HPLC-FLD Method Research Articles

Voriconazole therapeutic drug monitoring including analysis of CYP2C19 phenotype in immunocompromised pediatric patients with invasive fungal infections

PurposeTherapeutic drug monitoring (TDM) of voriconazole (VCZ) should be mandatory for all pediatric patients with invasive fungal infections (IFIs). The narrow therapeutic index, inter-individual variability in VCZ pharmacokinetics, and genetic polymorphisms cause achieving therapeutic concentration during therapy to be challenging in this population.MethodsThe study included 44 children suffering from IFIs treated with VCZ. Trough concentrations (Ctrough) of VCZ ware determined by the HPLC-FLD method. Identification of the CYP2C19*2 and CYP2C19*17 genetic polymorphisms was performed by PCR–RFLP. The correlation between polymorphisms and VCZ Ctrough was analyzed. Moreover, the effect of factors such as dose, age, sex, route of administration, and drug interactions was investigated.ResultsVCZ was administered orally and intravenously at a median maintenance dosage of 14.7 mg/kg/day for a median of 10 days. The VCZ Ctrough was highly variable and ranged from 0.1 to 6.8 mg/L. Only 45% of children reached the therapeutic range. There was no significant association between Ctrough and dosage, age, sex, route of administration, and concomitant medications. The frequencies of variant phenotype normal (NM), intermediate (IM), rapid (RM) and ultrarapid metabolizers (UM) were 41%, 18%, 28%, and 13%, respectively. Ctrough of VCZ were significantly higher in NM and IM groups compared with RM, and UM groups.ConclusionThe Ctrough of VCZ is characterized by inter-individual variability and a low rate of patients reaching the therapeutic range. The significant association exists in children between VCZ Ctrough and CYPC19 phenotype. The combination of repeated TDM and genotyping is necessary to ensure effective treatment.

Occurrence of ochratoxin A in cocoa beans and bean-to-bar chocolates.

The growing health consciousness of consumers has led to an increase in the consumption of artisanal chocolates, mainly due to their recognized health benefits. However, processing steps such as fermentation and drying of cocoa beans can favor the growth of ochratoxigenic fungi. This study aimed to assess the occurrence of ochratoxin A (OTA) in cocoa beans (purchased from e-commerce and post-harvest processing) and bean-to-bar chocolates sold in Brazil. An HPLC-FLD method was validated, with recovery values between 84 and 97% and limits of detection and quantification of 0.04 and 0.01 µg/kg, respectively. OTA was detected in 30% of the cocoa bean samples studied (n = 43), with values ranging from < 0.04 to 1.18 µg/kg. Regarding the bean-to-bar chocolates (n = 62), the OTA concentrations ranged from < 0.04 to 1.11 µg/kg, with a prevalence in semi-sweet and dark chocolates. Despite representing a growing market, to the best of our knowledge, this is the first study to report OTA concentrations in bean-to-bar chocolates and Brazilian cocoa beans used to produce this type of chocolate.

Bioanalytical HPLC method with fluorescence detector for determination of Entresto™ when co-administered with ibuprofen and fexofenadine: a pharmacokinetic study.

