation. The precipitated solid was filtered and washed twice with ethanol (2 × 30 mL). The product was dried in a vacuum oven at 60 °C to obtain 2.35g of gray solid. Yield: 81.03%. Mp. 188.0-189.9 ℃.

Synthesis of 4-(2-hydroxyethylamino)-N-methyl-1,8-naphthalene dicarboximide (HMNDI)

To a 100 mL round bottom flask, 4-Bromo-N-methyl-1,8-naphthalimide (2.03g, 7 mmol) and 2-Methoxyethanol (50 mL) were added and stirred for 10 min at room temperature, subsequently, ethanolamine (1.26 mL, 21 mmol) was added dropwise. The mixture was then heated to reflux for 6h. When the reaction was completed, the mixture was cooled and allowed to stand overnight to obtain complete precipitation. The precipitated solid was filtered and washed twice with ethanol (2 × 30 mL). The crude product was dried at room temperature and recrystallized from acetonitrile to give 1.60 g of red crystal. Yield: 84.56%. Mp: 228.3-229.8 ℃. MS: m/z 271.0 [M+H]⁺.

Synthesis of MNDMB

To a 50 mL round bottom flask, 15 mL of dried pyridine with anhydrous calcium chloride was added, and the flask was cooled to 0 ℃ in an ice-water bath. HMNDI (0.60 g, 2 mmol) and p-methylbenzenesulfonyl chloride (0.80 g, 4 mmol) were then added. The reactant solution was stirred in an ice-water bath for 4 h and then at room temperature for 6 h. After the reaction was completed, 15 mL of water was added in portions with stirring until the yellow precipitate was formed. The precipitated solid was collected by suction filtration and recrystallized twice with acetonitrile to give 0.66 g of yellow crystals. Yield: 70.01%. Mp:159.3-160.8 ℃. MS: m/z 425.2 [M+H]⁺. 1H NMR (500 MHz, DMSO) δ 8.52 (d, J = 8.5 Hz,1H), 8.45 (d, J = 7.2 Hz 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.79 (t, J = 5.7 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.58 (d, J = 8.2Hz, 2H), 7.00 (d, J =8.1 Hz, 2H), 6.65 (d, J = 8.6 Hz, 1H), 4.26 (d, J = 5.0 Hz, 2H), 3.70 – 3.67 (m, 2H), 2.51 (s, 3H), 2.06 (s, 3H). 13C NMR (126 MHz, DMSO) δ 164.48, 163.66, 150.17, 144.89, 134.19, 132.15, 131.05, 130.13 (2C, Ar), 129.60, 128.92, 127.89 (2C, Ar), 124.89, 122.31, 120.74, 108.70, 104.31, 68.11, 41.57, 26.85, 21.19. IR (KBr): 3386 (ν NH), 1683 and 1639 [ν N(C═O)₂], 1360 and 1174 (ν SO2) , 1281 (ν CN), 771 (ν Ar) cm^-1.

Preparation of Solutions

The concentration of 1.0 × 10^-3 mol/L derivative reagent was prepared by fixing 4.25 mg MNDMB to 10 mL with distilled analytically pure DMF solution. The seven unsaturated fatty acid standards were accurately weighed separately, and completely dissolved with distilled analytically pure DMF, and the corresponding standard stock solution with a concentration of 1.0 × 10^-3mol/L was obtained by dilution with chromatographically pure acetonitrile solution. Low-concentration solutions of individual unsaturated fatty acids were prepared by diluting standard stock solutions with acetonitrile. The mixed stock solution of the seven unsaturated fatty acids (total concentration of 7.0 × 10^-4 mol/L) was prepared by mixing an individual stock solution of each standard (1.0 × 10^-3 mol/L) and acetonitrile. The low concentration of the seven unsaturated fatty acid mixtures was prepared by dilution with acetonitrile. When not in use, all reagent solutions were stored in the dark at 4 °C.

