Conventional HPLC Method Research Articles

An Electrochemical Sensor Based on Simple Nano-Modification for Sensitive Detection of Ofloxacin Residue in Animal Derived Food

The residue of ofloxacin in animal derived food would seriously affect human health. This study reports an inexpensive, fast, and simple electrochemical sensor for the sensitive detection of OFL in animal derived food samples. A poly-L-glutamic acid/poly(sulfobutyl-β-cyclodextrin) CdTe quantum dot-modified electrode P(L-Glu)/SEB-β-CD@CdTeQDs/GCE was prepared by an electrochemical polymerization reaction. We investigated the electrochemical behavior of OFL on the sensing electrode by cyclic voltammetry and square wave voltammetry. The results showed that the sensitivity of the sensor was significantly improved under the synergistic effect of L-glutamic acid, sulfobutyl-β-cyclodextrin, and CdTe quantum dots. In addition, the electrochemical reaction process of OFL on the sensing electrode was investigated and the results showed that the transfers of two protons and two electrons were involved in the reaction. Subsequently, the electrochemical sensing method based on P(L-Glu)/SEB-β-CD@CdTeQDs/GCE was developed and validated for the determination of OFL, showing a good linear response in the range of 0.07 ∼ 1 μg·ml−1 with a detection limit of 3.4 ng·ml−1, and the precision and accuracy were high enough. Compared with the conventional HPLC method, the established electrochemical sensing method shows the merits of being eco-friendly, shorter detection time and being much more simple, sensitive, and convenient.

On-site detection of chloramphenicol in fish using SERS-based magnetic aptasensor coupled with a handheld Raman spectrometer

In recent years, the rapid detection of chloramphenicol (CAP) has become a market demand due to its high toxicity. In this study, for the first time, a portable surface-enhanced Raman scattering (SERS) aptasensor for the rapid and on-site detection of chloramphenicol (CAP) residues in fish was developed. Fe3O4@Au nanoflowers combined with sulfhydryl (SH)-CAP aptamer complementary DNA acted as capture probes. SH-CAP aptamer modified Au@Ag nanoparticles (Au@Ag NPs) embedded with 4-mercaptobenzoic acid (4-MBA) were served as reporter probes. The strongest Raman intensity was produced due to the coupling of Fe3O4@Au nanoflowers (Fe3O4@Au NFs) and Au@Ag NPs. For CAP detection, a wide linear range from 0.001 to 1000 μg/L, with an R2 of 0.9805, was obtained. The limit of detection was determined to be 0.87 ng/L. The SERS aptasensor showed excellent performance for analytical applications for real fish samples. Compared with the conventional HPLC method, the developed SERS aptasensor coupled with a handheld Raman spectrometer had flexible application and avoided the limitations of complex operating conditions. It should be a promising portable analytical tool for analysis of drug residues in the field.

Multiple chemiluminescence immunoassay detection of the concentration ratio of glycosylated hemoglobin A1c to total hemoglobin in whole blood samples

The concentration ratio of glycosylated hemoglobin A1c (HbA1c) to total hemoglobin (Hb) has long been used to accurately determine stagewise diabetes because this parameter represents a reliable and accurate biomarker of mean 90-day blood glucose values. In this paper, we report a time-resolved chemiluminescence assay that can detect both Hb and HbA1c. For the determination of Hb, the interaction of heme in Hb with H2O2 in NaOH solution was performed to generate a chemiluminescence peak. HbA1c was detected using a sandwich immunoassay based on an acridine ester-labeling method using the same Hb chemiluminescence trigger system. The results showed that the repeatability %CV of the proposed method for multiple detections of HbA1c and Hb ranged from 1.22 to 2.21%, with a median value of 1.73%, while the within-site reproducibility %CV ranged from 2.13 to 3.27%, with a median value of 2.81%. Compared with the conventional HPLC method (BIO-RAD D10 system), the correlation coefficient was 0.9959. In conclusion, a time-resolved multiple chemiluminescence immunoassay biosensor for HbA1c/Hb detection was established, and the method has excellent reproducibility and accuracy, thus demonstrating great potential for clinical application.