Entresto™ (LCZ696) has been approved globally for heart failure management. However, its lifelong use alongside over-the-counter (OTC) drugs like ibuprofen (IBU) and fexofenadine (FEX) necessitates an in-depth investigation of potential pharmacokinetic interactions, as they share the same metabolic and elimination pathways. This study aimed to develop a bioanalytical HPLC method with a fluorescence detector (FLD) to quantify LCZ696 analytes (valsartan, VAL; sacubitril, SAC; and sacubitril active metabolite, LBQ657) in rat plasma. Additionally, an in vivo study was performed to investigate the pharmacokinetic interactions of LCZ696 with IBU and FEX. Utilizing HPLC with a gradient-mode mobile phase of acetonitrile and 0.025 M phosphate buffer (pH 3), the study demonstrated a significant increase in the bioavailability of LCZ696 analytes (VAL and LBQ657) when co-administered with IBU (C max 0.23 ± 0.07 and 0.53 ± 0.21 μg mL-1, respectively) compared to the control (0.17 ± 0.03 and 0.33 ± 0.14 μg mL-1). A more significant increase in C max was noticed with FEX (0.38 ± 0.01 and 0.77 ± 0.18 μg mL-1, respectively). Moreover, a decrease in the clearance (Cl/F) of VAL and LBQ657 was observed (18.05 ± 1.94 and 12.42 ± 2.97 L h-1 kg, respectively) with a more pronounced effect in the case of FEX (30.87 ± 4.29 and 33.14 ± 9.57 L h-1 kg, respectively) compared to the control (49.99 ± 7.31 and 51.19 ± 9.12 L h-1 kg, respectively). In conclusion, our study underscores the importance of cautious administration and appropriate dose spacing of IBU and FEX in patients treated with LCZ696 to prevent elevated serum concentrations and potential toxicity. The novelty of this work lies in its dual contribution: developing a highly sensitive HPLC-FLD method and comprehensively elucidating significant pharmacokinetic interactions between LCZ696 and common OTC drugs.

Cannabinoid Modulation of Monoamine Levels in Mouse Brain: Unveiling Neurochemical Dynamics through an Innovative High-performance Liquid Chromatography-Fluorescence Detection Bioanalysis

Homeostasis of monoaminergic pathways is compromised in aging and neurodegenerative/neurological diseases such as Alzheimer’s disease and depression. On the other hand, their modulation has also been linked to the mechanism of action of several drugs. Therefore, monitoring the levels of noradrenaline (NA), adrenaline (AD), dopamine (DA), and serotonin (5-HT), as well as those of tryptophan (TRYP), the precursor of 5-HT, and DA metabolites, namely 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), is fundamental for assessing disease severity and progression. This work aimed to develop and validate the first High-Performance Liquid Chromatography (HPLC) coupled with fluorescence detector (FLD) method that simultaneously and accurately quantifies NA, AD, DA, DOPAC, HVA, 5-HT and TRYP in mouse brain and prefrontal cortex (PFC) matrices. Previous sample preparation by protein precipitation was required to extract the compounds. Calibration curves were plotted using the background subtraction approach to reduce the interference of the endogenous analytes. Intra and inter-day accuracy and precision were within the ranges defined by ICH (The International Council for Harmonisation) guideline for bioanalytical method validation. Following validation, the impact of cannabidiol (CBD), cannabigerol (CBG), and cannabidivarin (CBDV) was explored in mice brains post-administration, revealing significant alterations in specific neurotransmitter levels upon cannabinoid exposure and shedding light on the complex modulation of neurochemical dynamics by cannabinoids. This research highlights the fit-for-purpose of the HPLC-FLD method and provides insights into potential mechanisms underlying phytocannabinoid actions in the central nervous system (CNS).

Malondialdehyde levels and bioaccessibility in healthy diet bars: A gastrointestinal digestion model

Malondialdehyde (MDA), which is composed when n-6 and n-3 PUFAs are peroxidized, has been utilized as an indicator of lipid peroxidation and has been considered neurotoxic, cytotoxic, and mutagenic. This study aimed to determine the bioaccessibility level of MDA in diet bars sold as healthy snacks in the market using in vitro gastrointestinal digestive model. In our study, 28 different diet bar samples were bought from markets in Istanbul. MDA contents of the products were determined by the HPLC-FLD method. The investigation showed that diet bars had an average MDA concentration of 116.25 μg/100 g before digestion, while the average MDA concentration was 90.50 μg/100 g after in vitro digestion. In line with these data, the average MDA bioaccessibility of 28 diet bar samples was calculated as 77.3%. For this reason, more studies are needed to understand the relationship between both the MDA content and the reaction and nutritional components.