Preparation of the actual sample solution

Corn oil and soybean oil samples were obtained from the canteen of Qufu Normal University. 5 mL of KOH-methanol solution (0.6 mol/L) was added to a 15 mL EP tube containing 1 mL of corn oil (or soybean oil) sample. The EP tube was placed in a water bath at 60 °C and saponified for 40 min with continuous shaking. Subsequently, 2 mL of distilled water was added and the pH was adjusted to 3-4 with 5 mol/L of hydrochloric acid. The solution was extracted twice with 2 mL of n-hexane and the n-hexane phases were combined. The n-hexane phase was extracted twice with 2 mL of distilled water in the reverse phase and the n-hexane phase was combined. The n-hexane phase was nitrogen blown to no n-hexane smell and diluted to 5 mL with acetonitrile. The dilutions were filtered through a 0.22 μm nylon organic needle filter and stored in a 4°C refrigerator.

Derivatization Procedure

The derivatization scheme of MNDMB with UFAs was shown in Fig 1. To a 2 mL glass vial containing 2 mg K₂CO₃ (catalyst), 105 µL of derivative reagent MNDMB (concentration of 1.0 × 10^-3 mol/L, the amount of substance is 1.05 × 10^-7 mol), 50 µL of mixed Unsaturated fatty acids standard solutions (individual concentration of 1.0 × 10^-4 mol/L, the mixed concentration of 7.0 × 10^-4 mol/L, the amount of substance is 3.50 × 10^-8 mol) and 100 µL of distilled DMF solution were added. The vial was sealed and allowed to react at 95 °C for 40 min. After the reaction was completed, the reaction solution was cooled to room temperature and diluted to 2 mL with chromatographically pure acetonitrile. The dilution was filtered through a 0.22 μm nylon organic needle filter and 10 μL was injected into the HPLC-FLD system for analysis.

Method Validation

The feasibility of the analytical method was verified by linearity, limits of detection (LODs), repeatability, precision, recovery, and accuracy. The calibration curve was constructed for each compound by plotting peak area versus concentration in the range of 3.0 × 10^-8-1.0 × 10^-5 mol/L. The LOD was calculated at a signal-to-noise (S/N) ratio of 3. Quantitative analysis was performed by using external standard methods. Precision was expressed as a percentage of relative standard deviation (R.S.D. %). The reproducibility of the method was investigated by measuring the relative standard deviation (RSD) of peak area and retention time. Intraday precision was determined by using the same level of standards and performing six replicates. Inter-day precision was determined by repeating the same level of standard six times over three consecutive days. The accuracy of the analytical method was determined by spiking known amounts of standards into the food samples and analyzing their recoveries.

Fluorescence properties of MNDMB-UFA derivative

The study of the fluorescence properties of MNDMB-UFA derivatives is important for the establishment of efficient and sensitive HPLC-fluorescence detection methods. UFAs have no or poor fluorescence, and MNDMB-UFA derivatives have the same parent cyclic naphthalimide to produce strong fluorescence. The excitation and emission wavelengths of seven MNDMB-UFA derivatives are close to each other, so the fluorescence properties of the reaction solutions of derivative reagents with eicosapentaenoic acid (EPA) in different solvents were studied. The reaction solution of the derivatization reagent with EPA was obtained according to the derivatization procedure. The excitation and emission wavelengths of the MNDMB-EPA derivative in different solvents were measured by fluorescence spectrometry. The solvents selected for this study included ethyl acetate (EtOAc), ethanol (EtOH), methanol (MeOH), acetonitrile (ACN), N,N-dimethylformamide (DMF), formamide (MeON), and dimethyl sulfoxide (DMSO). As shown in Fig 2, under the same conditions, different kinds of solvents had a large influence on the fluorescence intensity. The maximum excitation and emission wavelengths were nearly unchanged, and the best fluorescence intensity of the reaction solution was obtained in acetonitrile solvent. Therefore, acetonitrile was chosen as the dilution solution after the derivatization reaction and the mobile phase in the chromatographic separation. The fluorescence intensity of the derivatives in DMF solvent was weaker compared with other solvents, but DMF was chosen to configure the standard solution of derivatization reagent because of the high solubility of K₂CO₃ and DMF at high temperature, which helps to accelerate the rate of derivatization reaction.