Smart sensor for assessment of oxidative/nitrative stress biomarkers using a dual-imprinted electrochemical paper-based analytical device

We present a novel dual-imprinted electrochemical paper-based analytical device (Di-ePAD) to simultaneously determine 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) and assess oxidative and nitrative biomarkers in urine and plasma samples. The Di-ePAD was designed with hydrophobic barrier layers formed on filter paper to provide three-dimensional circular reservoirs and assembled electrodes. The molecularly imprinted polymer (MIP) was synthesized using a silica nanosphere decorated with silver nanoparticles (SiO2@AgNPs) as a core covered with dual-analyte imprinted sites on the polymer to recognize selectively and bind the target biomarkers. This strategy drives monodispersity and enhances the conductivity of the resulting MIP core-shell products. 3-NT-MIP and 8-OHdG-MIP were synthesized by successively coating the surface of SiO2@AgNPs with l-Cysteine via the thiol group, then terminating with MIP shells. The dual imprinted core-shell composites possess attractive properties for the target biomarkers' sensing, including catalytic activity, selectivity, and good conductivity. The Di-ePAD revealed excellent linear dynamic ranges of 0.01–500 μM for 3-NT and 0.05–500 μM for 8-OHdG, with detection limits of 0.0027 μM for 3-NT and 0.0138 μM for 8-OHdG. This newly developed method based on the synergistic effects of SiO2@AgNPs combined with promising properties of MIP offers outstanding selectivity, sensitivity, reproducibility, simplicity, and low cost for quantitative analysis of 3-NT and 8-OHdG. The proposed Di-ePAD showed good accuracy and precision when applied to actual samples, including urine and serum samples validated by a conventional HPLC method.

Development and Validation of a UPLC-MS/MS Method for Ultra-Trace Level Determination of Acyl Chloride Potential Genotoxic Impurity in Mezlocillin.

3-Chlorocarbonyl-1-methanesulfonyl-2-imidazolidinone (CMI) is a critical intermediate used in the synthesis of mezlocillin drug substance and also a potential genotoxic impurity with acyl chloride moiety. The content of CMI in mezlocillin should be <0.16ppm to avoid the carcinogenicity and mutagenicity threats to patients. Therefore, a workable determination of CMI was critically crucial for ensuring the safety of mezlocillin drug products. However, the conventional HPLC method is insufficient for detection limits at ppm or lower levels. Besides, the high activity of acyl chloride also raises a challenge to the direct measurement of CMI. Thus, we explored a simple esterification approach, which converts CMI into methyl 3-(methylonyl)-2-oxoimidazolidine-1-carboxylate completely by optimizing the reaction temperature and time. Furthermore, the selected reaction monitoring model of triple quadrupole mass spectrometer optimized by the Box-Behnken design significantly enhanced the sensitivity of ultra-trace level determination. The limit of detection and limit of quantification of the method were reached 0.014 and 0.02ppm, respectively, in the following validation study. A sensitive and specific ultra-performance liquid chromatography tandem mass spectrometry method for ultra-trace level determination of acyl chloride potential genotoxic impurity in mezlocillin drug substance has been successfully established in this study, which will provide a practical quality control tool of mezlocillin.

A comparison of chiral separations by supercritical fluid chromatography and high-performance liquid chromatography

The supercritical fluid chromatography technique is one of the most promising and innovative technologies used in the analysis as well as the separation of various racemates. The various enantiomers have been separated using SFC due to its several merits over conventional HPLC methods such as low cost, wide polarity compatibility, higher column efficiency, and fast analyzing capability, which are desirable features for the pharmaceutical industry. Therefore, it was considered worthwhile to review the applications of SFC in achiral and chiral separations. This review encompasses the latest literature exploration of various practical aspects of SFC for enantiomeric separation, purification, and applications to the pharmaceutical industry. A detailed discussion of SFC was made in terms of productivity, stacked injections, sample solubility, particle size, green incentive, organic solvent, recycle solvents, and economical aspects. Besides, future perspectives of SFC have also been highlighted. A comparison was also made between SFC and HPLC. SFC has been found better technique than HPLC for the chiral separations on a preparative scale. This article will be highly useful for chiral chromatography in pharmaceutical industries and research laboratories.