Determination of norfloxacin, cipfloxacin, levofloxacin and moxifloxacin in urine by HPLC with a two-channel fluorescence detector

A sensitive HPLC-FLD method with two-channel fluorescence detection was established and validated for quantification of four fluoroquinolones (norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in urine samples. The fluorescent detector was set at two-channel excitation and emission wavelengths: 280/445 nm (for NOR, CIP) and 290/500 nm (for LEVO, MOXI). The measurement protocol was in compatible with both of the sample treatment procedures including the liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The method was well-validated with the limit of quantification: 0.027; 0.034; 0.024; 0.020 µg/mL (for LLE), and 0.028; 0.033; 0.024; 0.020 µg/mL (for SPE) for NOR, CIP, LEVO, MOXI, respectively. The method has been proved to be precise and accurate, with the relative standard deviation lower than 3% and the recovery ranging from 98.0%¸102.0%, for all fluoroquinolones. The proposed HPLC-FLD method provides an alternative approach for the simultaneous analysis of fluoroquinolones in urine. ® 2023 Journal of Science and Technology - NTTU

Determination of norfloxacin, cipfloxacin, levofloxacin and moxifloxacin in urine by HPLC with a two-channel fluorescence detector

A sensitive HPLC-FLD method with two-channel fluorescence detection was established and validated for quantification of four fluoroquinolones (norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in urine samples. The fluorescent detector was set at two-channel excitation and emission wavelengths: 280/445 nm (for NOR, CIP) and 290/500 nm (for LEVO, MOXI). The measurement protocol was in compatible with both of the sample treatment procedures including the liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The method was well-validated with the limit of quantification: 0.027; 0.034; 0.024; 0.020 µg/mL (for LLE), and 0.028; 0.033; 0.024; 0.020 µg/mL (for SPE) for NOR, CIP, LEVO, MOXI, respectively. The method has been proved to be precise and accurate, with the relative standard deviation lower than 3% and the recovery ranging from 98.0%¸102.0%, for all fluoroquinolones. The proposed HPLC-FLD method provides an alternative approach for the simultaneous analysis of fluoroquinolones in urine.&#x0D; ® 2023 Journal of Science and Technology - NTTU

Turmeric Powder Counteracts Oxidative Stress and Reduces AFB1 Content in the Liver of Broilers Exposed to the EU Maximum Levels of the Mycotoxin.

The most frequent adverse effects of AFB1 in chicken are low performance, the depression of the immune system, and a reduced quality of both eggs and meat, leading to economic losses. Since oxidative stress plays a major role in AFB1 toxicity, natural products are increasingly being used as an alternative to mineral binders to tackle AFB1 toxicosis in farm animals. In this study, an in vivo trial was performed by exposing broilers for 10 days to AFB1 at dietary concentrations approaching the maximum limits set by the EU (0.02 mg/kg feed) in the presence or absence of turmeric powder (TP) (included in the feed at 400 mg/kg). The aims were to evaluate (i) the effects of AFB1 on lipid peroxidation, antioxidant parameters, histology, and the expression of drug transporters and biotransformation enzymes in the liver; (ii) the hepatic accumulation of AFB1 and its main metabolites (assessed using an in-house-validated HPLC-FLD method); (iii) the possible modulation of the above parameters elicited by TP. Broilers exposed to AFB1 alone displayed a significant increase in lipid peroxidation in the liver, which was completely reverted by the concomitant administration of TP. Although no changes in glutathione levels and antioxidant enzyme activities were detected in any treatment group, AFB1 significantly upregulated and downregulated the mRNA expression of CYP2A6 and Nrf2, respectively. TP counteracted such negative effects and increased the hepatic gene expression of selected antioxidant enzymes (i.e., CAT and SOD2) and drug transporters (i.e., ABCG2), which were further enhanced in combination with AFB1. Moreover, both AFB1 and TP increased the mRNA levels of ABCC2 and ABCG2 in the duodenum. The latter changes might be implicated in the decrease in hepatic AFB1 to undetectable levels (<LOD) in the TP supplemented group. Overall, our findings further support the use of TP as an effective feeding strategy to prevent AFB1-related adverse effects in broilers.