Selection of catalyst

The derivatization reaction of UFAs with the derivatization reagent MNDMB needs to be conducted in the presence of a basic catalyst, thus, the option of this catalyst is critical. In this study, K₂Cr₂O₄, NaAc, K₂CO₃, and C₄H₄K₂O₆ were selected as catalysts to investigate the derivatization effect of UFAs. The derivatisation process was followed as described above and the catalyst dosages were all 2mg for the experiments. The results showed in Fig 3 that the maximum peak areas of all seven UFAs derivatives were obtained when K₂CO₃ was used as the catalyst, indicating that K₂CO₃ was the best catalyst for this derivatization reaction. Therefore, K₂CO₃ was selected as the basic catalyst for the subsequent research.

Stability of the reagent MNDMB and its unsaturated fatty acid derivatives

The anhydrous DMF solution of MNDMB could be stored at 4 °C in the dark for 1 month without obvious changes in the derivatization yields with UFAs. The stability of the corresponding MNDMB-UFA derivatives was subsequently investigated. The seven UFAs were derivatized under optimal derivatization conditions and repeatedly injected for HPLC-FLD analysis at room temperature for 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively. The results showed that the peak area of each MNDMB-UFA derivative changed rarely within 48 h, and the standard deviation of the peak area was less than 5.0%. It indicates that the stability of the derivatives is good and can satisfy the requirements of the following quantitative analysis.

Optimization of Derivatization Conditions

To enhance the derivatization efficiency, the single-factor optimization method was used to optimize the derivatization temperature, derivatization time, amount of catalyst, and amount of derivatization reagent for the derivatization reaction. Each reaction was repeated three times to decrease the error. The total volume was kept constant when optimizing. The optimization results are shown in Fig 4.

The temperature has a significant effect on reaction time and yields. As shown in Fig 4a, the effect of temperature on the derivatization reaction was investigated from 60 °C to 95 °C. The peak area of MNDMB-UFA derivatives gradually increased with the increase in temperature. The derivatization efficiency improved at least 6-fold with the increase of derivatization temperature from 60 °C to 95 °C. A higher temperature was no longer chosen for optimization to facilitate the experimental operation. Therefore, the derivatization temperature of 95 ℃ was selected for the subsequent experiments.

The derivatization peak area of the derivatization was optimized in the range of 20-60 min, as shown in Fig 4b. The MNDMB-UFA derivatives peak area reached its maximum value when the derivatization time reached 40 min and showed a slightly decreasing trend with the increase in the reaction time. Therefore, 40 min was sufficient in all subsequent experiments.

The effect of the amount of K₂CO₃ on the peak area of the derivatives was investigated, as shown in Fig 4c. The peak area of the MNDMB-UFA derivatives increased and then decreased in the K₂CO₃ dosage range of 1-8 mg, and the maximum peak area of the derivatives was reached when the dosage of K₂CO₃ was 2 mg. Therefore, the optimal amount of catalyst is 2 mg.

To ensure complete reactions of UFAs, it is necessary to ensure that sufficient concentrations of derivatization reagents are used. The molar ratio of derivatization reagent to UFAs was studied in the range of 1-8. As shown in Fig 4d, the fluorescence response increased when the concentration of the derivatization reagent increased. The fluorescence intensity of MNDMB-UFA derivatives reached a maximum and remained constant at a molar ratio of 3.