Simple methods of analyzing proteins and amino acids in small pollen samples

For many hymenopteran insects, floral pollen is essential for survival and development. Nutritional analysis of floral pollen is important to understand plant–pollinator interactions. Here, we evaluated a simple method for measuring proteins and amino acids in pollen; it requires samples far smaller than are required by conventional methods. We used a fluorometer for quantitative analysis of protein and investigated its accuracy with small samples (0.01–2.99 mg). Quantitative and qualitative analysis of amino acids was performed by conventional HPLC methods but with smaller samples (0.05–3.0 mg). The two analyses were highly accurate with samples of ≥1.0 mg. Our method allows nutritional analysis of pollen from a wide range of flower species, including those producing only small amounts of pollen, contributing to a better understanding of the interactions between flowers and flower-visiting insects.

Analytical quality by design approach versus conventional approach: Development of HPLC-DAD method for simultaneous determination of etizolam and propranolol hydrochloride

Considering the importance of robust analytics in the pharmaceutical industry, an analytical quality by design (AQbD) based reversed phase liquid chromatographic method was developed for the quantification of propranolol hydrochloride (PRO) and etizolam (ETZ). The developed method was validated and compared with an analytical method of conventional approach. In AQbD approach, analytical target profile (ATP) and risk assessment were tailored to identify critical method variables (CMVs) as inputs. Accordingly, pH (X1) % aqueous composition (X2), flow rate (X3) and % triethylamine (X4) were identified as CMVs and the respective retention time (Y1 and Y2) and theoretical plate (Y3 and Y4) for PRO and ETZ were set as method responses. The central composite design (CCD) predicted the scientific relationship between CMVs (Xn) and method responses (Yn). Then, the method operable design region (MODR) was arrived from systematic simulation and contour plots. There were six candidate methods selected from MODR and they demonstrated the r 2 value > 0.9, thus the model proved its consistency for prediction. The robust range for the selected CMVs was derived from MODR, thus the ranges were 3–4 for pH, 30–35 for % aqueous composition, 0.9–1.1 mL min−1 for flow rate and 0.1–0.2 for % TEA buffer. Finally, the target AQbD method was optimized with pH; 3.5 (X1), 30% of aqueous composition in mobile phase with acetonitrile (X2), 0.9 mL min−1 of flow rate of (X3) and 0. 15% trimethylamine as buffer (X4) and further validated as per ICH Q2 guidelines. The result was compared with the conventional LC method with respect to specificity, accuracy, precision and robustness. The conventional HPLC method failed for robustness and showed relatively higher % RSD values than AQbD method.

Development of Electrochemical Sensor Based on Thiolated Calixarene-Functionalized Gold Nanoparticles for the Selective Recognition of Anthracene

In this work, an electrochemical sensor for anthracene detection was constructed based on a modified electrode consisting of thiolated calixarene (TC4)-functionalized gold nanoparticles (AuNPs) and electrochemically reduced graphene oxide (ERGO). Firstly, graphene oxide GO) was reduced on screen printed carbon electrodes (SPCE), followed by the immobilization of AuNPs and TC4 through a self-assembly process to form TC4/AuNPs/ERGO-SPCE. The molecules’ structure was characterized using FTIR and Raman spectroscopy, the sample’s morphology and topology were characterized using Field Emission Scanning Electron Microscopy (FESEM), High Resolution Transmission Electron Microscopy (HRTEM) and electrochemical performance of developed sensor was characterized using electrochemical method. Owing to the outstanding supramolecular recognition of anthracene by TC4, the target analyte was detected accurately with a linear range of 1- $7~\mu \text{M}$ and limit of detection down to $0.0625~\mu \text{M}$ (S/N = 3). Additionally, the sensor was acknowledged for its excellent anti-interference properties in the presence of organic compounds as well as inorganic metal ions. The proposed sensor also yielded comparable results to conventional HPLC method, with a satisfactory recovery of 92–98% in environmental samples.