Dual-layers Raman reporter-tagged Au@Ag combined with core-satellite assemblies for SERS detection of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin identified in corn. A SERS-based immunosensor by constructing core-satellite assemblies was developed for ZEN detection. ZEN monoclonal antibody modified gold nanostars (AuNSs) were fabricated as the capture probe (core). The Raman signal probes (satellites) utilized ZEN antigen linked to the core-shell structures loaded with two layers of Raman reporter molecules (AuMBA@AgMBANPs). The coupling between AuNSs and AuMBA@AgMBANPs can produce a poweful electromagnetic field, thus considerably amplifying the Raman signal. The detection range of ZEN for corn samples under the optimal conditions was 5∼400μg/kg with a LOD of 3μg/kg, which completely satisfying the requirement of maximum residual level (60μg/kg). Moreover, the proposed SERS method was consistent with the HPLC-FLD method for the detection of ZEN in naturally contaminated corn samples (90.58% ∼ 105.29%). Conclusively, fabricated immunosensor with exceptional sensitivity and specificity broaden the application of SERS in mycotoxin detection.

Development and cross-validation of simple HPLC-fluorescence and UPLC-MS-UV methods for rapid determination of oleuropein in olive leaves.

Olive leaves, abundant by-products of the olive oil industry, are a rich source of oleuropein, an important polyphenol in olive leaves. So far, no published methods have been validated using matrix standards for oleuropein quantification in olive leaves. The study aimed to develop an HPLC method for oleuropein determination in olive leaves using spiked matrix standards prepared from a blank olive leaf matrix, to validate the method with respect to aqueous standards, and cross-validate the HPLC method with UPLC-MS and UPLC-UV techniques. Oleuropein was extracted into methanol and analysed by HPLC with fluorescence detection (FLD; excitation and emission wavelengths 281 and 316 nm, respectively) and by UPLC-MS-UV. For validation, calibration curves of spiked matrix standards (0.4 to 4.8 mg/g) were analysed by the three methods over several days. Oleuropein was then analysed in French olive varieties. For the HPLC-FLD method, repeatability and intermediate precision were less than 5% RSD and linearity was demonstrated by the Fischer test. Differences in results of the spiked placebos by the three methods were non-significant, as confirmed by ANOVA. Extraction recovery was >90%, and there was a strong linear relationship between authentic and spiked matrix standards. The determination of oleuropein in French olive varieties is reported, including analysis in "Olivière" cultivar for the first time, leaves of which contained twice the amount of oleuropein compared with "Picholine". Accurate quantification of oleuropein is possible using aqueous standards. Cross-validation indicates that selective analysis can equally be carried out by HPLC or by UPLC-MS techniques.

Molecularly imprinted dispersive micro solid-phase extraction coupled with high-performance liquid chromatography for the determination of four aflatoxins in various foods

A new dummy template-based molecularly imprinted dispersive micro solid-phase extraction (MI-d-µSPE) coupled with HPLC-FLD developed for the simultaneous determination of four aflatoxins (B1, B2, G1, G2) in various food matrices. The synthesized MIP was used as a dispersive solid-phase extraction (dSPE) sorbent for aflatoxins extraction. The chemometric approach was used to identify the optimum conditions of dSPE. The results showed the amount of MIP sorbent (55 mg), adsorption time (12.5 min), and %ACN (75%) were significant extraction parameters. The method has a detection limit in the range of 0.059–0.208 µg kg−1 and a quantification limit in the range of 0.197–0.694 µg kg−1 for aflatoxins. The intra- and inter-day precision was less than 5%, and recoveries were 79.1–109.4%. The expanded uncertainty of the developed method was found to be 2.9–22.8%. The new MI-d-µSPE with HPLC-FLD method was applied for 37 food matrices.