Chromatographic Separation

To improve the resolution of the separation of MNDMB-UFA derivatives, several chromatographic separation columns, such as Hypersil ODS C18 column (4.6 mm × 200 mm, 5 µm), Hypersil C18 column (4.6 mm × 200 mm, 5 µm), Agilent ZORBAX 300SB-C18 column (4.6 mm × 200 mm, 5 µm ), Eclipse XDB-C8 column (4.6 mm × 150 mm, 5 µm) and Hypersil BDS C8 column (4.6 mm × 200 mm, 5 µm) were evaluated for optimal chromatographic separation. The optimal chromatographic separation was evaluated on the Agilent ZORBAX 300SB-C18 column (4.6 mm × 200 mm, 5 μm), which was found to be able to separate the different MNDMB-UFA derivatives efficiently and with good separation. A gradient elution from (A) 30% acetonitrile in water to (B) 100% acetonitrile was used to achieve the best separation with the shortest retention time and the sharpest peaks. As can be seen in Fig 5, the baseline resolution of all unsaturated fatty acid derivatives was good.

Method Performance

Following the optimization of the derivatization conditions and chromatographic separation, the linear regression equation, correlation coefficient, LOD, reproducibility of retention time and peak area, and intra-day and inter-day precision of the established method were calculated and the results are shown in Table 1. The linear correlation coefficients (R²) of the seven UFAs ranged from 0.99980 to 0.99998, indicating that the method showed a good linear relationship. LODs were in the range of 4-21 nmol/L, which indicated that the proposed method has a high sensitivity. The intra-day precision of the method was in the range of 1.91-3.51% and the inter-day precision was in the range of 3.33-5.41%. RSD values of retention time and peak area of seven unsaturated fatty acids were less than 1.00% and 3.70%, respectively.

Sample Analysis

To assess the applicability of the proposed method to real samples with complex matrices, the established method was applied to determine the seven targeted analytes in corn oil and soybean oil. The representative chromatograms of corn oil and soybean oil are shown in Fig 6(a, b), the spiked chromatograms were obtained by adding 1.0 × 10^-6 mol/L UFA standards to the corn oil and soybean oil samples respectively as shown in Fig 6(c, d). Table 2 shows the contents of the analytes in the actual samples. The results showed that some of the target compounds were found in the measured samples, which indicated that the development of a new method for simultaneous screening of trace UFAs is of practical interest. As expecteccd, Corn oil contained the maximum amount of CLA (8.65 mg/mL), followed by OA (6.57 mg/mL), LNA (0.20 mg/mL), and EA (0.054 mg/mL), while soybean oil contained the maximum amount of CLA (19.83 mg/mL), followed by OA (11.62 mg/mL), LNA (5.01 mg/mL), DHA (2.21 mg/mL), and EA (0.037 mg/mL). As shown in Fig 7, the type and relative content of UFAs contained in the two vegetable oils can be visualized. The recovery tests were conducted by adding three concentrations of UFAs of 1.0 × 10^-6 mol/L, 2.0 × 10^-6 mol/L, and 5.0 × 10^-6 mol/L to the oil samples, respectively. The results (Table 2) showed that the spiked recoveries ocf the seven UFAs ranged from 90.2% to 103.5%, and the intra-day and inter-day precision of the two oil samples ranged from 2.10% to 3.19% and 3.40% to 6.34%, respectively, which demonstrated the high accuracy of the proposed method.

In this study, a novel fluorescent reagent was designed and synthesized for the rapid determination of 7 unsaturated fatty acids in food samples in combination with HPLC-FLD. The synthesis process of the derivative reagent MNDMB was simple and well-stabilized, and the reaction of MNDMB with UFAs was fast and could be accomplished under mild conditions. By introducing MNDMB with excellent fluorescence properties into the UFA molecules, the fluorescence properties of FA molecules can be improved. Meanwhile, the proposed method was successfully applied to the analysis of unsaturated fatty acids in oil samples. The method showed its advantages of rapidity, simplicity, low cost, high sensitivity, good selectivity, and high accuracy. The developed method has the powerful potential for practical application in the analysis of trace unsaturated fatty acids in complex samples.