A Rapid Method to Measure Serum Retinol Concentrations in Japanese Black Cattle Using Multidimensional Fluorescence

Vitamin A levels in fattening Japanese Black cattle affect meat quality; therefore, it is important to monitor serum retinol concentrations. To simplify and accelerate the evaluation of serum retinol concentrations in cattle, we developed a new predictive method using excitation-emission matrix (EEM) fluorescence spectrophotometry. For analytical comparison, the concentration of serum retinol was also measured using the conventional HPLC method. We examined excitation (Ex) and emission (Em) wavelengths of cattle serum, which were 250–450 and 250–600 nm, respectively. Parallel factor analysis separated four components from EEM data, one of which was related to retinol. Next, a partial least square regression model was created using the obtained EEMs as explanatory variables and accrual measurement values as objective variables. The determination coefficient value (R2), root mean squared error of prediction (RMSEP), and the ratio of performance to deviation (RPD) of the model were determined. A comparison with reference values found that R2, RMSEP, and RPD of the calibration model were 0.95, 6.4 IU/dl, and 4.2, respectively. This implies that EEM can estimate the serum retinol concentration with high accuracy. Additionally, the fluorescent peaks that contributed to the calibration, which were extracted from the regression coefficient and variable importance in projection plots, were Ex/Em = 320/390 and 330/520 nm. Thus, we assume that this method observes not only free retinol, but also retinol-binding protein. In conclusion, multidimensional fluorescence analysis can accurately and quickly determine serum retinol concentrations in fattening cattle.

A NOVEL VALIDATED UHPLC METHOD FOR ESTIMATION OF ASSAY AND ITS RELATED SUBSTANCES OF TRICHOSTATIN-A

Objective: The main objective of the research work is to develop and validate a rapid UHPLC method for the estimation of assay and its related substances of Trichostatin A (TSA) in pharmaceutical samples.&#x0D; Methods: The UHPLC method developed for chromatographic separation between TSA and its related compounds on Poroshell 120 SB C18(50×4.6) mm; 2.7 µm RRLC column using Agilent RRLC (UHPLC) system with linear gradient elution.&#x0D; Results: The developed UHPLC method has shown excellent chromatographic separation between TSA and its related compounds within 12 min run time, during validation experiments, specificity study revealed that the peak threshold was more than the peak purity and no purity flag was observed. Repeatability, intra, and inter-day precision results were well within the tolerable limits. Limits of detection concentrations were found between 0.075 to 0.077 ppm and the limit of quantitation is between 0.252 to 0.258 ppm for related compounds and TSA. The related substances method recoveries were found between 80 and 120 % and assay method recovery was found between 98.0 to 102.0%.&#x0D; Conclusion: The developed method capability was proven for the assay of TSA and its related compounds in pharmaceutical samples and the method shows eco-friendlier than routine, conventional HPLC methods in terms of analysis time, cost and HPLC effluent waste.

Impurity profiling high-performance-thin-layer chromatography method involving the assay of essential human micronutrient niacin with eco-scale assessment.

Currently, analytical scientists are paying special attention to reducing reliance on hazardous chemicals in various analytical methods. By embracing this concept, we developed an eco-friendly high-performancethin-layer chromatography (HPTLC) method as an alternative for the conventional HPLC method for the determination of an essential human micronutrient, niacin (NIA), which is used improve the lipid profile of patients. Furthermore, the proposed HPTLC method is capable of determining the structurally related impurities of NIA such as pyridine-2,5-dicarboxylic acid, isonicotinic acid, pyridine, and 5-ethyl-2-methylpyridine, which exhibit nephrotoxic and hepatotoxic effects. The separation of this challenging mixture was achieved on HPTLC sheets using a mixture of ethyl acetate/ethanol/ammonia solution (6:4:0.05, v/v/v), and then the dried plates were scanned at 254 nm. The analytical eco-scale assessment protocol was used to assess the greenness profile of the presented method and compare it with the reported HPLC method. The suggested method was found to be greener with regard to the consumption of solvents and the yielding of waste. The results suggest that the described method can be safely implemented for the routine analysis of NIA pharmaceutical dosage without the interference of potential impurities in quality control laboratories.

Laboratory-Scale Preparation and Characterization of Dried Extract of Muirapuama (Ptychopetalumolacoides Benth) by Green Analytical Techniques.