Kıbrıs’ın Kuzeyinde Üretilen Süt Örneklerinde Aflatoksin M1 Düzeyleri

Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 (AFB1), which is formed in the liver by cytochrome P450 enzymes and can be secreted into the urine, feces, and milk of mammals. AFM1 is a carcinogenic, cytotoxic, teratogenic, mutagenic and genotoxic agent that poses a significant health risk to both humans and animals. This study was conducted to determine the presence of AFM1 in both raw and ultra-high temperature (UHT) cow’s milk samples produced in the northern part of Cyprus, and to determine whether it poses a risk to public health. In this survey, a total of 20 UHT cow’s milk samples from 2 different milk brands produced in the northern part of Cyprus, and 22 raw cow’s milk samples collected from the different dairies were analyzed for the presence of AFM1 by high performance liquid chromatography (HPLC) with fluorescence detector after immunoaffinity cleanup. AFM1 could not be detected in any of the analyzed raw and UHT cow milk samples. The LOD and LOQ values of the HPLC-FLD method were 1.038 μg/kg and 3.145 μg/kg, respectively. The mean recovery and repeatability values of the method were 95.6% and 4.9%, respectively. Although the presence of AFM1 in milk samples produced in the northern part of Cyprus poses no major risk to public health, more milk samples and animal feed must be monitored on a regular basis to decrease potential consumer exposure.

Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma.

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C18 (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze-thaw cycles, and 2weeks at -20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.

HPLC methods for studying pharmacokinetics of tivozanib and in vitro metabolic interaction with dexamethasone in rat

Tivozanib is a recently approved tyrosine kinase inhibitor for the treatment of renal cell carcinoma. In this work, two new HPLC methods coupled with fluorescence (FLD), or photodiode array detectors (PDA) were developed and used for the first time for tivozanib quantification in rat plasma and liver microsomes. The described methods were efficient with a 4-min runtime employing a Gemini-NX C18 column (50 × 2.1 mm, 3 µm) and a mobile phase of acetonitrile and ammonium acetate buffer (pH 4.7, 10 mM) (40:60, v/v) delivered at a flow rate of 0.4 mL/min. The use of HPLC-FLD allowed the quantification of 50 ng/ mL tivozanib using only 100 µL rat plasma. The HPLC-FLD method was validated according to the US food and drug administration (FDA) bioanalytical guidelines and was applied successfully in a rat pharmacokinetic study (n = 7) following oral administration of 1 mg/ kg tivozanib. Furthermore, HPLC-PDA was used for monitoring the depletion of 1 μM (454.9 ng/mL) tivozanib in rat liver microsomes and was applied to study the effect of dexamethasone induction on tivozanib metabolism in vitro. Results showed that dexamethasone enhanced the intrinsic clearance of tivozanib by 60 % suggesting a potential drug-drug interaction at the metabolism level. Dexamethasone is commonly used in the management of cancer disease and thus coadministration with tivozanib therapy may cause treatment failure in patients. The simplicity, speed and cost-effectiveness of the reported methods are ideal for supporting in vivo and in vitro tivozanib studies, including drug-drug interaction studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities.

Detection of aflatoxin producing fungi in rice and peanut, and determination of aflatoxin by HPLC-FLD method

Detection of aflatoxin producing fungi in rice and peanut, and determination of aflatoxin by HPLC-FLD method

Extraction of bioactive coumarins from lime peel as sample pretreatment before chromatographic analysis

Abstract Analysis of lime peel was applied to assess their suitability for various intended purposes, e.g., application in perfumery, cosmetics, and cleaning products, or as a source of bioactive or other value-added compounds. Targeted analysis allows wider usability of the waste part of these natural materials. In the present study, a novel, efficient, lab-simple, time-saving analytical method for coumarins determination in lime peel, including sample pretreatment by extraction and quantification by HPLC with fluorescence detection (FLD), is introduced. Optimal conditions of ultrasound assisted extraction included water as extraction solvent, temperature of 40 °C, and extraction time of 10 min. A magnetic molecularly imprinted polymer was employed as solid phase extraction adsorbent for primary extract cleaning, isolation, and enrichment of coumarins before chromatographic analysis. Recovery of herniarin and umbelliferone was more than 86 % ((RSD ≤ 4.8 %). Linear range of 50—1000 ng/mL with correlation coefficient above 0.998 was obtained for the proposed HPLC-FLD method. The limit of quantification was 8.2 and 44 ng/mL for herniarin and umbelliferone, respectively. These results show that the proposed sample pretreatment procedure is suitable for analytical purposes and is perspective also for the analysis of other citrus samples, as well as for future scale-up preparative isolation of bioactive coumarins from citrus peel.