Funding

This work was financially supported by the Major Innovation Fund of Shandong Province (No. 2021ZDSYS23) and supported by the National Natural Science Foundation of China (No. 21976105).

Author Contributions Statement

Shuiqiang Yu: methodology, data collection and analysis, writing-original draft. Jinmao You: conceptualization, writing-review and editing. Zhihao Lu: methodology. Yu Wang: HPLC-FLD analysis. Xinxin Shi: data curation. Xiaocong Zou: synthesis. Jiangkun Tan: purifcation. Zhiwei Sun, Zan Li, Zhongyin Ji and Cuihua Song: conceptualization. All authors reviewed the manuscript.

Declarations

Conflict of Interest Shuiqiang Yu declares that he has no confict of interest. Jinmao You declares that he has no confict of interest. All authors have no conflict of interest.

Availability of Data

The datasets generated or analyzed for this study are available from reasonable requests to the corresponding author.

The results presented in this manuscript have neither been published before nor submitted to publication elsewhere.

Ethical Approval

Not applicable

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Table 1 Dynamic ranges, regression equations, and detection limits of the proposed method

Analytes	Regression equation^a	R²	RepeatabilityR.S.D(%) (n=6)		PrecisionR.S.D(%) (n=6)		LOD (μg/kg)^b
Analytes	Regression equation^a	R²	Retention time	Peak area	Intraday	Interday	LOD (μg/kg)^b
EPA	Y=34.94X-4.80	0.99984	0.42	2.78	1.91	3.83	20
LNA	Y=137.37X+39.45	0.99995	0.77	3.65	2.34	3.52	5.3
DHA	Y=19.90X+9.27	0.99980	0.63	1.97	2.28	3.33	21
ARA	Y=99.08X+10.48	0.99998	0.57	2.05	2.37	4.59	9.8
LA	Y=264.37X+83.62	0.99998	0.91	3.15	2.56	5.41	4
OA	Y=165.54X+40.15	0.99996	0.76	1.99	3.23	4.67	5.5
EA	Y=154.60X+46.88	0.99997	0.96	2.57	3.51	4.38	6

^aY=peak area (mV min); X=sample concentration (pmol)

^bS/N=3, injection volume: 10 μL

Table 2 Recovery, contents, intra-day and inter-day relative standard deviations

Samples	Analytes	Contents (mg/mL)	RepeatabilityRSD%^a		Recovery (%)^b
Samples	Analytes	Contents (mg/mL)	Intraday	Interday	1 μM	2 μM	10 μM
Corn oil	EPA	–	2.10	4.14	93.6	91.5	100.6
	LNA	0.20	2.43	3.40	98.7	95.2	91.9
	DHA	–	2.57	3.81	94.7	93.8	99.3
	ARA	–	2.97	3.98	102.8	98.7	98.9
	LA	8.65	2.89	4.76	97.8	101.2	103.1
	OA	6.57	2.67	4.23	93.3	94.9	97.0
	EA	0.054	2.73	3.75	96.8	101.4	102.5
Soybean oil	EPA	–	2.67	5.78	93.6	92.8	96.7
	LNA	5.01	3.16	5.53	99.8	102.3	103.5
	DHA	2.21	2.78	3.87	91.5	95.3	97.8
	ARA	–	2.56	4.59	90.2	92.4	96.5
	LA	19.83	3.19	6.34	90.8	95.9	95.7
	OA	11.62	2.54	4.47	94.1	95.6	101.3
	EA	0.037	3.01	3.91	101.2	97.6	98.0

a Intraday RSD%, n=6; interday RSD%, n=6×3;

b Recovery is given as mean (n=6, %); samples spiked at two different concentrations (1 μM, 2 μM and 10 μM).

No competing interests reported.

Supplementarymaterial.doc

New Fluorescence Derivatization Reagent for the Determination of 7 Unsaturated Fatty Acids in Oil Samples by Pre-Column Derivatization HPLC-FLD

Status:

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Abstract

Figures

Introduction

Reagents And Materials

Results And Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

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