This work reports on the preparation of a drying process from the ethanolic extract of Muirapuama and its characterization through green analytical techniques. The spray-drying processes were performed by using ethanolic extract in a ratio of 1:1 extract/excipient and 32 factorial design. The properties of dried powder were investigated in terms of total flavonoid content, moisture content, powder yield, and particle size distribution. An analytical eco-scale was applied to assess the greenness of the developed protocol. Ultra-high performance liquid chromatography (UHPLC)with reduced solvent consumption in the analysis was compared to the conventional HPLC method. A Fourier transform near-infrared (FT-NIR) spectroscopic method was applied based on the principal component scores for the prediction of extract/excipient mixtures and partial least squares regression model for quantitative analysis. NIR spectroscopy is an economic, powerful, and fast methodology for the detection of excipient in muirapuama dried extracts, generating no residue in the analysis. Scanning electron microscopy (SEM) images showed samples with a higher concentration of excipient, presenting better morphological characteristics and a lower moisture absorption rate. An eco-scale score value of 85 was achieved for UHPLC and 100 was achieved for NIR (excellent green analysis). Above all, these methods are rapid and green for the routine analysis of herbal medicines based on dried extracts.

Core-shell particles and monolithic columns; tools for simultaneous LC analysis of avanafil, sildenafil, apomorphine, trazodone, yohimbine, tramadol and dapoxetine in pharmaceutical dosage forms, counterfeit products and human plasma.

By 2025, it's estimated that 322 million males worldwide will suffer from sexual disorders. This can give an estimation for the size of the pharmaceutical and counterfeit products industry for the next few years. Meanwhile, green analytical chemistry forced itself to decrease the massive environmental pollution and hence new analytical methodologies are needed to replace the old ones that consume large amounts of hazardous solvents. In this research, two new methods were validated for determination of seven recognized drugs used in treatment of male impotence, premature ejaculation as well as enhancing sexual libido by HPLC on RP-C18 core–shell particulate and monolithic columns. The study was extended to compare the capabilities of those stationary phases to accommodate greener chromatography concepts without loss of efficiency. Both morphologies shortened the analysis time relative to the previously reported conventional HPLC methods by different approaches. Core–shell particles had higher efficiency in terms of theoretical plates' number and enhanced resolution power which enabled lower detection limits. However, the monolithic column had lower column backpressure which enabled the use of ethanol as a greener alternative solvent at even higher flow rates. The methods were finally applied successfully for the determination of drugs under study in pharmaceutical dosage forms, counterfeit products and in human plasma.

Simultaneous HPLC-DAD determination of pseudoephedrine HCl, sodium benzoate, sunset yellow, and methyl paraben in syrup preparation by use of partial least squares and principal component regression

The chromatographic methods (high performance liquid chromatography-diode array detection (HPLC-DAD)) combined with a partial least squares (type PLS-2) and principal component regression (PCR) were applied to the simultaneous determination of pseudoephedrine hydrochloride, sodium benzoate, sunset yellow, and methyl paraben in a syrup sample. By the aid of chromatographic data, the developed methods successfully solved the overlapped chromatographic peaks in HPLC analysis with the short elution time. A series of synthetic solutions containing different concentrations of analytes were used to check the prediction ability of the proposed methods. Satisfactory results were obtained by the PLS-2 and PCR methods for synthetic samples, with the average recoveries ranging from 98.33–100.20% to 98.44–100.07%, respectively. The developed methods were applied to a commercial syrup sample and the results were statistically compared with the conventional HPLC method. Considering the advantages of the chemometrics-assisted HPLC-DAD strategy, such as a short analysis time, simple and low-cost mobile phase, rapid sample preparation, the proposed methods could be an attractive alternative and green chromatography strategy for the determination of pseudoephedrine hydrochloride, sodium benzoate, sunset yellow, and methyl paraben in pharmaceutical samples.