Development and validation of HPLC-FLD method for aflatoxin M1 determination in milk and dairy products

Abstract Milk and dairy products are the most consumed foods in human diet and their safety is in the attention centre of control authorities. Aflatoxin M1 (AFM1) is a dangerous toxin that can occur in milk and dairy products as a metabolite formed from aflatoxin B1 contained in contaminated animal feed. Therefore, the aim of this study was to develop and validate a reliable method for the determination of AFM1 content in milk and dairy products based on HPLC with fluorescence detection employing immunoaffinity columns (IAC) pre-treatment. Optimal chromatographic separation of AFM1 was achieved using a water/acetonitrile mixture (80/20, v/v) as a mobile phase, column with C18 stationary phase maintained at 25 °C, and fluorescence detection at excitation wavelengths of 360 nm and emission of 440 nm. Efficacy of AFM1 extraction from the samples was found to be influenced by the elution agent composition. The best results were obtained using 1.25 mL of acetonitrile/methanol (3/2, v/v) and 1.25 mL of water. Validation parameters of the proposed method met the criteria set by the European legislation with the limits of detection and quantification at 0.002 and 0.007 µg/kg, respectively. Also, suitability of the method was confirmed by its application for AFM1 determination in certified reference material. Finally, the method was applied for AFM1 determination in 25 milk and dairy products collected in Slovakia; the AFM1 content was below the limit of quantification. It was concluded that the method is suitable for AFM1 content monitoring in milk and dairy products.

Glyphosate and aminomethylphosphonic acid levels in water and soil samples from Transylvanian Roma community analyzed by HPLC-FLD method

Glyphosate and aminomethylphosphonic acid levels in water and soil samples from Transylvanian Roma community analyzed by HPLC-FLD method

Development and Validation of an HPLC-FLD Method for the Determination of NDMA and NDEA Nitrosamines in Lisinopril Using Pre-Column Denitrosation and Derivatization Procedure

In order to meet the analytical requirements of the European Medicines Agency (EMA), a new HPLC-FLD method was successfully developed using dansyl chloride for the derivatization and determination of the genotoxic impurities N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) in Lisinopril API and its final product. Samples’ pretreatment includes liquid–liquid microextraction, denitrosation, and derivatization steps. To optimize the process, the parameters contributing to high sensitivity and yielding reliable results were thoroughly studied and optimized using one-factor-at-a-time and experimental design approaches. The analytes were pre-column derivatized with Dansyl-Cl and analyzed by HPLC-fluorescence (λem/λem = 340/530) using a C18 column and a mixture of phosphate buffer (pH = 2.8; 20 mM)/acetonitrile 55:45 v/v as the mobile phase. The six-level concentration calibration was shown to be linear, with R equal to 0.9995 for both analytes. The limit of detection (LOD) was satisfactory and equal to 4.7 and 0.04 ng/mL for NDMA and NDEA, respectively. Precision was less than 13.4% in all cases, and the average recoveries were equal to 109.2 and 98.1% for NDMA and NDEA, respectively. The proposed procedure is relatively easy, rapid, and suitable for the determination of the two nitrosamines in routine analysis tests.

Preparation of Monoclonal Antibody against Pyrene and Benzo [a]pyrene and Development of Enzyme-Linked Immunosorbent Assay for Fish, Shrimp and Crab Samples

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental and food pollutants that can cause cancer. In this work, a specific monoclonal antibody (mAb) to identify pyrene (PYR) and benzo [a]pyrene (BaP) was prepared, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was established to detect PYR and BaP residues in living aquatic products for the first time. The effects of complete antigens with different coupling ratios on the production of high-sensitivity mAb was explored. Under the optimal conditions, the IC50 value was 3.73 ± 0.43 µg/L (n = 5). The limits of detection (LODs) for PYR and BaP in fish, shrimp, and crab ranged from 0.43 to 0.98 µg/L. The average recoveries of the spiked samples ranged from 81.5–101.9%, and the coefficient of variation (CV) was less than 11.7%. The validation of the HPLC-FLD method indicated that the ELISA method set up in this experiment provided a trustworthy tool for PAHs residues detection in aquatic products.