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis

The currently available haemoglobin A1c (HbA1c) enzymatic assay consists of two specific steps: proteolysis of HbA1c and oxidation of the liberated fructosyl peptide by fructosyl peptide oxidase (FPOX). To develop a more convenient and high throughput assay, we devised novel protease-free assay system employing modified FPOX with HbA1c oxidation activity, namely HbA1c direct oxidase (HbA1cOX). AnFPOX-15, a modified FPOX from Aspergillus nidulans, was selected for conversion to HbA1cOX. As deduced from the crystal structure of AnFPOX-15, R61 was expected to obstruct the entrance of bulky substrates. An R61G mutant was thus constructed to open the gate at the active site. The prepared mutant exhibited significant reactivity for fructosyl hexapeptide (F-6P, N-terminal amino acids of HbA1c), and its crystal structure revealed a wider gate observed for AnFPOX-15. To improve the reactivity for F-6P, several mutagenesis approaches were performed. The ultimately generated AnFPOX-47 exhibited the highest F-6P reactivity and possessed HbA1c oxidation activity. HbA1c levels in blood samples as measured using the direct assay system using AnFPOX-47 were highly correlated with the levels measured using the conventional HPLC method. In this study, FPOX was successfully converted to HbA1cOX, which could represent a novel in vitro diagnostic modality for diabetes mellitus.

Highly Sensitive Detection of Carbendazim and Its Electrochemical Oxidation Mechanism at a Nanohybrid Sensor

Sensitive and rapid detection of pesticides in food has become an important and crucial research area due to the extensive use of agriculture residues and stringent environmental protection acts. In present work, an electrochemical nanohybrid sensor was facilely developed based on carboxymethyl cellulose (CMC) fuctionalized carbon nanotubes (CNTs). The sensor shows high sensitivity, excellent stability and good anti-interference capability in the detection of carbendazim (MBC) by differential pulse voltammetry method. Under optimized conditions, it exhibits a wide linearity of 0.03 μmol·L−1 – 10 μmol·L−1 with a low detection limit of 0.015 μmol·L−1. In real samples analyses for peer and kiwifruit, the proposed sensor shows good recoveries which are comparable with that of the conventional HPLC method. In addition, the oxidation mechanism of MBC at the nanohybrid sensor was studied using voltammetric methods and validated according to the density functional theory (DFT) calculation. The results reveal that the oxidation of carbendazim is a typical adsorption-controlled electrochemical oxidation process proceeding with four protons and four electrons. The proposed nanohybrid sensor is a promising alternative to the currently used methods for carbendazim detection and our results provide a new insight in MBC electrochemical oxidation mechanism.

Electrochemical detection of microcystin-LR based on its deleterious effect on DNA

This work reveals the deleterious effect of microcystin-LR (MC-LR) on the conformation of DNA and develops an electrochemical biosensor for detection of MC-LR. The biosensor is prepared by physically immobilizing calf thymus DNA (ctDNA) on gold electrode. In the presence of MC-LR, the conformation change of immobilized ctDNA decreases the electron transfer impedance, thus enhances the amperometric response. The proposed method shows a linear range of 4–512 ng/L and a detection limit of 1.4 ng/L, which is 700-fold lower than the guideline level suggested by the World Health Organization. The detection results are in good agreement with those from the conventional HPLC method. This biosensor possesses good stability against other components in natural water sample, and has been used for detection of MC-LR in local water bodies, indicating its application promise.

Antioxidant and anticancer activities of an Aporphine alkaloid isolated from Alphonsea sclerocarpa

Herein, we report on the anticancer and antioxidant activity of an Aporphine alkaloid isolated from medicinal plant Alphonsea sclerocarpa. Conventional column chromatography and preparative HPLC methods were used to isolate and purify the alkaloid. Based on NMR, FTIR and MS/MS spectroscopic techniques the compound was identified as Crebanine. This is the first report on the presence of Crebanine from the leaves of Alphonsea sclerocarpa. Antioxidant activity was evaluated using DPPH and FRAP assay. The antioxidant activity was dose-dependent and activity increased with the increase in the concentration. The cytotoxicity assay was carried out using MTT assay. The results showed remarkable cytotoxic activity against K562 (CML-chronic myeloid Leukaemia blood cancer) cells with a % inhibition of 37.545 at 500µm concentration after an incubation of 24h.

Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay.

Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.